AU703070B2

AU703070B2 - Method and product for regulating cell responsiveness to external signals

Info

Publication number: AU703070B2
Application number: AU80177/94A
Authority: AU
Inventors: Gary L Johnson
Original assignee: National Jewish Center for Immunology and Respiratory Medicine
Current assignee: National Jewish Health
Priority date: 1993-04-15
Filing date: 1994-10-14
Publication date: 1999-03-11
Anticipated expiration: 2014-04-15
Also published as: CA2186526C; JPH09511906A; DE69434650D1; JP3980609B2; CA2186526A1; JP3980635B2; AU8017794A; DE69434650T2; ATE319821T1; JP2006051026A

Description

WO 95/28421 PCT/US94/11690 METHOD AND PRODUCT FOR REGULATING CELL RESPONSIVENESS TO EXTERNAL SIGNALS FIELD OF THE INVENTION This invention relates to isolated nucleic acid molecules encoding MEKK proteins, substantially pure MEKK proteins, and products and methods for regulating signal transduction in a cell.

SUMMARY OF THE INVENTION The present invention relates to a substantially pure MEKK protein capable of phosphorylating mammalian MEK protein, in which the MEKK protein comprises a catalytic domain. The present invention includes a substantially pure MEKK protein capable of regulating signals initiated from a growth factor receptor on the surface of a cell by regulating the activity of MAPK protein, the ability to regulate being divergent from Raf protein signal regulation. In particular, the substantially pure MEKK protein comprises at least a portion of an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with a nucleic acid molecule encoding an amino acid sequence including SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10. The substantially pure MEKK protein capable of regulating the activity of MAPK protein, said protein having an amino acid sequence distinct from Raf protein.

The present invention also includes a formulation comprising at least one isolated protein having at least a portion of an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with a nucleic acid molecule encoding an amino WO 95/28421 PCT/US94/11690 -2acid sequence including SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID One aspect of the present invention includes an isolated nucleic acid molecule having a sequence encoding a protein capable of phosphorylating mammalian MEK independent of Raf protein and capable of regulating the activity of MAPK protein. In particular, the present invention includes an isolated nucleic acid molecule capable of hybridizing under stringent conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9.

Another aspect of the present invention includes a recombinant molecule, comprising a nucleic acid molecule capable of hybridizing under stringent conditions with a nucleic acid sequence including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9, in which the nucleic acid molecule is operatively linked to an expression vector.

Yet another aspect of the present invention is a recombinant cell transformed with a recombinant molecule, comprising a nucleic acid molecule operatively linked to an expression vector, the nucleic acid molecule comprising a nucleic acid sequence capable of hybridizing under stringent conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9 the nucleic WO 95/28421 PCT/US94/11690 -3acid sequence shown in Table 1, Table 2, Table 3, Table 4 and Table The present invention also includes a method for regulating the homeostasis of a cell comprising regulating the activity of an MEKK-dependent pathway relative to the activity of a Raf-dependent pathway in the cell. In particular, the method comprises regulating the apoptosis of the cell. Such a method is useful for the treatment of a medical disorder. In particular, the method is useful for inhibiting tumorigenesis and autoimmunity.

According to the present invention, the method for treatment of a disease, comprises administering to a patient an effective amount of a therapeutic compound comprising at least one regulatory molecule including a molecule capable of decreasing the activity of a Rafdependent pathway, a molecule capable of increasing the activity of an MEKK-dependent pathway, and combinations thereof, in which the effective amount comprises an amount which results in the depletion of harmful cells involved in the disease.

Also included in the present invention is a therapeutic compound capable of regulating the activity of an MEKK-dependent pathway in a cell identified by a process, comprising: contacting a cell with a putative regulatory molecule; and determining the ability of the putative regulatory compound to regulate the activity of an MEKK-dependent pathway in the cell by measuring the -4activation of at least one member of said MEKK-dependent pathway.

One embodiment of the present invention includes a substantially pure protein, in which the protein is isolated using an antibody capable of selectively binding to an MEKK protein capable of phosphorylating mammalian MEK protein and capable of regulating the activity of MAPK protein independent of Raf protein, the antibody capable of being produced by a method comprising: administering to an animal an effective amount of a substantially pure MEKK protein of the present invention; and (b) recovering an antibody capable of selectively binding to the MEKK protein.

Another embodiment of the present invention includes an isolated antibody capable of selectively binding to an MEKK protein, the antibody capable of being produced by a method comprising administering to an animal an effective amount of a substantially pure protein of the present invention, and recovering an antibody capable of selectively binding to the MEKK protein.

According to a first aspect the present invention provides an isolated and/or recombinant protein comprising an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ i SID NO: 8 aiftdSEQ ID According to a second aspect the present invention provides a formulation 20 comprising at least one isolated and/or recombinant protein having at least a portion of an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 20936-00.DOC 4a- According to a third aspect the present invention provides a formulation comprising at least one isolated and/or recombinant protein having at least a portion of an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6.

According to a fourth aspect the present invention provides an isolated nucleic acid molecule capable of hybridizing under stringent conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQIDNO: 9.

According to a fifth aspect the present invention provides an isolated nucleic acid molecule capable of hybridizing under stringent conditions with the nucleic acid sequence of SEQ ID NO: 1.

According to a sixth aspect the present invention provides A recombinant 1. 15 molecule, comprising a nucleic acid molecule capable of hybridizing under stringent •conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, or the complementary •oe• sequence there f, said nucleic acid molecule being operatively linked to an expression *o vector.

o •a 20 According to a seventh aspect the present invention provides a recombinant molecule, comprising a nucleic acid molecule capable of hybridizing under stringent conditions with the nucleic acid sequence of SEQ ID NO: 1, or the complementary conditions with the nucleic acid sequence of SEQ ID NO: 1, or the complementary 20936-o00 DOC 4bsequence thereof, said nucleic acid molecule being operatively linked to an expression vector.

According to an eighth aspect the present invention provides a recombinant cell transoformed with the recombinant molecule of either of the sixth or seventh aspect.

According to a ninth aspect the present invention provides an isolated nucleic acid molecule which selectively hybridizes under stringent conditions to a MEKK nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, or the complementary sequence thereof, which nucleic acid molecule can specifically detect the MEKK nucleic acid sequence.

According to a tenth aspect the present invention provides an isolated nucleic acid molecule which selectively hybridizes under stringent conditions to the MEKK nucleic acid sequence of SEQ ID NO: 1, or the complementary sequence thereof, which nucleic acid molecule can specifically detect the MEKK nucleic acid sequence.

According to an eleventh aspect the present invention provides an isolated and/or 15 recombinant protein comprising an amino acid sequence encoded by a nucleic acid •sequence that is capable of hybridizing under stringent conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, and SEQID NO: 9, or the complementary sequence thereof, which protein modulates activation of map kinase kinases.

20 According to a twelfth aspect the present invention provides a fusion protein

S.

°0 comprising all or a portion of a MEKK protein, which fusion protein further comprises a second polypeptide sequence having an amino acid sequence distinct from an MEKK amino acid sequence.

20936-00. DOC 4c- According to a thirteenth aspect the present invention provides an isolated and/or recombinant protein comprising an amino acid sequence at least 80% identical to a MEKK sequence of SEQ ID NO: 8, or SEQ ID NO: According to a fourteenth aspect the present invention provides an isolated and/or recombinant protein encoded by a nucleic acid sequence having at least 80% homology with the nucleic aicd sequence of SEQ ID NO: 7 or SEQ ID NO: 9.

According to a fifteenth aspect the present invention provides a nucleic acid vector comprising a coding sequence for a recombinant protein, which coding sequence includes a nucleotide sequence which hybridizes under stringent conditions to a coding sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, or the complementary sequence thereof.

According to a sixteenth aspect the present invention provides a nucleic acid vector comprising a coding sequence for a recombinant protein, which coding sequence includes a nucleotide sequence which hybridizes under stringent conditions to the coding sequence SEQ ID NO: 1, or the complementary sequence thereof.

According to a seventeenth aspect the present invention provides an isolated and/or 000 0 recombinant protein comprising the amino acid sequence, as set forth in SEQ ID NO: 8, or SEQ ID NO: 10, or a functional fragment thereof.

According to an eighteenth aspect the present invention provides an method for 0 *0 20 regulating homeostasis of a cell comprising introducing into said cell a nucleic acid molecule according to the fourth aspect or a recombinant molecule according to the sixth aspect, which encodes a MEKK protein such that homeostasis of said cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein.

20936-00. DOC 4d According to an ninteenth aspect the present invention provides an method for regulating homeostasis of a cell comprising introducing into said cell a nucleic acid molecule according to the fifth aspect or a recombinant molecule according to the seventh aspect, which encodes a MEKK protein such that homeostasis of said cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein.

According to a twentieth aspect the present invention provides a method for regulating apoptosis of a cell comprising introducing into said cell a nucleic acid molecule according to the fourth aspect or a recombinant molecule according to the sixth aspect, which encodes an MEKK protein such that apoptosis of the cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein.

According to a twentyfirst aspect the present invention provides a method for regulating apoptosis of a cell comprising introducing into said cell a nucleic acid molecule according to the fifth aspect or a recombinant molecule according to the seventh aspect, which encodes an MEKK protein such that apoptosis of the cell is i regulated, wherein said MEKK protein regulates the activity of a MAPK signal transductior protein.

According to a twenty second aspect the present invention provides a method for 20 regulating mitogen activated protein kinase (MAPK) activity in a cell, comprising contacting the cell with a nucleic acid molecule according to the fourth aspect or a recombinant molecule according to the sixth aspect, which encodes a MEKK protein *that MAPK activity is regulated in the cell.

such that MAPK activity is regulated in the cell.

20936-0O.DOC 4e According to an ninteenth aspect the present invention provides an method for regulating homeostasis of a cell comprising introducing into said cell a nucleic acid molecule according to the fifth aspect or a recombinant molecule according to the seventh aspect, which encodes a MEKK protein such that homeostasis of said cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein.

According to a twentyfirst aspect the present invention provides a method for regulating apoptosis of a cell comprising introducing into said cell a nucleic acid molecule according to the fifth aspect or a recombinant molecule according to the seventh aspect, which encodes an MEKK protein such that apoptosis of the cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction-protein.

o According to a twenty second aspect the present invention provides a method for regulating mitogen activated protein kinase (MAPK) activity in a cell, comprising contacting the cell with a nucleic acid molecule according to the fourth aspect or a recombinant molecule according to the sixth aspect, which encodes a MEKK protein such that MAPK activity is regulated in the cell.

20936-00DOC 20936-00.DOC 4f- According to a twenty third aspect the present invention provides a method for regulating mitogen activated protein kinase (MAPK) activity in a cell, comprising contacting the cell with a nucleic acid molecule according to the fifth aspect or a recombinant molecule according to the seventh aspect, which encodes a MEKK protein such that MAPK activity is regulated in the cell.

According to a twenty fourth aspect the present invention provides a method for regulating mitogen ERK kinase kinase (MEKK) protein activity in a cell comprising transforming said cell with an antisense nucleic acid molecule such that MEKK protein activity is regulated in said cell.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

BACKGROUND OF THE INVENTION Mitogen-activated protein kinase (MAPKs) (also called extracellular signalregulated kinases or ERKs) are rapidly activated in response to ligand binding by both growth factor receptors that are tyrosine kinases (such as the epidermal growth factor 0 (EGF) receptor) and receptors that are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) such as the thrombin receptor. The o 20936-00 DOC WO 95/28421 PCT/US94/11690 MAPKs appear to integrate multiple intracellular signals transmitted by various second messengers. MAPKs phosphorylate and regulate the activity of enzymes and transcription factors including the EGF receptor, Rsk phospholipase A 2 c-Myc, c-Jun and Elk-1/TCF. Although the rapid activation of MAPKs by receptors that are tyrosine kinases is dependent on Ras, G protein-mediated activation of MAPK appears to occur through pathways dependent and independent of Ras.

Complementation analysis of the pheromone-induced signaling pathway in yeast has defined a protein kinase system that controls the activity of Spkl and Fus3-Kssl, the Schizosaccharomyces pombe and Saccharomyces cerevisiae homologs of MAPK (see for example, B.R. Cairns et al., Genes and Dev. 6, 1305 (1992); B.J. Stevenson et al., Genes and Dev. 6, 1293 (1992); S.A. Nadin-Davis et al., EMBO J.

7, 985 (1988); Y. Wang et al., Mol. Cell. Biol. 11, 3554 (1991). In S. cerevisiae, the protein kinase Ste7 is the upstream regulator of Fus3-Kssl activity; the protein kinase Stell regulates Ste7. The S. pombe gene products Byrl and Byr2 are homologous to Ste7 and Stell, respectively. The MEK (MAPK Kinase or ERK Kinase) or MKK (MAP Kinase kinase) enzymes are similar in sequence to Ste7 and Byrl. The MEKs phosphorylate MAPKs on both tyrosine and threonine residues which results in activation of MAPK.

The mammalian serine-threonine protein kinase Raf phosphorylates and activates MEK, which leads to activation of MAPK. Raf is activated in response to growth factor WO 95/28421 PCT/US94/11690 -6receptor tyrosine kinase activity and therefore Raf may activate MAPK in response to stimulation of membraneassociated tyrosine kinases. Raf is unrelated in sequence to Stell and Byr2. Thus, Raf may represent a divergence in mammalian cells from the pheromone-responsive protein kinase system defined in yeast. Cell and receptor specific differences in the regulation of MAPKs suggest that other Raf independent regulators of mammalian MEKs exist.

Certain biological functions, such as growth and differentiation, are tightly regulated by signal transduction pathways within cells. Signal transduction pathways maintain the balanced steady state functioning of a cell. Disease states can arise when signal transduction in a cell breaks down, thereby removing the tight control that typically exists over cellular functions. For example, tumors develop when regulation of cell growth is disrupted enabling a clone of cells to expand indefinitely.

Because signal transduction networks regulate a multitude of cellular functions depending upon the cell type, a wide variety of diseases can result from abnormalities in such networks. Devastating diseases such as cancer, autoimmune diseases, allergic reactions, inflammation, neurological disorders and hormone-related diseases can result from abnormal signal transduction.

Despite a long-felt need to understand and discover methods for regulating cells involved in various disease states, the complexity of signal transduction pathways has precluded the development of products and processes for WO 95/28421 PCT/US94/11690 -7regulating cellular function by manipulating signal transduction pathways in a cell. As such, there remains a need for products and processes that permit the implementation of predictable controls of signal transduction in cells, thus enabling the treatment of various diseases that are caused by abnormal cellular function.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a schematic representation of the signal pathways of vertebrates and yeast.

Fig. 2 is a schematic representation of the dual MEKK and Raf pathways divergent from Ras protein pathway.

Fig. 3A shows a Northern (RNA) blot of a single 7.8 kb MEKK mRNA in several cell lines and mouse tissues.

Fig. 3B shows a Southern (DNA) blot of the MEKK gene.

Fig. 3C shows an immunoblot showing expression of the 78 kD and 50 kD forms of MEKK in rodent cell lines.

Fig. 4 shows immunprecipitates of MEKK protein using MEKK antiserum.

Fig. 5 shows immunoblotting of MEKK protein in immunoprecipitates and cell lysates.

Fig. 6A shows the activation of MAPK in COS cells transfected with MEKK.

Fig. 6B is an immunoblot showing expression of MEKK in cells either treated or not treated with EGF.

Fig. 7 shows the activation and phosphorylation of MEK in COS cells transfected with MEKK.

WO 95/28421 PCT/US94/11690 -8- Fig. 8A shows the phosphorylation of MEK-1 by MEKK.

Fig. 8B shows the time course of phosphorylation of MEK-1 by MEKK expressed in COS cells.

Fig. 8C is an immunoblot of MEKK overexpressed in COS cells.

Fig. 9A shows the phosphorylation of MAPK by activated MEK-1.

Fig. 9B shows phosphorylation of MEK-1 by immunoprecipitated MEKK.

Fig. 10A shows the phosphorylation of MEK-1 by activated Raf.

Fig. 10B shows the phosphorylation state of Raf isolated from COS cells which are overexpressing MEKK and have been treated with EGF.

Fig. 11 shows the relative ability of immunoprecipitated MEKK and Raf-B to phosphorylate kinase inactive MEK-1.

Fig. 12 shows a time course of EGF-stimulated MEKK and Raf-B activation.

Fig. 13 shows that the immunodepletion of Raf-B from MEKK immunoprecipitates has no effect on MEKK activity.

Fig. 14 shows that the immunodepletion of Raf-B from MEKK immunoprecipitates decreases Raf-B activity.

Fig. 15 shows MEKK activity in FPLC Mono Q ionexchange column fractions of PC12 cell lysates.

Fig. 16 shows inhibition of MEKK and Raf-B activation by dominant negative N 17 RAS expression.

Fig. 17 shows activation of MEK protein by 98 kD MEKK.

WO 95/28421 PCT/US94/11690 -9- Fig. 18 shows inhibition of EGF activation of MEKK by forskolin.

Fig. 19 shows improved MEKK activity by truncated MEKK molecules.

Fig. 20 shows JNK activation by MEKK protein.

Fig. 21 shows regulation of c-Myc controlled transcription and not CREB controlled transcription by MEKK protein.

Fig. 22 is a schematic representation of MEKK regulation of c-Myc controlled transcription.

Fig. 23 shows induction of p38 MAPK phosphorylation by MEKK 3.

Fig. 24 shows induction of cellular apoptosis in swiss 3T3 and REF52 cells by beauvericin.

Fig. 25 shows induction of cellular apoptosis in REF52 cells by MEKK.

Fig. 26 shows induction of cellular apoptosis in Swiss 3T3 and REF52 cells by MEKK.

Fig. 27 shows 3 representative microscopic views of apoptotic REF52 cells expressing MEKK protein.

Fig. 28 shows 3 representative microscopic views of apoptotic Swiss 3T3 cells expressing MEKK protein.

Fig. 29 shows similar stimulation of MAPK activity by MEKK protein and Raf protein.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel mitogen ERK kinase kinase protein (MEKK) capable of regulating signal WO 95/28421 PCT/US94/11690 transduction in cells. The present invention includes a novel method for treating disease by regulating the activity of cells involved in such disease. The present invention is particularly advantageous in that the novel product and method of the present invention is capable of regulating a signal transduction pathway that can lead to cellular apoptosis.

One embodiment of the present invention is an isolated MEKK protein. According to the present invention, an isolated protein is a protein that has been removed from its natural milieu. An isolated MEKK protein can, for example, be obtained from its natural source, be produced using recombinant DNA technology, or be synthesized chemically. As used herein, an isolated MEKK protein can be a full-length MEKK protein or any homologue of such a protein, such as an MEKK protein in which amino acids have been deleted a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the modified protein is capable of phosphorylating mitogen ERK kinase (MEK) and/or Jun ERK kinase (JEK). A homologue of an MEKK protein is a protein having an amino acid sequence that is sufficiently similar to a natural MEKK protein amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to with) a WO 95/28421 PCT/US94/11690 -11nucleic acid sequence encoding the natural MEKK protein amino acid sequence. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. A homologue of an MEKK protein also includes a protein having an amino acid sequence that is sufficiently cross-reactive such that the homologue has the ability to elicit an immune response against at least one epitope of a naturallyoccurring MEKK protein.

The minimal size of a protein homologue of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. As such, the size of the nucleic acid molecule encoding such a protein homologue is dependent on nucleic acid composition, percent homology between the nucleic acid molecule and complementary sequence, as well as upon hybridization conditions per se temperature, salt concentration, and formamide concentration). The minimal size of such nucleic acid molecules is typically at least about 12 to about nucleotides in length if the nucleic acid molecules are GCrich and at least about 15 to about 17 bases in length if they are AT-rich. As such, the minimal size of a nucleic WO 95/28421 PCT/US94/11690 -12acid molecule used to encode an MEKK protein homologue of the present invention is from about 12 to about 18 nucleotides in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof. Similarly, the minimal size of an MEKK protein homologue of the present invention is from about 4 to about 6 amino acids in length, with preferred sizes depending on whether a full-length, multivalent protein fusion protein having more than one domain each of which has a function), or a functional portion of such a protein is desired.

MEKK protein homologues can be the result of allelic variation of a natural gene encoding an MEKK protein. A natural gene refers to the form of the gene found most often in nature. MEKK protein homologues can be produced using techniques known in the art including, but not limited to, direct modifications to a gene encoding a protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. The ability of an MEKK protein homologue to phosphorylate MEK and/or JEK protein can be tested using techniques known to those skilled in the art. Such techniques include phosphorylation assays described in detail in the Examples section.

In one embodiment, an MEKK protein of the present invention is capable of regulating an MEKK-dependent WO 95/28421 PCT/US94/11690 -13pathway. According to the present invention, an MEKKdependent pathway refers generally to a pathway in which MEKK protein regulates a pathway substantially independent of Raf, and a pathway in which MEKK protein regulation converges with common members of a pathway involving Raf protein, in particular, MEK protein (see Fig. A suitable MEKK-dependent pathway includes a pathway involving MEKK protein and JEK protein, but not Raf protein. One of skill in the art can determine that regulation of a pathway by an MEKK protein is substantially independent of Raf protein by comparing the ability of an MEKK protein and a Raf protein to regulate the phosphorylation of a downstream member of such pathway using, for example, the general method described in Example 16. An MEKK protein regulates a pathway substantially independently of Raf protein if the MEKK protein induces phosphorylation of a member of the pathway downstream of MEKK proteins including JEK, JNK, Jun and/or ATF-2) by an amount significantly greater than that seen when Raf protein is utilized. For example, MEKK induction of phosphorylation of JNK is preferably at least about fold, more preferably at least about 20-fold and even more preferably at least about 30-fold, greater phosphorylation of JNK protein than the phosphorylation induced when using Raf protein. If MEKK induction of phosphorylation is similar to Raf protein induction of phosphorylation, then one of skill in the art can conclude that regulation of a pathway by an MEKK protein includes members of a signal WO 95/28421 PCT/US94/11690 -14transduction pathway that could also include Raf protein.

For example, MEKK induction of phosphorylation of MAPK is of a similar magnitude as induction of phosphorylation with Raf protein.

A "Raf-dependent pathway" can refer to a signal transduction pathway in which Raf protein regulates a signal transduction pathway substantially independently of MEKK protein, and a pathway in which Raf protein regulation converges with common members of a pathway involving MEKK protein. The independence of regulation of a pathway by a Raf protein from regulation of a pathway by an MEKK protein can be determined using methods similar to those used to determine MEKK independence.

In another embodiment, an MEKK protein is capable of regulating the activity of signal transduction proteins including, but not limited to, mitogen ERK kinase (MEK), mitogen activated protein kinase (MAPK), transcription control factor (TCF), Ets-like-1 transcription factor (Elk- Jun ERK kinase (JEK), Jun kinase (JNK), stress activated MAPK proteins, Jun, activating transcription factor-2 (ATF-2) and/or Myc protein. As used herein, the "activity" of a protein can be directly correlated with the phosphorylation state of the protein and/or the ability of the protein to perform a particular function phosphorylate another protein or regulate transcription).

Preferred MEK proteins regulated by an MEKK protein of the present invention include MEK-1 and/or MEK-2. Preferred MAPK proteins regulated by an MEKK protein of the present WO 95/28421 PCT/US94/11690 invention include p38 MAPK, p42 MAPK and/or p44 MAPK. A preferred MEKK protein that is capable of phosphorylating p38 MAPK protein includes a protein encoded by the nucleic acid sequence represented by SEQ ID NO:5 with a protein having the amino acid sequence represented by SEQ ID NO:7 being more preferred. Preferred stress activated MAPK proteins regulated by an MEKK protein of the present invention include Jun kinase (JNK), stress activated MAPK-a and/or stress activated MAPK-B. An MEKK protein of the present invention is capable of increasing the activity of an MEK protein over basal levels of MEK levels found in nature when not stimulated). For example, an MEKK protein is preferably capable of increasing the phosphorylation of an MEK protein by at least about 2-fold, more preferably at least about 3-fold, and even more preferably at least about 4-fold over basal levels when measured under conditions described in Example 9.

A preferred MEKK protein of the present invention is also capable of increasing the activity of an MAPK protein over basal levels of MAPK levels found in nature when not stimulated). For example, an MEKK protein of the present invention is preferably capable of increasing MAPK activity at least about 2-fold, more preferably at least about 3-fold, and even more preferably at least about 4fold over basal activity when measured under the conditions described in Example 3.

Moreover, an MEKK protein of the present invention is capable of increasing the activity of a JNK protein. JNK WO 95/28421 PCT/US94/11690 -16regulates the activity of the transcription factor JUN which is involved in controlling the growth and differentiation of different cell types, such as T cells, neural cells or fibroblasts. JNK shows structural and regulatory homologies with MAPK. For example, an MEKK protein of the present invention is preferably capable of inducing the phosphorylation of JNK protein at least about times more than Raf, more preferably at least about times more than Raf, and even more preferably at least about 50 times more than Raf, when measured under conditions described in Example 16.

A preferred MEKK protein of the present invention is additionally capable of inducing the phosphorylation of a c-Myc transcriptional transactivation domain protein in such a manner that the phosphorylated transcriptional transactivation domain of c-Myc is capable of regulating gene transcription. The ability of an MEKK protein to regulate phosphorylation of a c-Myc transcriptional transactivation domain protein exceeds the ability of Raf protein or cyclic AMP-dependent protein kinase to regulate a c-Myc protein. For example, an MEKK protein of the present invention is preferably capable of inducing luciferase gene transcription by phosphorylated c-Myc transcriptional transctivation domain protein at least about 25-fold, more preferably at least about 35-fold, and even more preferably at least about 45-fold, over Raf induction when measured under the conditions described in Example 17.

WO 95/28421 PCT/US94/11690 -17- Another aspect of the present invention relates to the ability of MEKK activity to be stimulated by growth factors including, but not limited to, epidermal growth factor (EGF), neuronal growth factor (NGF), tumor necrosis factor (TNF), C5A, interleukin-8 monocyte chemotactic protein 1 (MIPla), monocyte chemoattractant protein 1 (MCPplatelet activating factor (PAF), N-Formyl-methionylleucyl-phenylalanine (FMLP), leukotriene B 4

(LTB

4 gastrin releasing peptide (GRP), IgE, major histocompatibility protein (MHC), peptide, superantigen, antigen, vasopressin, thrombin, bradykinin and acetylcholine. In addition, the activity of an MEKK protein of the present invention is capable of being stimulated by compounds including phorbol esters such as TPA. A preferred MEKK protein is also capable of being stimulated by EGF, NGF and TNF (especially TNFa).

Preferably, the activity of an MEKK protein of the present invention is capable of being stimulated at least 2-fold over basal levels levels found in nature when not stimulated), more preferably at least about 4-fold over basal levels and even more preferably at least about 6-fold over basal levels, when a cell producing the MEKK protein is contacted with EGF under the conditions described in Example 3.

Similarly, the activity of an MEKK protein of the present invention is capable of being stimulated at least 1-fold over basal levels, more preferably at least about 2fold over basal levels and even more preferably at least WO 95/28421 PCT/US94/11690 -18about 3-fold over basal levels by NGF stimulation, when a cell producing the MEKK protein is contacted with NGF under the conditions described in Example 9.

Preferably, an MEKK protein of the present invention is capable of being stimulated at least 0.5-fold over basal levels, more preferably at least about 1-fold over basal levels and even more preferably at least about 2-fold over basal levels by TPA stimulation when a cell producing the MEKK protein is contacted with TPA under the conditions described in Example 9.

TNF is capable of regulating cell death and other functions in different cell types. The present inventor discovered that MEKK stimulation by TNF is independent of Raf. Similarly, the present inventor is the first to appreciate that an MEKK protein can be directly stimulated by ultraviolet light (UV) damage of cells while a Rafdependent pathway cannot. Therefore, both TNF and UV stimulate MEKK activity without substantially activating Raf. In addition, both UV and TNF activation of MEKK is Ras dependent.

Another aspect of the present invention is the recognition that an MEKK protein of the present invention is capable of regulating the apoptosis of a cell, an ability not shared by Raf protein. As used herein, apoptosis refers to the form of cell death that comprises: progressive contraction of cell volume with the preservation of the integrity of cytoplasmic organelles; condensation of chromatin, as viewed by light or electron

I

WO 95/28421 PCT/US94/11690 -19microscopy; and DNA cleavage, as determined by centrifuged sedimentation assays. Cell death occurs when the membrane integrity of the cell is lost and cell lysis occurs.

Apoptosis differs from necrosis in which cells swell and eventually rupture.

A preferred MEKK protein of the present invention is capable of inducing the apoptosis of cells, such that the cells have characteristics substantially similar to cytoplasmic shrinkage and/or nuclear condensation as shown in Figs. 24, 25, 26, 27 and 28. The apoptotic cells in Figs. 24 through 28 were obtained when cells were microinjected with expression plasmids encoding MEKK protein. Injected cells were identified using anti-B-Gal antibody and the DNA of the cells were stained with propidium iodide. Cytoplasmic organization was monitored using an anti-tubulin antibody. The cells were then imaged by differential fluorescent imaging microscopy using techniques standard in the art. The cells demonstrated apoptosis by displaying a morphology having cytoplasmic shrinkage and nuclear condensation.

A schematic representation of the cell growth regulatory signal transduction pathway that is MEKK dependent is shown in Fig. 2. An MEKK protein of the present invention is capable of regulating the activity of JEK protein, JNK protein, Jun protein and/or ATF-2 protein, and Myc protein, such regulation being substantially, if not entirely, independent of Raf protein. Such Rafindependent regulation can regulate the growth WO 95/28421 PCT/US94/11690 characteristics of a cell, including the apoptosis of a cell. In addition, an MEKK protein of the present invention is capable of regulating the activity of MEK protein, which is also capable of being regulated by Raf protein. As such, an MEKK protein of the present invention is capable of regulating the activity of MAPK protein and members of the Ets family of transcription factors, such as TCF protein, also referred to as Elk-1 protein.

Referring to Fig. 2, an MEKK protein of the present invention is capable of being activated by a variety of growth factors capable of activating Ras protein. In addition, an MEKK protein is capable of activating JNK protein which is also activated by Ras protein, but is not activated by Raf protein. As such, an MEKK protein of the present invention comprises a protein kinase at a divergence point in a signal transduction pathway initiated by different cell surface receptors. An MEKK protein is also capable of being regulated by TNF protein independent of Raf, thereby indicating an association of MEKK protein to a novel signal transduction pathway which is independent of Ras protein and Raf protein. Thus, an MEKK protein is capable of performing numerous unique functions independent of or by-passing Raf protein in one or more signal transduction pathways. An MEKK protein is capable of regulating the activity of MEK and/or JEK activity. As such, an MEKK protein is capable of regulating the activity of members of a signal transduction pathway that does not substantially include Raf activity. Such members include, WO 95/28421 PCT/US94/11690 -21but are not limited to, JNK, Jun, ATF and Myc protein. In addition, an MEKK protein is capable of regulating the members of a signal transduction pathway that does involve Raf, such members including, but are not limited to, MEK, MAPK and TCF. An MEKK protein of the present invention is thus capable of regulating the apoptosis of a cell independent of significant involvement by Raf protein.

In addition to the numerous functional characteristics of an MEKK protein, an MEKK protein of the present invention comprises numerous unique structural characteristics. For example, in one embodiment, an MEKK protein of the present invention includes at least one of two different structural domains having particular functional characteristics. Such structural domains include an NH 2 -terminal regulatory domain that serves to regulate a second structural domain comprising a COOHterminal protein kinase catalytic domain that is capable of phosphorylating an MEK protein and/or JEK protein.

According to the present invention, an MEKK protein of the present invention includes a full-length MEKK protein, as well as at least a portion of an MEKK protein capable of performing at least one of the functions defined above.

The phrase "at least a portion of an MEKK protein" refers to a portion of an MEKK protein encoded by a nucleic acid molecule that is capable of hybridizing, under stringent conditions, with a nucleic acid encoding a full-length MEKK protein of the present invention. Preferred portions of MEKK proteins are useful for regulating apoptosis in a WO 95/28421 PCT/US94/11690 -22cell. Additional preferred portions have activities useful for regulating MEKK kinase activity. Suitable sizes for portions of an MEKK protein of the present invention are as disclosed for MEKK protein homologues of the present invention.

In another embodiment, an MEKK protein of the present invention includes at least a portion of an MEKK protein having molecular weights ranging from about 70 kD to about 250 kD as determined by Tris-glycine SDS-PAGE, preferably using an 8% polyacrylamide SDS gel (SDS-PAGE) and resolved using methods standard in the art. A preferred MEKK protein has a molecular weight ranging from about 75 kD to about 225 kD and even more preferably from about 80 kD to about 200 kD.

In yet another embodiment, an MEKK protein of the present invention comprises at least a portion of an MEKK protein encoded by an mRNA (messenger ribonucleic acid) ranging from about 3.5 kb to about 12.0 kb, more preferably ranging from about 4.0 kb to about 11.0 kb, and even more preferably ranging from about 4.5 kb to about 10.0 kb.

Particularly preferred MEKK proteins comprise at least a portion of an MEKK protein encoded by an mRNA having a size ranging from about 4.5 kb to about 5.0 kb, a size ranging from about 6.0 kb to about 6.5 kb, a size of about 7.0 kb, or a size ranging from about 8.0 kb to about 10.0 kb.

In another embodiment, an NH 2 -terminal regulatory domain of the present invention includes an NH 2 -terminal comprising about 400 amino acids having at least about WO 95/28421 PCT/US94/11690 -23serine and/or threonine residues, more preferably about 400 amino acids having at least about 15% serine and/or threonine residues, and even more preferably about 400 amino acids having at least about 20% serine and/or threonine residues.

A preferred an NH 2 -terminal regulatory domain of the present invention includes an NH 2 -terminal comprising about 360 amino acids having at least about 10% serine and/or threonine residues, more preferably about 360 amino acids having at least about 15% serine and/or threonine residues, and even more preferably about 360 amino acids having at least about 20% serine and/or threonine residues.

Another preferred an NH 2 -terminal regulatory domain of the present invention includes an NH 2 -terminal comprising about 370 amino acids having at least about 10% serine and/or threonine residues, more preferably about 370 amino acids having at least about 15% serine and/or threonine residues, and even more preferably about 370 amino acids having at least about 20% serine and/or threonine residues.

In one embodiment, an MEKK protein of the present invention is devoid of SH2 and SH3 domains.

In another embodiment, an MEKK protein of the present invention includes at least a portion of an MEKK protein homologue preferably having at least about 50%, more preferably at least about 75%, and even more preferably at least about 85% amino acid homology (identity within comparable regions) with the kinase catalytic domain of a naturally occurring MEKK protein. Another MEKK protein of WO 95/28421 PCT/US94/11690 -24the present invention also includes at least a portion of an MEKK homologue of the present invention has at least about 10%, more preferably at least about 20%, and even more preferably at least about 30% amino acid homology with the NH 2 -terminal regulatory domain of an MEKK protein of a naturally occurring MEKK protein.

The sequences comprising the catalytic domain of an MEKK protein are involved in phosphotransferase activity, and therefore display a relatively conserved amino acid sequence. The NH 2 -terminal regulatory domain of an MEKK protein, however, can be substantially divergent. The lack of significant homology between MEKK protein NH 2 -terminal regulatory domains is related to the regulation of each of such domains by different upstream regulatory proteins.

For example, an MEKK protein can be regulated by the protein Ras, while others can be regulated independent of Ras. In addition, some MEKK proteins can be regulated by the growth factor TNFa, while others cannot. As such, the NH2-terminal regulatory domain of an MEKK protein provides selectivity for upstream signal transduction regulation, while the catalytic domain provides for MEKK substrate selectivity function.

A preferred MEKK homologue has at least about more preferably at least about 75% and even more preferably at least about 85% amino acid homology with the kinase catalytic domain of an MEKK protein having an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10. Another preferred MEKK WO 95/28421 PCT/US94/11690 homologue has at least about 10%, more preferably at least about 20% and even more preferably at least about 30% amino acid homology with the NH 2 -terminal regulatory domain of an MEKK protein having an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID In a preferred embodiment, an MEKK protein of the present invention includes at least a portion of an MEKK protein homologue of the present invention that is encoded by a nucleic acid molecule having at least about 50%, more preferably at least about 75%, and even more preferably at least about 85% homology with a nucleic acid molecule encoding the kinase catalytic domain of an MEKK protein.

Another preferred MEKK protein homologue is encoded by a nucleic acid molecule having at least about 10%, more preferably at least about 20%, and even more preferably at least about 30% homology with a nucleic acid molecule encoding the NH 2 -terminal regulatory domain of an MEKK protein.

Still another preferred MEKK homologue is encoded by a nucleic acid molecule having at least about 50%, more preferably at least about 75% and even more preferably at least about 85% amino acid homology with the kinase catalytic domain of an MEKK protein encoded by a nucleic acid sequence represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. An MEKK homologue also includes those encoded by a nucleic acid molecule having at least about 10%, more preferably at least about WO 95/28421 WO 95/842 1PCTIUS94/1 1690 -26and even more preferably at least about 30% amino acid homology with the NH 2 -terminal regulatory domain of an MEKK protein encoded by a nucleic acid sequence represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.

An MEKK protein of the present invention, referred to here as MEKK 1, includes an MEKK protein having including) at least a portion of the nucleic acid and/or an amino acid sequence shown in Table 1 and represented by SEQ ID NO:1 and SEQ ID NO:2, respectively.

Table 1.

TACACTCCTT GCCACAGTCT GGCAGAAAGA ATCAAACTTC AGAGACTCCT CCGGCCAGTT GTAGACACTA TCCTTGTCAA GTGTGCAGAT CCAACAGCCG CACGAGTCAG CTGTCCATAT 120 CTACAGTGCT GGAACTCTGC AAGGGCCAAG CAGGAGAGCT GGCGGTTGGG AGAGAAATAC 180 TTAAAGCTGG GTCCATCGGG GTTGGTGGTG TCGATTACGT CTTAAGTTGT ATCCTTGGAA 240 ACCAAGCTGA ATCMAACMAC TGGCAAGAAC TGCTGGGTCG CCTCTGTCTT ATAGACAGGT 300 TGCTGTTGGA ATTTCCTGCT GAATTCTATC CTCATATTGT CAGTACTGAT GTCTCACAAG 360 CTGAGCCTGT TGAAATCAGG TACAAGAAGC TGCTCTCCCT CTTAACCTTT GCCTTGCAAT 420 CCATTGACMA TTCCCACTCG ATGGTTGGCA AGCTCTCTCG GAGGATATAT CTGAGCTCTG 480 2 0 CCAGG ATG GTG ACC GCA GTG CCC GCT GTG TTT TCC MAG CTG GTA ACC 527 Met VaL Thr Ata Vat Pro Ata VaL Phe Ser Lys Leu Vat Thr 1 5

ATG

Met 15

CTG

Lau CTT MAT GCT TCT GGC TCC ACC Leu Asn Ala Ser GLy Ser Thr 20 ATG GCT ATC GCG GAT GAG GTA Met ALa lie Ata Asp GLu Vat CAC TTC ACC AGG His Phe Thr Arg 25 GMA ATT GLu Ile 40 GCC GAG Ala GLu ATG CGC CGG CGT Met Arg Arg Arg GTC ATC CAG CTG VaL lie Gin Leu TTA CAG GCC GTG Leu Gin Ala Val GGT GTG GAG GAC ACT GTG GAT GGG Gty VaL G~u Asp Thr VaL Asp Gty GCC CCC ACC AGC TGT CTA GMA MC Ala Pro Thr Ser Cys Leu Glu Asn 70 CAG GAC AGC Gin Asp Ser

AGA

Arg

TCG

Ser 95

AGC

Ser

AGA

Arg

GAG

GLu ACT GGA MAA GGA CTA Thr Gly Lys Gty Leu 85 AGC TCC CTT Ser Ser Lau AGT GCT ACG Ser ALa Thr GCC GGC GTC ALa GLy Vat 105 MAG CCA GCG Lys Pro Ata GAG GAC ATT TCT GAC AGA CTG GLu Asp lie Ser Asp Arg Leu 100 TCA ACA ACA ACA GMA CMA CCA Ser Thr Thr Thr Gtu Gin Pro 115 CCC CAC AGT CAG TGT TTG MAC Pro His Ser Gin Cys Leu Asn 130 GAG CAC ACA GTC CAT Gtu His Thr VaL His AGA CTG AGT GCC AGC Arg Leu Scr Ata Ser TCT GTA GGA CTT CCC Ser VaL GLy Leu Pro 110 GTT CMA ACA MAA GGC VaL Gin Thr Lys Gty 125 TTG TCT CAT GCT CMA Leu Ser His Ata Gin 140 120 TCC TCC CCT Ser Ser Pro 135 WO 95/28421 WO 9528421PCTJUS94/1 1690 -27- TTA ATG TTC CCA GCA CCA TCA GCC CCT TGT TCC TCT GCC CCG Leu Met Phe Pro Ala Pro Ser Ala Pro Cys Ser 9cr Ala Pro 145 150 155 CCA GAT ATT TCT MAG CAC AGA CCC CAG GCA TTT GTT CCC TGC Pro Asp lie Ser Lys His Arg Pro Gin Ata Ph. Vat Pro Cys 160 165 170 CCT TCC GCA TCT CCT CAG ACA CAG CGC MAG TTC TCT CTA CAA Pro Ser Ata Ser Pro Gin Thr Gin Arg Lys Ph. 9cr Lau Gin 175 180 185 AGG MAC TGC TCT GMA CAC CGA GAC TCA GAC CAG CTC TCC CCA Arg Asn Cys 9cr Gtu His Arg Asp Ser Asp Gin Leu Ser Pro 195 200 ACT CAG TCA AGA CCC CCA CCC TCC AGT MAC ATA CAC AGG CCA Thr Gin Ser Arg Pro Pro Pro Ser Ser Asn lie His Arg Pro 210 215 220 TCC CGA CCC GTT CCG GGC AGT ACA AGC AMA CTA GGG GAC GCC Ser Arg Pro Vat Pro GLy Ser Thr Ser Lys Lau GLy Asp Ala 225 230 235 AGT AGC ATG ACA CTT GAT CTG GGC AGT GCT TCC AGG TGT GAC 2 0 Ser Ser Met Thr Leu Asp Leu Giy 9cr Ala Ser Arg Cys Asp 240 245 250 TTT GGC GGC GGC GGC MAC AGT GGC MAC 6CC GTC ATA CCC AGC Phe Gly Gly Gly Gly Asn 9cr Gly Asn Ala Vat lie Pro Ser 255 260 265 2 5 ACA GTG TTC ACG CCG GTG GAG GAC MAG TGC AGG TTA GAT GTG Thr Vat Ph. Thr Pro Vat Giu Asp Lys Cys Arg Lau Asp Vat 275 280 GAG CTC MAC TCC AGC ATC GAG GAC CTT CTT GMA GCA TCC ATG Giu Lau Asn 9cr 9cr lie GLu Asp Lau Leu Giu Ala 9cr Met 290 295 300 AGT GAC ACG ACA GTC ACT TTC MAG TCC GMA GTC GCC GTC CTC 9cr Asp Thr Thr Vat Thr Phe Lys 9cr Giu Vat Ala Vat Lau 305 310 315 GMA MG GCC GMA MT GAC GAC ACC TAC AMA GAC GAC GTC MAT 3 5 Giu Lys Ala Giu Asn Asp Asp Thr Tyr Lys Asp Asp Vat Asn 320 325 330 TCT GTC Scr Vat AMA ATA Lys lie TTC CAG Ph. Gin 190 GTC TTC Vat Ph.

205 MAG CCA Lys Pro ACA AMA Thr Lys GAC AGC Asp 9cr GAC GAG Asp Giu 270 MAC ACC Asn Thr 285 CCT TCA Pro 9cr TCT CCG Ser Pro CAT MAT His Asn TTA GCG Lcu Ala 350 GTC CCT Vat Pro 365 CAG GAC Gin Asp TAC AGA Tyr Arg 959 1007 1055 1103 1151 1199 1247 1295 1343 1391 1439 1487 1535 1583 1631 1679 1727 1775 1823

CMA

Gin 335

ATC

lie

CAG

Gin

ACA

Thr

GMA

Giu

TCT

9cr 415

GTG

Vat AAG TGC AMA GMA MG ATG GMA GCT GMA GAG GAG GAG Lys Cys Lys Giu Lys Met Giu Ala Giu Giu Giu Giu 340 345 6CC ATG GCG ATG TCA GCG TCT CAG GAT GCC CTC CCC Ala Met Ala Met 9cr Ala Ser Gin Asp Ala Lcu Pro 355 360 CTG CAG GTG GMA MT GGA GMA GAT ATT ATC ATC ATT Leu Gin Vat Giu Asn Gly Giu Asp lie lie lie lie 370 375 CCA GMA ACT CTT CCA GGA CAT ACC AMA GCG AMA CAG Pro Giu Thr Lau Pro Gly His Thr Lys Ala Lys Gin 385 390 395 GAC GCT GAG TGG CTG AMA GGC CAG CAG ATA GGC CTC Asp Aia Glu Trp Lcu Lys Gly Gin Gin lie Gly Lau 400 405 410 TCC TGT TAC CMA GCA CAG GAT GTG GGG ACT 666 ACT 9cr Cys Tyr Gin Ala Gin Asp Vat Giy Thr Gly Thr 420 425 AMA CAG GTG ACG TAC GTC AGA MAC ACA TCC TCC GAG Lys Gin Vat Thr Tyr Vat Arg Asn Thr 9cr 9cr Giu

GCT

Ala

ATC

lie

CAG

Gin 380

CCT

Pro GGA GCA TTT Gly Ala Ph.

TTA ATG GCT Lcu Met Ala 430 CAG GAG GAG Gin Glu GLu WO 95/28421 WO 95/842 1PCTIUS94/1 1690 -28-

GTG

VatI

CAT

His

TAC

Tyr

TTG

Lau 495

GAG

Gtu

CAC

His

AGG

Arg

GGA

G Ly

TTC

Phe 575

GAT

Asp

CCA

Pro

MAG

Lys

CCG

Pro

GAC

GTG GMA GCG Vat Gtu ALa 450 CCA MAC AIC Pro Asn lie 465 MAC CTC TTC Asn Leu Phe 480 AGT AMA TAC Ser Lys Tyr CAG TTA CTG Gin Lau Leu AGA GAC GTC Arg Asp Vat 530 CTG AGA ATT Lau Arg Ile 545 ACC GGT GCA Thr Gly Ala 560 ATG GCG CCT Met Ala Pro GTA TGG AGT Vat Trp Ser AGO GMA GAG ATC CG Arg GLu Glu ie Arg 455 CGG ATG CTG GGG CC Arg Met Lau GLy Ala 470 GAG TOG ATO GCG GGA Giu Trp Met Ala Gly 485 GCT TTC MAG GAG TCA ALa Ph. Lys GLu Ser 500 GGC CTT TCC TAT CTC Gly Lau Ser Tyr Lau 520 GGT GCC MAC CTG CTC GLy ALa Asn Lau Lau 535 GAC TTT GGA GCT GCT Asp Ph. OLy Ala ALa 550 GAG TTC CAG GGA CAG GLu Ph. Gin Gly Gin 565 GTC CTA AGA GOT CAG Vat Lou Arg GLy Gin 580 GGC TGC 0CC ATT ATA GLy Cys ALa lie lie 600 GMA AMA CAC TCC MAT GLu Lys His Ser Asn 615 ACT ACT GCA CCG TCC Thr Thr Ala Pro Ser 630 GTG GCC OTO CGC TGC VaL Ala Vat Arg Cys 645 AGA GAG CTG CTG AMA Arg G~u Lou Lou Lys 660 ATG ATG GOT CAC CTC MAC Met Met Gly His Lou Asn 460 ACG TGC GAG MAG AOC MAC Thr Cys Gtu Lys Ser Asn 475 GGA TCT GTG GCT CAC CTC GLy Ser Vat Ala His Leu 490 GTC GTC ATT MAC TAC ACT Vat Vat lite Asn Tyr Thr 505 510 CAC GAG MAC CAG ATC ATT His Gtu Asn Gin lie Ie 525 ATT GAC AGC ACC GGT GAG IIe Asp Ser Thr Gly Gin 540 GCC AGG TTG OCA TGA AMA Ala Arg Lou Ala Ser Lys 555 TTA CTG GGG ACA ATT GGA Lou Lou GLy Thr lIo ALa 570 CAG TAT GGT AGG AGC TGT Gin Tyr Giy Arg Ser Cys 585 590 GMA ATG OCT TGT GCA AMA Gtu Met Ala Cys Ala Lys 605 CAT CTC GCC TTG ATA TTT His Lou Ala Lau lIe Phe 620 ATC CCG TCA CAC CTG TCC lie Pro Ser His Lou Ser 635 TTA GMA CTT GAG CCT CAG Lou Gtu Lou Gin Pro Gin 650 CAT CCG GTC TTC CGT ACC His Pro Vat Ph. Arg Thr 665 670 GACAGGATAT CGAACCGGGA 1871 1919 1967 2015 2063 2111 2159 2207 2255 2303 2351 2399 2447 2495 2551 2611 2671 2731 2791 2851 2911 2971 3031 3091 3151 3211 3260 CCT TGG MAT GCA Pro Trp Asn Ala 610 ATT GCT AGC GCA lie Ala Sor Ala 625 GGT CTG CGC GAC Gly Leu Arg Asp 640 CGG CCT CCG TCC Arg Pro Pro Ser ACG TGO TAOTTMATTG TTCAGATCAO CTCTMTGGJ Thr Trp GAGAGAAAAG AGMACTTGTG GGCGACCATG CCGCTMACCG AGCCAGAAAC GGGGCCAGCG GGGMACCGTA CCTMGCATG GTACCTMAGC TCGATATOCA GACATCTACA GCTCGTGCAG 0 CACAGGACTG OCTCTGGGGG ACCAGGMAGG CGATGGAGTT GCATAMATTT ATTTTTGGAG CACTTTTTCA GCTMATCAOT GCCCGCCACA TTTCAMACTC AGACTGTCCC AGATGTCMAG TTTGCAOTTC CCTCAGCTTO CTGGTAATTG TGGTGTTTTO TAATATTCTT ATTTTCTTTG GATCAMAGCT GGACTGAAAA 5 TGTGTTTTTA ATGTTATTTG OTACTCGMAT TOTAATMAC GTTTCTACTA CCTCAGGTGT CCTATAGATT TTTCTTCTAC AMATTCTACG TOCTOTGTGA CTATGACTCC TMAGACTTCC TATTAGCACC TTACTATOTA AGCAMATGCT ACAAAAAAAA CAOCCCTCAC GCCACTGMAC TGATTGACMA ATCATGACCT GMACTGCACA CCGTGCCTTT TGCATGACTA MAGMCAGMA ATTACCATOT ACATCMACAT ATCCACTGTO TTTGAGTTTO TTTTCGATGC AAMTGTGATG TTGTACTGTG TMATTATTTT GTCTACTGCT GTTTATTCCA CAMAGTTCAC TCTCGAMTG AGGGCTTMAG GOCTMACTCC

MMAMA

I

WO 95/2842 1 PCTfUS94/1 1690 -29- An MEKK protein of the present invention, referred to here as MEKK 2, includes an MEKK protein having at least a portion of the nucleic acid and/or amino acid sequence shown in Table 2 and represented by SEQ ID NO:3 and SEQ ID NO:4, respectively.

Table 2.

GGTGGCGGCC GCTCTAGAAC TAGTGGATCC CCCGGGCTGC AGGAATTCGG CACGAGGGAC GATCCAGCGG CAGAGTCGCC GCTTCCGCTT CGCTGCTTCT CCGGTCGGCG ACGCGGGCCC 120 GGGGCTTCCT TTTCATCGGC CCAGCTTATT CCGCGGGCCC CGGGGCTGCA GCTACCCAGA 180 AGCGGCGAAG AGGCCCTGGG CTGCGCGCCC GCTGTCCCAT GTGAAGCAGG TTGGGCCTGG 240 TCCCCGGCCC GTGCCCGGTT GTCTGCGGCC CTTCAGGCCT CAGGGACCCC CGCGAGGCGC 300 TGCTCCTGGG GGGCGCGGTG ACAGGCCGTG CGGGGGCGGA GGGGCCAGCT CGGTGGCCTC 360 CTCTCGGCCC TCGCGTCCGC GATCCCGCCC AGCGGCCGGG CAATAAAGAA TGTTGATGGG 420 AGAACCATTT TCCTAATTTT CAAATTATTG AGCTGGTCGC GCATA ATG GAT GAT 474 Met Asp Asp CAG CAA GCT TTG MAT TCA ATC ATG CMA GAT TTG GCT GTC CTT CAT MAG 522 Gin Gin Ata Leu Asn Ser lie Met Gin Asp Leu ALa VaL Lau His Lys 10 2 0 CCA GTC GGC CAG CAT TAT CTT TAC MAG AAA CCA GGA MAG CAA MAC CTT 570 Pro Vat GLy Gin His Tyr Leu Tyr Lys Lys Pro Gly Lys Gin Asn Leu 25 30 CAT CAC CMA AMA MC AGA ATG ATG TTC GAG TCA MAT TTG MAC ATA GAG 618 His His Gin Lys Asn Arg Met Met Phe GLu Ser Asn Leu Asn iLe GLu 40 45 GAG GMA AMA AGG ATC CTG CAG GTT ACT AGA CCA GTT AMA CTA GAA GAC 666 GLu GLu Lys Arg lie Leu Gin Val Thr Arg Pro VaL Lys Lau GLu Asp 60 CTG AGA TCT MAG TCT MAG ATC GCC TTT GGG CAG TCT ATG GAT CTA CAC 714 3 0 Lau Arg Ser Lys Ser Lys lie ALa Phe GLy Gin Ser Met Asp Lau His 75 TAT ACC MAC MAT GAG TTG GTA ATT CCG TTA ACT ACC CMA GAT GAC TTG 762 Tyr Thr Asn Asn Giu Lou VaL lIe Pro Lou Thr Thr Gin Asp Asp Lou 90 3 5 GAC AMA GCT GTG GMA CTG CTG GAT CGC AGT ATT CAC ATG MAG AGT CTC 810 Asp Lys ALa VaL Glu Lou Leu Asp Arg Ser lie His Met Lys Ser Lau 100 105 110 115 MAG ATA TTA CTT GTA GTA MAT GGG AGT ACA CAG GCT ACT MAT TTA GMA 858 Lys ILe Leu Lou Vai VaL Asn GLy 5cr Thr Gin Ate Thr Asn Lou GLu 120 125 130 CCA TCA CCG TCA CCA GMA GAT TTG MAT MAT ACA CCA CTT GGT GCA GAG 906 Pro 5cr Pro 5cr Pro GLu Asp Lou Asn Asn Thr Pro Lau GLy ALa Giu 135 140 145 AGG AMA MG CGG CTA TCT GTA GTA GGT CCC CCT MAT AGG GAT AGA AGT 954 4 5 Arg Lys Lys Arg Lou Ser VaL Vat GLy Pro Pro Asn Arg Asp Arg Ser 150 155 160 TCC CCT CCT CCA GGA TAC ATT CCA GAC ATA CTA CAC CAG ATT GCC CGG 1002 Ser Pro Pro Pro GLy Tyr lILe Pro Asp lie Lou His Gin lie Ata Arg 165 170 175 WO 95/28421 WO 9528421PCTfUS94/1 1690 DAT GGG TCA TTC ACT Asn Gty 9cr Phe Thr 180 AGC ATG GAC CAA ATG 9cr Met Asp Gin Met 200 TCT GGC TCA GGA AGC Ser Gly Ser GLy Ser 215 AGC TAC CCA AAA TCA 9cr Tyr Pro Lys Ser 230 CAT CAG GAG TTT ACA His Gin Gtu Phe Thr 245 AAA GGA GGA ACA TAT Lys Gty Giy Thr Tyr 260 GAG TAT MAT GAC GGT 2 0 GLu Tyr Asn Asp GLy 280 OGC ACC AGT TTC CGG Gly Thr Ser Phe Arg 295 2 5 TTA AGC ACT AGT AGT Leu Ser Thr Ser Ser 310 AGC ATC MAC AGT GMA GGA GAG TTC ATT LCA GAG 9cr lie Asn 9cr Gtu Giy Gtu Phe lie Pro GLu 185 190 195 CTG GAT CCA TTG TCT TTA AGC AGC CCT GMA MT Leu Asp Pro Lau 9cr Leu 9cr 9cr Pro Gtu Asn 205 210 TGT CLO TCA CTT OAT AGT CCT TTG GAT GGA GMA Lys Pro Ser Lau Asp Ser Pro Lou Asp Gty OLu 220 225 CGG ATG CCT AGG GCA CAG AGC TAC CCA GAT MAT Arg Met Pro Arg ALa Gin Ser Tyr Pro Asp Asn 235 240 GAC TAT GAT MAC CCC ATT TTT GAG MAA TTT GGA Asp Tyr Asp Asn Pro lie Phe Giu Lys Ph. Gty 250 255 CCA AGA AGG TAC CAC GTT TCC TAT CAT CAC LAG Pro Arg Arg Tyr His Vai Ser Tyr His His Gin 265 270 275 COG MAG ACT TTT CCA AGA GCT AGA AGG ACC CAG Arg Lys Thr Phe Pro Arg Ala Arg Arg Thr Gin 285 290 TCT CCT GTG AGC TTC AGT CCT ALT OAT CAC TCC 9cr Pro Val 9cr Phe 9cr Pro Thr Asp His Ser 300 305 GGA AGC AGT GTL TTT ALL CLA GAG TAT GAL GAC Gly 9cr 9cr Val Phe Thr Pro Giu Tyr Asp Asp 315 320 1050 1098 1146 1194 1242 1290 1338 1386 1434 1482 1530 1578 1626 1674 1722 1770 1818 1866 AGT LGA ATA AGA AGA LGG GOG AGT GAC ATA GAL MAT LLT ALT TTG ALT Ser Arg lie Arg Arg Arg Oiy 9cr Asp lie Asp Asn Pro Thr Lou Thr 3 0 325 330 335 GTL ALA GAL ATL AGC CLA CCC AGL CGT TCA LLT CGA GCT LLG ALL MAC Vai Thr Asp lie 9cr Pro Pro 9cr Arg 9cr Pro Arg Aia Pro Thr Asn 340 345 350 355 TOG AGA LTG GGLC MG CTG CTT GGL CAA OGA GCT TTT GOT AGG GTL TAC 3 5 Trp Arg Lau Giy Lys Lou Lou Giy Gin Oiy Aia Phe Giy Arg Vat Tyr 360 365 370 LTC TGL TAT OAT GTT GAT ALL GGA AGA GAG LTG GCT OTT MAG LMA OTT Lau Lys Tyr Asp Vai Asp Thr Gly Arg Giu Lou Ala Vai Lys Gin Vai 375 380 385 4 0 LAG TTT MLC CCT GAG AOL CLA GAG ALL AOL MAG GMA GTA MAT OLA LTT Gin Phc Asn Pro Giu 9cr Pro Gtu Thr 9cr Lys Giu Vai Asn Ala Lou 390 395 400 GAG TOT GMA ATT LAG TTG TTG AAA MLC TTG TTG CAT GAG CGA ATT OTT Giu Lys Glu lie Gin Lau Lou Lys Asn Lou Lou His Oiu Arg lie Vat 405 410 415 LAG TAT TAT GOL TOT TTG AGO OAT CCT LAG GAG MAA ALA CTT TCL ATL Gin Tyr Tyr GLy Cys Lou Arg Asp Pro Gin Giu Lys Thr Lou 9cr lie 420 425 430 435 TTT ATG GAG CTC TLG CLA 000 GOT TCA ATT MAG GAL CMA LTA MAA OLL 0 Phe Met Giu Leu 9cr Pro Oly Giy 9cr lie Lys Asp Gin Lau Lys ALa 440 4"5 450 TAL GGA OCT CTT ACT GAG MLC OTO ACG AGO MAG TAL ALL COT LAO ATT Tyr Gty Ala Lou Thr Oiu Asn Vat Thr Arg Lys Tyr Thr Arg Gin lie 455 460 465 WO 95/28421 WO 9528421PCTJUS94I111690 -31- CTG GAG GGG GTC CAT TAT TTG CAT AGT MAT ATC ATT GTC CAT AGA GAT Lau Gtu GLy Vat His Tyr Lau His Ser Asn Met Ile VaL His Arg Asp 470 475 480 ATC AAA GGA GCA MAT ATC TTA AGG GAT TCC ACA GGC MAT ATC MAG TTA Ile Lys GLy Ata Asn Ile Leu Arg Asp Ser Thr GLy Asn Ile Lys Lau 485 490 495 GCA CAC TTT GGG GCT AGT AAA CCC CTT GLy Asp Ph. Gly MA Ser Lys Arg Leu 500 505 ACA GGA ATG MAG Thr Cty Met Lys GTC ATC ACT GGA Val Ile Ser GLy 535 GCA TGT ACT GTC Ata Cys Thr VaL 550 TTT GMA GCA ATC 2 0 Ph. CLu Ala Met 565 CCA MAG CTG CCA Pro Lys Lau Pro 580 2 5 CCC ATT TTT GTA Arg R~e Ph. VaL CCC CAC ATG TTT Arg His Met Phe 615 TCT GTC ACA Ser Val Thr 520 CMA CCC TAT Gtu GLy Tyr CTA CMA ATG VaL GLu Met GCT CCC ATC Mla Ata Ile 570 CCT CAT CTC Pro His Vat 585 GAG CCC A GLu Ata Lys 600 GTG CAT TAT CCC ACGI GLy Thr CGA AGA CLy Arg 540 CTA ACT Leu Thr 555 TTT MAG Ph. Lys TCA GAC Set Asp CTT CGA Lau Arg CAC ACC ATC TGT CTC TCA GCC Gin Thr Ile Cys Leu Ser GLy 510 515 CCA TAC TGC ATC ACT CCT GAG Pro Tyr Trp Met Ser Pro GLu 525 530 MAA GCA GAC ATC TCG ACT GTA Lys Ata Asp Ite Trp Ser Vat 545 CMA MC CCA CCT TGG CCT CMA CLu Lys Pro Pro Trp Ata GLu 560 ATC CCC ACT CAC CCA ACC MAC Ite Ata Thr Cin Pro Thr Asn 575 TAT ACT CCC GAC TTC CTC A Tyr Thr Arg Asp Ph. Lau Lys 590 595 CCT TCA C GAG GAG CTC TTG Pro Set Ata CLu Ctu Lau Leu 2010 2058 2106 2154 2202 2250 2298 2352 2412 2472 2503 605 610 CAC TAGCAGCGCC GGCTTCGGTC CTCCACCAGC Vat His Tyr His TCCATCCTCG CGGCCACCTT CTCTCTTACT GCACTTTCCT TTTTTATMAA AMAGAGAGAT GGCAGAAAA AGACMAGAGG GAAAATATTT CTCTTGATTC TTGGTTAAAT TTCTTTMATA ATMATAGTMA ACTAAAAAAA MAAAAAAAAA A An MERK protein of the present invention, referred to here as MEKK 3, includes an MEKK protein having at least a portion of the nucleic acid and/or amino acid sequence shown in Table 3 and represented by SEQ ID NO:5 and SEQ ID NO: 6, respectively.

Table 3.

4 0 AGGMACAM ACCTGGAGCT CCACCGCGGT GCGCCCGCT CTACMACTAG TGGATCCCCC GGCCTCCAGC MATTCGGCAC GACCMACAGT GCCCGCTCGG ACCGTCTTCT CGACTTCAGG 120 ACTCCCAGGC GCCCCCTCG AGTCCCGCCG CCGAGCCCG GTTGGCCCGA CCCTGCGACC 180 GCCGCGGATG TAGCGCCCCA ACCTGCTCAT GCCACAGCCIC CCGGCCGCGG CCGAGCCGGA 240 CCCTGGCGA GCCGCGGG CCCCGACC ACCCCACGGC CCCCGCGCGG ACCCAGGCCC 300 4 5 GCTCCCGTCC CCCCCCCC CTCCCCCGGC ATGCACCCCC CCCTCGAG GTGACACTTC 360 TGCCCTCTAC TCCCCACCCC CGCCTCCGCC ATCCCCACC ATC GAT GMA CMA GAG 414 Met Asp GLu Gin GLu 1 WO 95/28421 WO 95/842 1PCTJUS94/1 1690 -32- GCA TTA GAC TCG ATC ATG MAG GAC CTG GTG GCC CTC CAG ATG AGC CGA AMa Leu Asp Ser IL. Met Lys Asp Leu Vat Ala Lau Gin Met Ser Arg 15 CGA ACC CGG TTG TCT GGA TAT GAG ACC ATG MAG MAT MG GAC ACA GGT Arg Thr Arg Leu Ser GLy Tyr Giu Thr Met Lys Asn Lys Asp Thr GLy 30 CAC CCA MAC AGG CAG AGT GAC GTC AGA ATC MAG TTT GMA CAC MAT GGG His Pro Asn Arg Gin Ser Asp Vat Arg Ito Lys Phe GLu His Asn GLy 45 GAG AGA CGA ATT ATA GCA TTC AGC CGG CCT GTG AGA TAC GMA GAT GTG GLu Arg Arg Ile R~e Ata Phe Ser Arg Pro VaL Arg Tyr GLu Asp Vat 60 GAG CAC MAG GTG ACA ACA GTC TTT GGG CAG CCT CTT GAT TTG CAT TAT Giu His Lys Vat Thr Thr VaL Pho GLy Gin Pro Lau Asp Lau His Tyr 70 75 8o ATG MAT MAT GAG CTC TCC ATC CTG TTG MAA MAC CMA GAT GAT CTC GAT Met Asn Asn GLu Leu Ser Ito Lou Lau Lys Asn Gin Asp Asp Lou Asp 95 100 MAA GCC ATT GAC ATT TTG GAT AGA AGC TCA AGT ATG AMA AGC CTT AGG 2 0 Lys Ala Re. Asp Ile Lau Asp Arg Ser Ser Ser Met Lys Ser Lou Arg 105 110 115 ATA CTA CTG TTA TCC CMA GAC AGA MAC CAT ACT AGT TCC TCT CCC CAC ILe Lau Leu Lou Ser Gin Asp Arg Asn His Thr Ser Ser Ser Pro His 120 125 130 2 5 TCT GGA GTG TCC AGG CAG GTT CGG ATC MAG CCT TCC CAG TCT GCA GGG Ser GLy Vat 5cr Arg Gin Vat Arg Ilie Lys Pro Ser Gin Ser Ala GLy 135 140 145 GAT ATA MAT ACC ATC TAC CMA GCT CCT GAG CCC AGA AGC AGG CAC CTG Asp Ite Asn Thr lie Tyr Gin ALa Pro GLu Pro Arg Ser Arg His Lou 150 155 160 165 TCT GTC AGC TCC CAG MAC CCT Ser Vat Ser Ser Gin Asn Pro 170 GTA CCT GAG CGA CMA CAG CAC Vat Pro Giu Arg Gin Gin His 185 ATC MAC AGC GMA GGT GMA TTC Ilie Asn Ser Gtu GLy Giu Ph, 200 4 0 CTA GAT CCC CTC AGC AGT GCC Lou Asp Pro Lau Ser 5cr Aia 215 220 TCC TTG GAC AGG TCA GCA GAC Ser Lou Asp Arg Ser Aia Asp 230 235 TCC CGA GCC CGG AGC TTC CCA Ser Arg Ala Arg Scr Phe Pro 250 GAG ACC CAG CTC TAT GAT AMA Gtu Thr Gin Lau Tyr Asp Lys 265 CGC TAC CAT GTG TCT GTG CAT Arg Tyr His Vat Scr Vat His 280 GGC CGA AGC TCT CCT CCC CCG GGA TAT Gty Arg 5cr Scr Pro Pro Pro Giy Tyr 175 180 ATT GCC CGG CMA GGA TCC TAT ACG AGC Ite Ala Arg Gin GLy Sor Tyr Thr Ser 190 195 ATC CCA GAG ACC AGC GMA CAG TGT ATG Rte Pro Gtu Thr 5cr GLu Gin Cys Met 205 210 GMA MT TCC TTG TCA GGA AGC TGC CMA Gtu Asn Ser Lou Ser Gty 5cr Cys Gin 225 AGC CCA TCC TTC AGG AMA TCA CMA ATG Scr Pro 5cr Ph. Arg Lys Ser Gin Met 240 245 GAC MAC AGA MAG GMA TGC TCA GAT CGG Asp Asn Arg Lys Giu Cys Ser Asp Arg 255 260 GGT GTC AMA GGT GGA ACC TAT CCC AGG GLy Vat Lys Gly Gty Thr Tyr Pro Arg 270 275 CAC AMA GAC TAC MAT GAT GGC AGA AGA His Lys Asp Tyr Asn Asp Gly Arg Arg 285 290 WO 95/28421 WO 95/842 1PCTIUS94/1 1690 -33- ACA TTT CCC CGA ATA CGA CGG CAT CAA GGC MAC CTA TTC ACT CTG GTG 1326 Thr Ph. Pro Arg ILe Arg Arg His Gin Gly Asn Lou Ph. Thr Lau Vat 295 300 305 CCC TCA ACT C6C TCC TTG AGC ACA MAT CCC GAG MAC ATG GGT GTA GCT 1374 Pro Ser Ser Arg Ser Lau Ser Thr Asn GLy GLU Asn Met GLy VaL ALa 310 315 320 325 GTG CMA TAC CTG GAC CCC CGT GGG CGC CTA CGG AGT GCA CAC AGT GAG 1422 Vat Gin Tyr Lau Asp Pro Arg Gty Arg Leu Arg Ser Ala Asp 9cr GLu 330 335 340 MAT GCC CTC ACT GTG CAG GMA AGG MAT GTG CCA ACC AAA TCT CCT ACT 1470 Asn AMa Lau Thr Vat Gin Gtu Arg Asn Vat Pro Thr Lys Ser Pro Ser 345 350 355 GCT CCC ATC MAT TGG CGT CGG GGG MAG CTC CTG GGT CMA GGT GCC TTC 1518 ALa Pro Ile Asn Trp Arg Arg GLy Lys Leu Leu GLy Gin GLy Ala Ph.

360 365 370 GGC AGG GTC TAC TTG TGC TAT GAT CTG GAC ACA GGA CGT GMA CTT GCT 1566 Gty Arg VaL Tyr Lau Cys Tyr Asp VaL Asp Thr Cly Arg Gtu Lau Ata 375 380 385 TCT MAG CAG GTC CAG TTT GAC CCA GAT ACT CCT GAG ACA AGC MAG GAG 1614 2 0 Ser Lys Gin Vat Gin Ph. Asp Pro Asp 9cr Pro Ctu Thr Ser Lys GLu 390 395 400 405 CTG AGT GCT CTG GAG TGT GAG ATC CAG TTG CTG MAG MAC CTG CAG CAT 1662 Vat 9cr ALa Lau Gtu Cys GLu Ite Gin Leu Leu Lys Asn Lau Gin His 410 415 420 2 5 GAG CCC ATT CTG CAG TAC TAC CCC TGC CTG CCC GAC CGT GCT GAG MCG 1710 Giu Arg IL. Vat Gin Tyr Tyr Gly Cys Lou Arg Asp Arg ALa GLu Lys 425 430 435 ATC CTC ACC ATC TTT ATG GAG TAT ATG CCA GCG CCC TCT CTA MAA GAC 1758 lie Leu Thr IL. Ph. Met Gtu Tyr Met Pro Giy GLy Scr Vat Lys Asp 440 445 450 CAC TTG MAG GCC TAC GGA GCT CTG ACA GAG ACT GTG ACC CGC MAG TAC 1806 Gin Leu Lys Ata Tyr Cty Ata Leu Thr Ctu Ser Vat Thr Arg Lys Tyr 455 460 465 ACC CCC GAG ATT CTC GAG CCC ATG TGA TAG CTG CAC AGC MAC ATC ATT 1854 3 5 Thr Arg Gtn It. Leu Gtu GLy Met 9cr Tyr Lau His Ser Asn Met Ite 470 475 480 485 GTC CAT CCC GAC ATC MAG CGA CCC MAT ATC CTC CGA GAG TGA CCT GGG 1902 Vat His Arg Asp lite Lys GLy Ata Asn lie Leu Arg Asp 9cr Ata CLy 490 495 500 4 0 MAT CTG MAG CTT CCC GAT TTT CCC CCC AC MAA CCC CTA GAG ACC ATC 1950 Asn Vat Lys Lau Gty Asp Ph. GLy ALa Ser Lys Arg Leu Gin Thr It.

505 510 515 TGC ATC TCA CCC ACA CCC ATT CCC TCT GTC ACT CCC ACA CCC TAC TGG 1998 Cys Met Sor Gty Thr Gly Ile Arg 9cr Vat Thr Gty Thr Pro Tyr Trp 520 525 530 ATG ACT CCT CMA CTC ATC ACT CCC GAG CCC TAT GGA AGA MAG CA GAC 2046 Mot Ser Pro Glu Vat It. 9cr Gty Ciu Gty Tyr Gty Arg Lys Ata Asp 535 540 545 GTG TGG ACC CTG CCC TGT ACT CTG GIG GMA ATG CTG ACA GAG MAA CCA 2094 0 Vat Trp 9cr Lou Gty Cys Thr Vat Vat Ctu Met Lau Thr Ctu Lys Pro 550 555 560 565 CCT TCG GCA GAG TAT GMA CCT ATG GGT CCC ATT TTC MAG ATT CCC ACC 2142 Pro Trp Ala Glu Tyr GLu Ata Met Ala Ata Ito Ph. Lys Ie Ala Thr 570 575 580 WO 95/2842 1 PCT/US94/1 1690 -34- CAG CCT ACC MAT CCT CAG CTG CCC TCT CAC ATC TCA GMA CAC GGC AGG Gin Pro Thr Asn Pro Gin Lau Pro Ser His lie Ser GLu His Gly Arg 585 590 595 GAC TTC CTG Asp Ph. Lau 600 GAG GAG CTG GLu Gtu Leu 615 AGG CGC ATA TTT Arg Arg lie Ph.

GMA GCT CGT CAG Giu Ata Arg Gin AGA CCC TCA GCT Arg Pro Ser Ate 610 TAC TGAGCTCTCA Tyr CTC ACA CAC Leu Thr His TTT GCA CAG CTA GTG Ph. Ata Gin Lau Vat 625 AGGCTATCAG GCTGCCAGCT GCCACCTGCT GAGCAGGCMA GGGGCTGCTG TCAGGCTCAG TGMAGTTGCT GCTTCTTCCA GGCAAGGCTA TGACCAGTGG AGCATCGGTC CAGCCATTGT TTGTCTGTGC CCCATCTGCC ACTGGGACTC AAAGCCAGGA TGGGATAGCT CTGGCATCAA GACTGGGAGC TCCAGCCTGT MAGACCCAAG AGCTTTAGCA CCTTMAGCTC AGTATGGCGG GMAGGGCTGG AMCAGTATG CMAGACTGCC ATGGGTCCTG CCTACCCTCA GATGTGTCCT MCACTGCAG ACAGCACTGA AGTCMAGAGG GACTGGGGCA CAGGAGGTCC TCAAGGGTAT GMATAGTGTT ACTTCATTCA GAGTGTTACT TTGTTTCTCT CCCMATGTTT GGAGACCACC AGCCTGTCTC TGGGCTGCMA GCCTGAGGTA MAGCCCAGCA TCCCCCAGCC AACAGAAGGT AGAGGTTTGG GCTACCCCAC TATAGCTTCC AGGTATTCGG TGTCAGTCCT GTCTTACCMA AGATGMATGA AGCAAATGTT ACACTGCCTT ATTCTGGGMA GGAGGAGCTA CTCGGATMAG 2 0 CAGGGCCTGA GAGATGGAGC TGCCTCCAGA MACTGGGGAG ACCCAGTCTT GTCMTGCAA TTGTCTCTGT TTTACMAGTT GGAGTCACTC TTATGCTGTT CCCAGTTTTA AAACTGGAGA CTTTGCCCTC TGAGCTCTGG AGACCCATGT GGGCTTAGGC TTGGACTGGA TGGMAGAGCT GATGGCCTCT GCCCCTGGCC TG 2238 2287 2347 2407 2467 2527 2587 2647 2707 2767 2827 2887 2947 3007 3067 3089 An MEKK protein of the present invention can also include an MEKK protein having at least a portion of the nucleic acid and/or amino acid sequence shown in Table 4 and represented by SEQ ID NO:7 and SEQ ID NO:8, respectively, and is referred to as MEKK 4.

Table 4.

3 0 MTTCGGCAC GAGMACCTAT CAGACATTGG GCCGTCCMAG GGCTACGAGC CAGAGGACGA GCTGGAGAGC GGGACGGAGG AGAGTGACGA GCTCAGGCTG TCCACAGACA CCATCTTGGA GCTGGAGAGG CTCGAGTCAG AGGMAGATTC 3 5 TGMAGCCAGC AGGCATTGTT TGACTTCTAT GCAMATGGGG CTMAGAMAGT TMTTTTACG MAGAGCTCGT GTAGCTCTGG TGMAGGACGA CCCATGTGGG GCTCGGATTA TGTGCAGTTG TGTAGCGCTG TGTCCTGGGA AGMCTGAGA 4 0 TTCCTGGTGC TCTGTCGGGT CCTGCTGMAC GAACAGAGGC TGCCGGGGAG CCTTCCCTCT MAGAGGTCCT AMAGGGCGGG CTCCTG ATG Met 1 CTGGCCAGTG TTTGAAATCC CCTCCCCTCG GGTCGAGGAC ACGGAGGTTG AGCTGAGGGA GGAGCCAACC CCCAGTCCGA GGGTGCCAGA CAGTCGCTCC CAGGGCTGCG TCTCCAGGAA CATAGGCTGG GGGACAGCGG ACTGTGGCCC CTATAGACCA TTCGTGGACA MAGCACTGMA ACTTCATMAG CTTATGAATG GGTCCTTGCA CCGTCAGTGG AGTTCTCTGA CTTTCCAGGT TCGGGMACAC CTCCTTCCTC AGAGCAGAAG GCCATGGACC TGCCTTCCTT TGAGCCCGCC GTGATCCACG AGTGCCTGMA GCTGCGGCTG TGAGTATCAA ACAGCTAGTG CGAGAGTGTA MAG CAG TAT TAC CAG TTC ATG CTG Lys Gin Tyr Tyr Gin Ph. Met Leu 4 5 CAG GAG GTC CTG GGC GGA CTG GAG MAG ACC GAC TGC MAC ATG GAT GCC Gin Gtu VaL Leu Gty Gty Leu Glu Lys Thr Asp Cys Asn Met Asp Ala 15 20 ITT GAG GAG GAC Ph. GLu Gtu Asp CAG MAG ATG CTG ATG GTG TAT TTT GAT TAC ATG Gin Lys Met Lau Met Vol Tyr Ph. Asp Tyr Met AGA AGC TGG ATC CMA ATG CTA CAG CAG TTA CCT CAG GCT TCC CAT AGC Arg Ser Trp lie Gin Met Lau Gin Gin Leu Pro Gin Ala Ser His Ser 50 WO 95/28421 WO 95/842 1PCTJUS94/1 1690 TTA AMA Leu Lys CAT TAT His Tyr ATC GCA lie AMa CTG CAG Lou Gin GCT GCC ALa ALa MAG GAG Lys GLu TTT GCT Phe Ala 155 MAC CTG CTA GMA GAG Asn Lau Lau GLu GLu ATC CGT GGC GGA GMA lie Arg GLy Giy GLu 80 GGG ATG CTG CTG AMA GLy Met Lou Lou Lys 95 GAG AGC TGT GCT GAG Gtu Ser Cys AMa GLu 110 GAC GAG CTA AGG AGA Asp GLu Lou Arg Arg 125 CTC TTC CAC GMA GCC Leu Phe His GLu Mla 140 AMA ATG CTG AGG MAG Lys Met Lou Arg Lys 160 GMA TGG GLu Trp 65 GCG CAG Mla Gin TCC ACA Ser Thr CTG TGG Lau Trp TCT GTC Ser Vat 130 AGG GMA Arg Gtu 145 GAC CTA Asp Lou TTC ACC Phe Thr GGA MAG GLy Lys AGC TTT Ser Phe 100 AGH GCC Xaa ALa GAG ATC GLu lie AMA GMA ATA ACC Lys Giu lie Thr CTT TTC TGT GAC Leu Phe Cys. Asp CTG GMA TCC GGC Lou Gtu Ser GLy 105 GAC GAC MAC GGT Asp Asp Asn GLy 120 AGC CGA GCA CTC Ser Arg Ala Lou 135 MAG GCC CTG GGC Lys ALa Lou Gty 150 GCA GAG TTC GTG Ata GLu Phe VaL CTA TCT GCA TCA GCC CGA GAG CTC CTG GAC GCT CTG AMA GCA MAG CAG Leu Ser Ala Ser ALa Arg Gtu Lou Lau Asp AMa Lou Lys ALa Lys Gin 170 175 180 185 2 5 TAT GTT MAG GTA CAG ATT CCC GGG TTA GAG MAT TTG CAC GTG TTT GTC Tyr VaL Lys VaL Gin lie Pro Gly Lau Gtu Asn Lou His VaL Phe Vat 190 195 200 CCC GAC AGIC CTC GCT GAG GAG MAG AMA ATT ATT TTG CAG CTA CTC MAT Pro Asp Ser Lou ALa Gtu Gtu Lys Lys lie lie Lou Gin Lou Lou Asn 205 210 215 GCT GCC ACA GGA MAG GAC TGC TCA MAG GAT CCA GAC GAC GTC TTC ATG Ala ALa Thr GLy Lys Asp Cys Ser Lys Asp Pro Asp Asp Vat Pho Met 220 225 230 GAT GCC TTC CTG CTC CTG ACC MAG CAT GGG GAC CGA GCC CGT GAC TCA 3 5 Asp ALa Phe Lou Lou Lou Thr Lys His GLy Asp Arg Ala Arg Asp Sor 235 240 245 GMA GAT GGC TGG GGC ACA TGG GMA GCT CGG GCT GTC AMA ATT GTG CCT GLu Asp GLy Trp GLy Thr Trp GLu Ala Arg ALa VaL Lys lio Vai Pro 250 255 260 265 4 0 CAG GTG GAG ACT GTG GAC ACC CTG AGA AGC ATG CAG GTG GAC MAC CTT Gin Val GLu Thr Vat Asp Thr Lou Arg Ser Met Gin VaL Asp Asn Lou 270 275 280 CTG CTG GTT GTC ATG GAG TCT GCT CAC CTC GTA CTT CAG AGA AMA GCC Lou Lau Vat VaL Met GLu Ser Ala His Lou Vat Lou Gin Arg Lys ALa 285 290 295 TTC CAG CAG TCC ATT GAG GGG CTG ATG ACT GTA CGC CAT GAG CAG ACA Phe Gin Gin Ser lie Glu GLy Lou Met Thr VaL Arg His Gtu Gin Thr 300 305 310 TCT AGC CAG CCC ATC ATC GCC AMA GGT TTG CAG CAG CTC MAG MAC GAT 0 Ser Ser Gin Pro lite lie Ala Lys GLy Lou Gin Gin Lou Lys Asn Asp 315 320 325 GCA CTT GAG CTA TGC MAC AGA ATC AGC GAT 0CC ATC GAC CGT GTG GAC ALa Lou Giu Lou Cys Asn Arg lie Ser Asp ALa lie Asp Arg VaL Asp 330 335 34.0 345 1301 1349 1397 1445 1493 1541 1589 1637 1685 1733 1781 WO 95/28421 WO 95/842 1PCTJUS94/1 1690 -36- CAC ATG TTC ACC CTG GAG TTC GAT GCT GAG GTC GAG GAG TCT GAG TCG 1829 His Met Ph. Thr Lau Gtu Phe Asp ALa GLu Vat Gtu Gtu Ser Gtu Ser 350 355 360 GCC ACG CTG CAG CAG TAC TAC CGA GMA GCC ATG ATT CAG GGC TAC MAC 1877 ALa Thr Leu Gin Gin Tyr Tyr Arg Glu ALa Met Ile Gin GLy Tyr Asn 365 370 375 TTT GGG TTT GAG TAT CAT AAA GMA GTT GTT CGT TTG ATG TCT GGG GMA 1925 Phe GLy Phe Giu Tyr His Lys GLu Vat Vat Arg Lau Met 9cr Gly Giu 380 385 390 TTC AGG CAG MAG ATA GGA GAC AAA TAT ATA ACG TTC GCC CAG MAG TGG 1973 Phe Arg Gin Lys ILe GLy Asp Lys Tyr lie Thr Ph. Ala Gin Lys Trp 395 400 405 ATG MAT TAC GTG CTG ACC MAA TGC GAG AGC GGC AGA GGC ACA AGA CCC 2021 Met Asn Tyr VaL Lou Thr Lys Cys GLu 9cr Gly Arg GLy Thr Arg Pro 410 415 420 425 AGA TGG GCC ACC CMA GGA TTT GAT TTC CTA CMA GCC ATT GMA CCT GCC 2069 Arg Trp ALa Thr Gin Gly Ph. Asp Ph. Leu Gin Ala lie GLu Pro Ala 430 435 440 TTT ATT TCA GCT TTA CCA GMA GAT GAC TTC TTG AGT TTG CAA GCC CTG 2117 2 0 Ph. lie Ser ALa Lau Pro Giu Asp Asp Ph. Lau 9cr Lau Gin Ala Lou 445 450 455 ATG MAT GAG TGC ATC GGG CAC GTC ATA GGA MAG CCA CAC AGC CCT GTC 2165 Met Asn Glu Cys, Ile Gly His Vat lie Giy Lys Pro His 9cr Pro Val 460 465 470 2 5 ACA GCT ATC CAT CGG MAC AGC CCC CGC CCT GTG MAG GTG CCC CGA TGC 2213 Thr Ala lie His Arg Asn 9cr Pro Arg Pro VaL Lys VaL Pro Arg Cys 475 480 485 CAC AGT GAC CCT CCT MAC CCT CAC CTC ATC ATC CCG ACT CCA GAG GGA 2261 His 9cr Asp Pro Pro Asn Pro His Lou lie lie Pro Thr Pro Giu Gly 490 495 500 505 TTC AGG GGT TCC AGT GTC CCT GMA MC GAC CGC TTG GCC TCC ATA GCT 2309 Ph. Arg Gty 9cr 9cr VaL Pro GLu Asn Asp Arg Lau ALa 9cr lie Ala 510 515 520 GCA GMA CTG CAG TTC AGG TCT CTG AGT CGG CAC TCA AGC CCC ACG GMA 2357 3 5 Ala GLu Leu Gin Ph. Arg Ser Lou Ser Arg His Ser Ser Pro Thr Glu 525 530 535 GAG CGA GAC GAG CCA GCG TAT CCT CGG AGT GAC TCA AGT GGA TCA ACT 2405 GLu Arg Asp Glu Pro Ala Tyr Pro Arg Ser Asp Ser Ser GLy 9cr Thr 540 545 550 4 0 CGG AGA AGC TGG GMA CTT CGA ACA CTC ATC AGC CAG ACC MAA GAC TCG 2453 Arg Arg Ser Trp Giu Lou Arg Thr Lou lie Ser Gin Thr Lys Asp 9cr 555 560 565 GCC TCT MG CAG GGG CCC ATA GMA GCT ATC CAG MAG TCA GTC CGA CTG 2501 Ala Ser Lys Gin Giy Pro lie Giu Ala lie Gin Lys Ser Val Arg Lou 570 575 580 585 TTT GMA GAG AGG AGG TAT CGA GAG ATG AGG AGA MAG MAT ATC ATC GGC 2549 Ph. Gtu GLu Arg Arg Tyr Arg Giu Met Arg Arg Lys Asn lie lie Gly 590 595 600 CMA GTG TGC GAT ACC CCT MAG TCC TAT GAT MAC GTC ATG CAT GTT GGA 2597 0 Gin Vat Cys Asp Thr Pro Lys Ser Tyr Asp Asn Vat Met His Vai Gly 605 610 615 CTG AGG MAG GTG ACA TTT MAG TGG CMA AGA GGA MAC MAA ATT GGA GMA 2645 Lou Arg Lys Vat Thr Ph. Lys Trp Gin Arg GLy Asn Lys lie Gly GLU 620 625 630 WO 95128421 WO 9528421PCTIUS94/1 1690 -37- GGA CAG GLy Gin 635 CTG ATG Lau Net 650 ATC MAG Ile Lys CCC AC Pro Asn AGT GTT GAC ACA GGG GAG Ser VaL Asp Thr Gty GLu 645 CCT MAC GAC CAC MAG ACT Pro Asn Asp His Lys Thr 660 665 TTT GMA GGC ATC MAG CAC Phe Giu Gty ILe Lys His 680 CTT CAC AGG GMA GAG ATG Leu His Arg GLu Gtu Net 695 ACA CTA GAG GAG GTG TCA Thr Lau GLu GLu Vat Ser 710 TTA TAT ACC MAG CAG ATC Lou Tyr Thr Lys Gin Ite 725 GGC ATC GTT CAC CGA GAC GLy ILe Vat His Arg Asp 740 745 TCT GGA CTA ATC MAG CTG Ser GLy Lou Iie Lys Leu TAC ATC TTC ATG GAG TAC TGT Tyr ILe Phe Net Giu Tyr Cys 700 CGA CTG GGC CTG CAG GAG CAC Arg Lou GLy Leu Gin Glu His 715 720 ACT GTC GCC ATC MAC GTC CTC 2 0 Thr VaL ALa ILe Asn VaL Leu 730 735 ATC AAA GGT GCC MAT ATC TTC Ile Lys Gty ALa Asn Ito Ph.

750 2 5 GGA GAT TTT GGA TGC TCT GTA Gly Asp Ph. GLy Cys Ser Vat 765 CCC GGA GAG GTG MAC AGC ACC Pro GLy GLu Vat Asn Ser Thr 780 GMA GTT ATT ACC CGA GCC AAA Gtu Vat Ile Thr Arg Ala Lys 795 800 ATC TGG AGT CTG GGG TGC GTC 3 5 Ile Trp Sor Leu Gty Cys Vat 810 815 CCT TGG CAT GAG TAT GAA CAC Pro Trp, His Gtu Tyr Gtu His 830 4 0 ATG GGA CAC MAG CCA CCA ATC Net Gly His Lys Pro Pro Ile 845 6CC TTT CTC TCG CAC TGC CTG Ala Phe Leu Ser His Cys Lou 860 GCC AGC CAG CTC CTC GAC CAC ALa Ser Gtn Lou Lou Asp His 875 880 GAG T GMAGTGAACC AGTCCGTGGC Gtu 890 AA MC MAC GCC CAG Lys Asn Asn Ala Gin 775 ACA GCA GCT TAC ATG Thr ALa Ala Tyr Not 790 GGC CAC GGA CGT GCG Gly His GLy Arg ALa 805 GAG ATG GTG ACT GGC Gtu Met Vat Thr GLy 820 CAG ATT ATG TAC MAG Gin Ite Not Tyr Lys 835 AGG CTA AGC CCT GMA Arg Lou Ser Pro GLu 855 GAC CCG MAG ATA CGG Asp Pro Lys Ite Arg 870 GTC MAG GTT TGC ACA GCC CCT ALa Pro GCA GAT Ata Asp MAG CGG Lys Arg 825 GTG 666 Vat GLy 840 GGA MAG GLy Lys TGG ACA Trp, Thr GAT GMA 2693 2741 2789 2837 2885 2933 2981 3029 3077 3125 3173 3221 3269 3317 3365 3413 3467 3527 3587 3647 3707 3767 3827 Pho Vat Lys Vat 885 Cys Thr Asp GLu CTAGTAGTGT GTGGACAGMA TCCCGTGATC ACTACTGTAT GTMATATTTA CATMAAGACT GCAGCGCAGG CGGCCTTCCT MACCTCCCAG GACTGMAGAC TACAGGGGTG ACMAGCCTCA CTTCTGCTGC TCCTGTCGCC TGCTGAGTGA CAGTGCTGAG GTTMAAGGAG CCGCACGTTA AGTGCCATTA CTACTGTACA CGGCCACCGC CTCTGTCCCC TCCGACCCTC TCGTGACTGA GMACCMACCG TGTCATCAGC ACAGTGTTTT TGAGCTCCTG GGGTTCAGMA GAACATGTAG TGTTCCCGGG TGTCCGGGAC GTTTATTTCA ACCTCCTGGT CGTTGGCTCT GACTGTGGAG CCTCCTTGTT CGAAAGCTGC AGGTTTGTTA WO 95128421 WO 9528421PCTJUS94/1 1690 -38- TGCAAAGGCT CGTAAGTGAA GCTGAAGAAA AGGTTCTTTT TCAATAAATG GTTTATTTTA 3887 GGAAAGCGAA AAAAAAAAAA AAAAAW 3913 An MEKK protein of the present invention, referred to here as MEKK 5, includes an MEKK protein having at least a portion of the nucleic acid and/or amino acid sequence shown in Table 5 and represented by SEQ ID NO:9 and SEQ ID NO: 10, respectively.

Table AAGAAGAAGG ACAGGGAGCA GAGGGGACAA GAAAACACGG CTGCTTTCTG GTTCAACCGA TCGAACGAAC TGATCTGGTT AGAACTGCAG GCCTGGCACG CGGGCCGCAC CATCAATGAC 120 CAGGACCTCT TTCTCTACAC AGCCCGCCAG GCCATCCCAG ACATCATCAA TGAGATCCTC 180 ACCTTCAAAG TTAACTACGG GAGCATTGCC TTCTCCAGCA ATGGAGCCGG TTTCAACGGG 240 CCCTTGGTAG AAGGCCAGTG CAGAACCCCT CAGGAGACAA ACCGTGTGGG CTGCTCATCG 300 TACCACGAGC ACCTCCAGCG CCAGAGGGTC TCGTTTGAGC AGGTGAAGCG GATA ATG 357 Met 1 GAG CTG CTG GAG TAC ATG GAG GCA CTT TAC CCA TCC TTG CAG GCT CTG 405 GLu Lou Leu Gtu Tyr Met Giu AMa Leu Tyr Pro Ser Lou Gin ALa Lou 10 2 0 CAG MAG GAC TAT GMA CGG TAC GCC GCC MAG GAC TTT GAG GAC AGA GTG 453 Gin Lys Asp Tyr GLu Arg Tyr ALa ALa Lys Asp Ph. GLu Asp Arg Vat 25 CAG GCG CTC TGC CTG TGG CTC MAC ATC ACG AAA GAT CTA MAT CAG MAG 501 Gin ALa Lou Cys Lou Trp Leu Asn lie Thr Lys Asp Lou Asn Gin Lys 35 40 CTG CGG ATC ATG GGC ACC GTG Lou Arg lie Met Gty Thr VaL 55 GGC TGG CCA GTG MAA GMA ATC 3 0 GLy Trp Pro Vat Lys GLu lie GAG CCA GAG GAC GAG GTC GAG GLu Pro Giu Asp GLu VaL GLu 3 5 GAG AGC GGG ACG GAG GAG AGT GLu Ser Gty Thr GLu Giu Ser 100 GTG CCA GAG CTC AGG CTG TCC Vai Pro Gtu Lou Arg Lou Ser 115 120 CTG GGC ATC MAG TTC CTA TCA GAC ATT Lou GLy lie Lys Phe Lou Ser Asp lie 60 CCC TCC CCT CGG CCG TCC MAG GGC TAC Pro Ser Pro Arg Pro Ser Lys GLy Tyr 75 GAC ACG GAG GTT GAG CTG AGG GAG CTG Asp Thr Giu Vai GLu Lou Arg GLu Lou 90 GAC GAG GAG CCA ACC CCC AGT CCG AGG Asp Giu GLu Pro Thr Pro Ser Pro Arg 105 110 ACA GAC ACC ATC TTG GAC AGT CGC TCC Thr Asp Thr lie Lou Asp Ser Arg Ser 125 CAG GOC Gin GLy 130 TCC ATA Ser lIe TGC GTC TCC AGG Cys Vat Ser Arg 135 GGC TGG GGG ACA Giy Trp Gty Thr 150 MAG CTG GAG Lys Leu Giu GCG GAC TGT Ala Asp Cys AGA CCA TTC Arg Pro Phe 170 AGG CTC GAG TCA GAG Arg Lou Gtu Ser GLu 140 GGC CCT GMA GCC AGC Gty Pro Gtu Ala Ser 155 GMA GAT GLu Asp 145 AGG CAT Arg His 160 TGT TTG ACT TCT ATC TAT Cys Lou Thr Ser lie Tyr 165 GTG GAC MAA GCA CTG MAG CMA Vai Asp Lys Ala Lou Lys Gin 175 WO 95/28421 PCT/US94/1 1690 ATG GGG CTA AGA MG TTA ATT Met GLy Lau Arg Lys Lau Ii.

180 TCC TTG CM AGA GCT CGT GTA Ser Leu Gin Arg Ala Arg VaL 195 200 GAG TTC TCT GAC TTT CCA GGT GLu Ph. Ser Asp Phe Pro GLy 210 215 TTG TCG GGA ACA CCT CCT TCC Leu Ser SLy Thr Pro Pro Ser 230 TGG GM GM CTG AGA GCC ATG Trp GLu GLu Lou Arg ALa Met 245 CTG GTG CTC TGT CGG GTC CTG Leu VaL Lau Cys Arg Vat Leu 260 CTG CGG CTG GM CAG AGG CCT Lou Arg Lou Glu Gin Arg Pro 275 280 AAA CAG CTA GTG CGA GAG TGT Lys Gin Lou VaL Arg Giu Cys 290 295 -39- TTA CGA CTT CAT MG CTT ATG MT GGG Leu Arg Lou His Lys Lou Met Asn GLy 185 190 GCT CTG GTG MG GAC GAC CGT CCA GTG Ate Lou Vai Lys Asp Asp Arg Pro Vat 205 CCC ATG TGG GGC TCG GAT TAT GTG CAG Pro Met Trp Gty Ser Asp Tyr Vat Gin 220 225 TCA GAG CAG MG TGT AGC GCT GTG TCC Ser GLu Gin Lys Cys Ser ALa Vat Ser 235 240 GAC CTG CCT TCC TTT GAG CCC GCC TTC Asp Lou Pro Sor Ph. GLu Pro ALa Ph.

250 255 CTG MC GTG ATC CAC GAG TGC CTG MG Leu Asn VaL Ie His GLu Cys Lou Lys 265 270 GCC GGG GAG CCT TCC CTC TTG AGT ATC ALa Gty GLu Pro Ser Leu Lou Ser lie 285 MA GAG GTC CTA MG GGC GGG CTC CTG Lys Gtu Vat Lou Lys Gly SLy Lou Leu 300 305 ATG MG CAG TAT TAC CAG TTC ATG CTG CAG GAG GTC CTG GGC GGA CTG Met Lys Gin Tyr Tyr Gin Phe Met Lou Gin GLu Val Lou Sly SLy Lou 310 315 320 GAG AAG ACC GAC TGC MC ATG GAT GCC TTT GAG GAG SAC CTG CAG MG GLu Lys Thr Asp Cys Asn Met Asp ALa Ph. Gtu GLu Asp Lou Gin Lys 325 330 335 ATG CTG ATG GTS TAT TTT GAT TAC ATG AGA AGC TGG ATC CM ATG CTA Met Lou Mot Vat Tyr Ph. Asp Tyr Mot Arg Sor Trp Ite Gin not Lou 340 345 350 CAG CAG TTA CCT CAG GCT TCC CAT AGC TTA MA MC CTG CTA GM GAG Gin Gin Lou Pro Gin Ate Scr His Ser Lou Lys Asn Lou Lou Giu Slu 355 360 365 GM TGG MT TTC ACC AM GM ATA ACC CAT TAT ATC CGT GGC GSA GM Giu Trp Asn Ph. Thr Lys GLu Ito Thr His Tyr ie Arg Giy Giy Slu 370 375 380 385 GCG CAS GCT GSA MG CTT TTC TGT SAC ATC GCA GGG ATG CTG CTG AAA Ala G~n Aia GLy Lys Lou Ph. Cys Asp IUe Ala SLy Met Lou Lou Lys 390 395 400 TCC ACA GGG AGC TTT CTG GM TCC SGC CTG CAS GAG AGC TGT GCT GAG Ser Thr Gly Ser Ph. Lou Giu Sor Gly Lou Gin Giu Sor Cys Ala Gtu 405 410 415 CTG TGG ACC AGC GCC GAC SAC MC GGT GCT GCC GAC GAG CTA AGG AGA Leu Trp Thr Ser Ala Asp Asp Asn GLy Ala Ala Asp Giu Lou Arg Arg 420 425 430 TCT GTC ATC GAG ATC AGC CGA SCA CTC AAG GAG CTC TTC CAC GM GCC ser Vat Ie GLu Ito Ser Arg Ala Lou Lys Glu Lou Ph. His Giu Ala 435 440 445 AGG GM AGA GCC TCC MG GCC CTG GGC TTT GCT MA ATG CTG ASS MG Arg GLu Arg Ala Sor Lys Ala Lou Gly Phe Ala Lys not Lou Arg Lys 450 455 460 465 933 981 1029 1077 1125 1173 1221 1269 1317 1365 1413 1461 1509 1557 1605 1653 1701 1749 WO 95/28421 PCTJUS94/11690 GAC CTA GAA ATA Asp Leu GLu Ile CTC CTG GAC GCT Leu Leu Asp Ala 485 GGG TTA GAG AAMT Gly Leu GLu Asn 500 AAG AAA ATT ATT Lys Lys ILe ILe 515 TCA MAAG GAT CCA Ser Lys Asp Pro 530 MAAG CAT GGG GAC Lys His Gly Asp GAA GCT CGG GCT GLu Ala Arg Ala 565 CTG AGA AGC ATG Leu Arg Ser Met 580 GCT CAC CTC GTA Atea His Leu Vat 595 GCA GCA GAG TTC Ala Aate GLu Phe 470 CTG AAA GCA AAMG Leu Lys Aate Lys TTG CAC GTG TTT Leu His Vat Phe 505 TTG CAG CTA CTC Leu Gin Leu Leu 520 GAC GAC GTC TTC Asp Asp VaL Phe 535 CGA GCC CGT GAC Arg Aate Arg Asp 550 GTC AAA ATT GTG Vat Lys ILe Vat CAG GTG GAC AAC GLn Vat Asp Asn 585 CTT CAG AGA AAA Leu GiLn Arg Lys 600 CTA TCT GCA TCA GCC CGA GAG Leu Ser Aate Ser Ala Arg GLu 475 480 TAT GTT MAAG GTA CAG ATT CCC Tyr Vat Lys Vat Gin Ile Pro 495 CCC GAC AGC CTC GCT GAG GAG Pro Asp Ser Leu Aate Gtu Gtu 510 GCT GCC ACA GGA AAG GAC TGC Alate Atela Thr Gly Lys Asp Cys 525 GAT GCC TTC CTG CTC CTG ACC Asp Atela Phe Leu Leu Leu Thr 540 545 GMAA GAT GGC TGG GGC ACA TGG Gtu Asp Gly Trp Gly Thr Trp 555 560 CAG GTG GAG ACT GTG GAC ACC Gin Vat GLu Thr Vat Asp Thr 575 CTG CTG GTT GTC ATG GAG TCT Leu Leu Vat Vat Met GLu Ser 590 TTC CAG CAG TCC ATT GAG GGG Phe GLn GLn Ser ILe GLu GLy 605 TCT AGC CAG CCC ATC ATC GCC Ser Ser Gin Pro Ite Ite Atela 620 625 1797 1845 1893 1941 1989 2037 2085 2133 2181 2229 2277 2325 2373 2421 2469 2517 2565 2613 CTG ATG ACT GTA CGC CAT GAG CAG ACA Leu Met Thr Vat Arg His GLu Gin Thr 610 615 AAA GGT TTG CAG CAG CTC MAAG MAAC GAT GCA CTT GAG CTA TGC MAAC AGA Lys GLy Leu GLn Gin Leu Lys Asn Asp Aate Leu GLu Leu Cys Asn Arg 630 635 640 ATC AGC GAT GCC ATC GAC CGT GTG GAC CAC ATG TTC ACC CTG GAG TTC Ite Ser Asp Atela Ile Asp Arg Vat Asp His Met Phe Thr Leu Glu Phe 645 650 655 GAT GCT GAG GTC GAG GAG TCT GAG TCG GCC ACG CTG CAG CAG TAC TAC Asp Atela Gtu Vat Glu Gtu Ser Gtu Ser Ala Thr Leu Gin GLn Tyr Tyr 660 665 670 CGA GMAA GCC ATG ATT CAG GGC TAC MAAC TTT GGG TTT GAG TAT CAT AAA Arg GLu Aate Met Ile GLn Gly Tyr Asn Phe Gty Phe GLu Tyr His Lys 675 680 685 GAA GTT GTT CGT TTG ATG TCT GGG GMAA TTC AGG CAG MAAG ATA GGA GAC GLu Vat Vat Arg Leu Met Ser Gly GLu Phe Arg Gin Lys ILe Gly Asp 690 695 700 705 AMAA TAT ATA AGC TTC GCC CAG MAAG TGG ATG MAAT TAC GTG CTG ACC AAA Lys Tyr Ite Ser Phe Aate GLn Lys Trp Met Asn Tyr Vat Leu Thr Lys 710 715 720 TGC GAG AGC GGC AGA GGC ACA AGA CCC AGA TGG GCC ACC CMAA GGA TTT Cys GLu Ser Gly Arg Gly Thr Arg Pro Arg Trp AteLa Thr Gin GLy Phe 725 730 735 GAT TTC CTA CMAA GCC ATT GMAA CCT GCC TTT ATT TCA GCT TTA CCA GMAA Asp Phe Leu GLn Atela Ite GLu Pro Ala Phe Ite Ser Atela Leu Pro GLu 740 745 750 WO 95/28421 WO 95/842 1PCTJUS94/1 1690 GAT GAC TTC TTG AGT Asp Asp Phe Leu Ser 755 GTC ATA GGA MAG CCA Vat lie Gly Lys Pro 770 CCC CGC CCT GTG MAG Pro Arg Pro VaL Lys 790 CAC CTC ATC ATC CCG His Leu lie lie Pro 805 TCC GAC GCT CCC ACC -41- TTG CMA GCC CTG ATG MAT GAG TGC ATC GGG CAC Lau Gin Ala Leu Met Asn GLu Cys lie GLy His 760 765 CAC AGC CCT GTC ACA GCT ATC CAT CGG MAC AGC His Ser Pro VaL Thr Ala lie His Arg Asn Ser 775 780 785 GTG CCC CGA TGC CAC AGT GAC CCT CCT MAC CCT VaL Pro Arg Cys His Ser Asp Pro Pro Asn Pro 795 goo ACT CCA GAG GGA TTC AGC ACC CGG AGC GTG CCT Thr Pro GLu GLy Phe Ser Thr Ara Ser VaL Pro 810 815 CAT n=C AC TET QTT GET CCT QCT nCT QCT QTT Ser Asp MAt Ara Thr His Gtv Asn Scr Vat Ata Ate Ata Ata Ata Vat 820 825 830 CGT GCC GCC GCC ACC ACT GCT GCT GGC CGC CCT GGC CCA GGT GGT GGT Arg Ata Ata Ata Thr Thr Ata Ata GLY Ara Pro MlY Pro GtivM GLC 835 840 845 GAC TCT GTG CCA GCC AMA CCT GTC MAC ACT GCC CCT GAT ACC AGG GGT 2 0 Aso Scr Vat Pro Ata Lys Pro Vat Asn Thr Ala Pro Aso Thr Arg GMy 850 855 860 865 TCC AGT GTC CCT GMA MC GAC CGC TTG GCC TCC ATA GCT GCA GMA CTG Ser Ser Vat Pro GLu Asn Asp Arg Lau Ala Ser lie Ala Ala GLu Lou 870 875 880 2 5 CAG TTC AGG TCT CTG AGT CGG CAC TCA AGC CCC ACG GMA GAG CGA GAC Gin Phe Arg Ser Lau Ser Arg His Ser Ser Pro Thr Glu Gtu Arg Asp 885 890 895 GAG CCA GCG TAT CCT CGG AGT GAC TCA AGT GGA TCA ACT CGG AGA AGC GLu Pro Ata Tyr Pro Arg Ser Asp Ser Ser Gty Ser Thr Arg Arg Ser 3 0 900 905 910 TGGI GMA CTT CGA ACA CTC ATC AGC CAG ACC AMA GAC TCG GCC TCT MAG Trp Giu Leu Arg Thr Leu lie 8cr Gin Thr Lys Asp Ser Ala Ser Lys 915 920 925 CAG GGG CCC ATA GMA GCT ATC GAG MAG TCA GTC CGA CTG TTT GMA GAG 3 5 Gin Gly Pro lie Ciu Ala lie Gin Lys 8cr Vat Arg Lou Phe Ciu GLu 930 935 940 945 AGG AGG TAT CGA GAG ATG AGG AGA MAG MAT ATC ATC GGC CMA GTG TGC Arg Arg Tyr Arg Giu Met Arg Arg Lys Asn lie lie Gty Gin Vat Cys 950 955 960 4 0 GAT ACC CCT MAG TCC TAT GAT MAC GTC ATG CAT GTT GGA CTG AGG MAG Asp Thr Pro Lys Ser Tyr Asp Asn Vat Met His Vat Gly Lou Arg Lys 965 970 975 GTG ACA TTT MAG TGG CAA AGA GGA MAC AMA ATT GGA GMA GGA GAG TAT Vat Thr Phe Lys Trp Gin Arg Giy Asn Lys lie GLy Giu Gty Gin Tyr 980 985 990 CGA AMA GTA TAC ACC TGC ATC ACT GTT GAC ACA GCG GAG CTG ATG GCC Gly Lys Vat Tyr Thr Cys lie 5cr Vat Asp Thr GLy Glu Lou Met Ata 995 1000 1005 ATG MAG GAG ATT CGA TTT CAG CCT MAC GAC CAC MAG ACT ATC MAG GAG 0 Met Lys Giu lie Arg Phe Gin Pro Asn Asp His Lys Thr lie Lys Giu 1010 1015 1020 1025 ACT GCA GAC GAG TTG AMA ATA TTT GMA GGC ATC MAG CAC CCC MAC CTG Thr Ala Asp Giu Leu Lys lie Phe Giu Ciy lie Lys His Pro Asn Lou 1030 1035 1040 2661 2709 2757 2805 2853 2901 2949 2997 3045 3093 3141 3189 3237 3285 3333 3381 3429 3477 WO 95/28421 WO 9528421PCTJUS94/1 1690 -42- GTC CGG TAT TTT GGC GTG GAG CTT CAC AGG GMA GAG ATG TAC ATC TTC 3525 Vat Arg Tyr Phe Gly Vat Gtu Leu His Arg Giu GLu Met Tyr ILe Phe 1045 1050 1055 ATG GAG TAC TGT GAT GAG GGT ACA CTA GAG GAG GTG TCA CGA CTG GGC 3573 Met GLu Tyr Cys Asp GLu Gly Thr Leu GLu Gtu VaL Ser Arg Leu GLy 1060 1065 1070 CTG CAG GAG CAC GTC ATC AGG TTA TAT ACC MAG CAG ATC ACT GTC GCC 3621 Leu Gin Gtu His Vat lie Arg Leu Tyr Thr Lys Gin ILe Thr Vat ALa 1075 1080 1085 ATC MAC GTC CTC CAT GAG CAC GGC ATC GTT CAC CGA GAC ATC AMA GGT 3669 Ile Asn VaL Lau His GLu His GLy Ile Vat His Arg Asp ILe Lys Gty 1090 1095 1100 1105 GCC MAT ATC TTC CTT ACG TCA TCT GGA CTA ATC MAG CTG GGA GAT TTT 3717 Ala Asn Ile Phe Lau Thr Ser Ser GLy Lau Ilie Lys Lau Gly Asp Phe 1110 1115 1120 GGA TGC TCT GTA MAA CTT MAA MAC MC GCC CAG ACC ATG CCC GGA GAG 3765 GLy Cys Ser Vat Lys Leu Lys Asn Asn Ala Gin Thr Met Pro Gly Gtu 1125 1130 1135 GTG MAC AGC ACC CTA GGG ACA GCA GCT TAC ATG GCC CCT GMA GTT ATT 3813 Vat Asn Ser Thr Leu Giy Thr Ala ALa Tyr Met Ala Pro Glu VaL lie 1140 1145 1150 ACC CGA GCC MAA GGA GMA GGC CAC GGA CGT GCG GCA GAT ATC TGG AGT 3861 Thr Arg Ala Lys GLy Giu Gly His Giy Arg Ala ALa Asp lie Trp Ser 1155 1160 1165 2 5 CTG GGG TGC GTC GTC ATA GAG ATG GTG ACT GGC MAG CGG CCT TGG CAT 3909 Leu Giy Cys Vat Vat lie Giu Met Vat Thr GLy Lys Arg Pro Trp His 1170 1175 1180 1185 GAG TAT GMA CAC MAC TTT CAG ATT ATG TAC MAG GTG GGG ATG GGA CAC 3957 GLu Tyr Giu His Asn Phe Gin Ilie Met Tyr Lys Vat Gly Met GLy His 1190 1195 1200 MAG CCA CCA ATC CCC GMA AGG CTA AGC CCT GMA GGA MAG CC TTT CTC 4005 Lys Pro Pro lie Pro Giu Arg Leu Ser Pro Giu GLy Lys Ala Phe Leu 1205 1210 1215 TCG CAC TGC CTG GMA AGT GAC CCG MAG ATA CGG TGG ACA GCC AGC CAG 4053 3 5 9cr His Cys Leu Gtu 9cr Asp Pro Lys Ile Arg Trp Thr Ala Ser Gin 1220 1225 1230 CTC CTC GAC CAC GCT TTT GTC MAG GTT TGC ACA GAT GMA GAG 4095 Leu Leu Asp His Ala Phe Vai Lys Vat Cys Thr Asp Giu Giu 1235 1240 1245 4 0 TGMAGTGMAC CAGTCCGTGG CCTAGTAGTG TGTGGACAGA ATCCCGTGAT CACTACTGTA 4155 TGTMATATTT ACATMAAGAC TGCAGCGCAG GCGGCCTTCC TMACCTCCCA GGACTGMAGA 4215 CTACAGGGGT GACAAGCCTC ACTTCTGCTG CTCCTGTCGC CTGCTGAGTG ACAGTGCTGA 4275 GGTTAMAGGA GCCGCACGTT MAGTGCCATT ACTACTGTAC ACGGCCACCG; CCTCTGTCCC 4335 CTCCGACCCT CTCGTGACTG AGAACCMACC GTGTCATCAG CACAGTGTTT TTGAGCTCCT 4395 4 5 GGGGTTCAGA AGMACATGTA GTGTTCCCGG GTGTCCGGGA CGTTTATTTC MACCTCCTGG 4455 TCGTTGGCTC TGACTGTGGA GCCTCCTTGT TCGAAAGCTG CAGGTTTGTT ATGCAAAGGC 4515 TCGTMAGTGA AGCTGMAGM MAGGTTCTTT TTCMATMAT GGTTTATTTT AGGMAAGCGA 4575 AAMAAAAAA AAAAMW 4592 MEKK 5 represents a splice variant of MEKK 4. The splice insert is shown by the underlined portion of the sequence shown in Table WO 95/2842 1 PCTJUS94/1 1690 -43- The amino acid sequences for MEKK 2 and MEKK 3 compared with the amino acid sequence of MEKK 1 are shown in Table 6.

Table 6.

NVTAVPAVFSKLVTNLNASGSTHFTRRUINAIADEVEIAEVJQLGVEDTVDGHDSL NEWK I NDQQALSINMI 2 MDEQALDSIMMcLVALuQNMTRL- 3 AVAPTSCLEUSSLEHTVHREKTGKGLSATRLSASSEDISDRLGVSVGLPSSTTEWPKP 1 AVLHKPVGQHYLYKKPKMLMHQJRJFESNLMIEEEKRILQVTRPVKLEDLRSKSKI 2 S--GYEMNKDTIPUQSDVRIKFEHUGERRI -IAFSRPWRYEDVEHICVTTVFGWPLD 3 AVUTKGRPHSCLNSS-PLSNALNFPAPSAPCSSAPSVPDISKaIRPQAFVPCIPSASP 1 AFGUSLHYTNELVIPLTTQWLDKAVELLDRSIHNKSL-KILLVVNGSTQA-TULEP 2 LHY1UNELSILLKJIODLDKAIDILDRSSSNJSLIRILLLSWPJHTSSSPHSGVSQRMI 3 OTQRKFSLQFQRUCSEHRDSDQLSPVFTQS-RPPPSSN IHRPKPSRPVPGSTSKLGDATK 1 SPSPEDLNNTPLGAkEEKKRLSVWPPNR--DRSSPPPGYIPDILHQIARNGSFTSINSEG 2 KPSQSAWDINTIYQAPEPRSRHLSVSSQHPGSSPPPGYVPERQQH IAROGSYTS INSEG 3 SSMTLDLGSASRCDDSFGGGGNSGNAVI PSDETVFTPVEDKCRLDVNTELNSSI EDLLEA 1 EF IPESNDQ-I4LDPLSLSSPENSGSGSCPSLDSPLDGESYPKSRMPRAgSYPtDNHgEFTD 2 EF IPETSEQCMLDPLSSAENSLSGSCQSLDRSADSPSFRKSHSRARSFPDNR- -ECSD 3 2 0 SMPSSDTTVTFKSEVAVLSPEKAENDDTYKDDVNHNQKCKEKIEAEEEEALAIAJWISAS 1 YDNPI FEKFGKGGTYPRRYHVSYHHOEYNDGRKTFPRARRTQGTSFRSPVSFSPTDHSLS 2 K- RETQLYDKGVKGGTYPRRYHVSVHHKDYNDGRRTFPRI RRHQGNLFTLVPSSRSL 3 QDALPIVPQLQVENGEDI II IQQOTPETLPGHTKAKQPYREDAEULKGGG!GLGAFSSCY 1 TSSGSSVFTPEYDDSR IRRRGSO IDNPTIT VTD ISPPSRSPRAPTNURLGICLLGOGAFGR 2 2 5 STNGENMGVAVQYLDPRGRLRSADSENALTVQERNVPTKSPSAPINWRRGKLLGQGAFGR 3 QAMVGTGTLNAVKQYVRMTSSEQEEVVEALREEIhJUIHLNHPNI IRNLGAiTCEKSU I VYLCYDVDTGRELAVKQVQFNPESPETSKEVMALECEIQLLKNLLHERIVQYYGCLRDPO 2 VYLCYDVDTGRELASKQVQFDPDSPETSKEVSALECEIQLLKNLQHERIVQYYGCLRDRA 3 YNLFJEIUAGGSVAHLLSKYGAFKESVVINYTEQLLRGLSYLHEN--Q- IIHRDVKGANL 1 EKTLSI FNELSPGGSIDLKAYGALTENVTRKYTRQILEGVHTLHSUMIVHRDIKGANI 2 EKI ITI FNEYIUCGSVKDQLKAYGAiLTESVTRKYTRQILEGWISYLHSNMIVHRDIKGANI 3 LIDSTGQ-RLRIADFGAAARLASK-GTGAkGEFQIGQLLGTIAFNAPEVLRGAQYGRSCDW 1 LRDSTGNIKLWDFGASKRLQTICLSGTQKSVG-PY IIISEVISGEGYGRUADIU 2 LRDSAGNVKLDFGASRLQTIOSGTGIRSTGTPY WqSMEISIEGYGRKOV 3 3 5 SVGCAI IENACAKPPUNAKHSNHILALI FKIASATTAPSIPSHLSPGLRDVAVRCLELQP I SYACTVVENLTEKPPU-AEFEA-MAA- IFKIATQPTNPKLPPHVSDYTRDFLKRI EVEAK 2 SLGCTWENLTEKPPV-AEYEA-MAA- IFKIATQPTNPQLPSHISEHGRDFLRRI FVEAR 3 ODRPPSRE-LLKHPVFRTTN I L-RP-SAEELLRHNFVHYH 2 Q-RP-SAEELLTHHFAQLVY 3 Bold Amino Terminus- Regulatory Domain Underline seauence- Regrulatory hingre Seqruence Bold Italics- Catalytic Domain Table 7 shows the amino acid sequence of the kinase domain of MEKK 4 compared with the kinase domains of MEKK 1, MEKK 2 and MEKK 3.

WO 95/28421 PCT/US94/11690 -44- Table 7.

IRFQPNDHKTIKETADEELKIFEGIKHPNLVRYFGV..ELHREEM.YI NEKK4 TYVRNTSSEQEEWEALREEIRMMGHLNHPNIIRNLGATCEKSNYNLFIE MEKK1 QVQFNPESPETSKEVNALECEIQLLKNLLHERIVQYYGCLRDPQEKTLSI MEKK2 QVQFDPDSPETSKEVSALECEIQLLKNLQHERIVQYYGCLRDRAEKILTI EKK3 IFNEYCDEGTLEEVSRLGLQEHV.I.RLYTKQITVAINVLHEHGNV NEKK4 .MMAGGSVAHLLSKYGAFKESW.IN..YTEQLLRGLSYLHENQII MEKK .FMELSPGGSIKDQLKAYGALTENVTRKYTRQILEGVHYLHSNMIV NEKK2 .FMEYMPGGSVKDQLKAYGALTESVTRKYTRQILEGNSYLHSNMIV MEKK3 The foregoing SEQ ID NO's represent sequences deduced according to methods disclosed in the Examples. It should be noted that since nucleic acid and amino acid sequencing technology is not entirely error-free, the foregoing SEQ ID NO's, at best, represent apparent nucleic acid and amino acid sequences of MEKK proteins of the present invention.

According to the present invention, an MEKK protein of the present invention can include MEKK proteins that have undergone post-translational modification. Such modification can include, for example, glycosylation including addition of N-linked and/or O-linked oligosaccharides) or post-translational conformational changes or post-translational deletions.

Another embodiment of the present invention is an isolated nucleic acid molecule capable of hybridizing, under stringent conditions, with an MEKK protein gene encoding an MEKK protein of the present invention. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu that has been subject to human manipulation). As such, "isolated" does not reflect the extent to which the nucleic acid molecule WO 95/28421 PCTIJUS94/11690 has been purified. An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.

An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire complete) gene or a portion thereof capable of forming a stable hybrid with that gene. As used herein, the phrase "at least a portion of" an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity. For example, at least a portion of a nucleic acid sequence, as used herein, is an amount of a nucleic acid sequence capable of forming a stable hybrid with a particular desired gene MEKK genes) under stringent hybridization conditions. An isolated nucleic acid molecule of the present invention can also be produced using recombinant DNA technology polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated MEKK protein nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode an MEKK protein of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates of MEKK.

WO 95/28421 PCT/US94/11690 -46- Preferred modifications to an MEKK protein nucleic acid molecule of the present invention include truncating a full-length MEKK protein nucleic acid molecule by, for example: deleting at least a portion of an MEKK protein nucleic acid molecule encoding a regulatory domain (examples illustrated in Table 6) to produce a constitutively active MEKK protein; deleting at least a portion of an MEKK protein nucleic acid molecule encoding a catalytic domain (examples illustrated in Table 6) to produce an inactive MEKK protein; and modifying the MEKK protein to achieve desired inactivation and/or stimulation of the protein, for example, substituting a codon encoding a lysine residue in the catalytic domain phosphotransferase domain) with a methionine residue to inactivate the catalytic domain.

A preferred truncated MEKK nucleic acid molecule encodes a form of an MEKK protein containing a catalytic domain but that lacks a regulatory domain. Preferred catalytic domain truncated MEKK nucleic acid molecules encode residues from about 352 to about 672 of MEKK 1, from about 352 to about 619 of MEKK 2, from about 358 to about 626 of MEKK 3, from about 811 to about 1195 of MEKK 4 or from about 863 to about 1247 of MEKK Another preferred truncated MEKK nucleic acid molecule encodes a form of an MEKK protein comprising an NH 2 -terminal regulatory domain a catalytic domain but lacking a catalytic domain. Preferred regulatory domain truncated MEKK nucleic acid molecules encode residues from about 1 to WO 95/28421 PCT/US94/11690 -47about 369 for MEKK i, from about 1 to about 335 for MEKK 2, from about 1 to about 360 for MEKK 3, from about 1 to about 825 for MEKK 4 and from about 1 to about 875 for MEKK thereby removing the regulatory domain to form the truncated MEKK molecule.

An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one MEKK protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides that comprise the nucleic acid molecule, the two phrases can be used interchangeably. As heretofore disclosed, MEKK proteins of the present invention include, but are not limited to, proteins having full-length MEKK protein coding regions, portions thereof, and other MEKK protein homologues.

As used herein, an MEKK protein gene includes all nucleic acid sequences related to a natural MEKK protein gene such as regulatory regions that control production of an MEKK protein encoded by that gene (including, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. A nucleic acid molecule of the present invention can be an isolated natural MEKK protein nucleic acid molecule or a homologue thereof. A nucleic acid molecule of the present invention can include one or more regulatory regions, full- WO 95/28421 PCT/US94/11690 -48length or partial coding regions, or combinations thereof.

The minimal size of an MEKK protein nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a corresponding natural gene.

An MEKK protein nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid the ability of a homologue to phosphorylate MEK protein or JEK protein) and/or by hybridization with isolated MEKK protein nucleic acids under stringent conditions.

One embodiment of the present invention is an MEKK protein nucleic acid molecule capable of encoding at least WO 95/28421 PCT/US94/11690 -49a portion of an MEKK protein, or a homologue thereof, as described herein. A preferred nucleic acid molecule of the present invention includes, but is not limited to, a nucleic acid molecule that encodes a protein having at least a portion of an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10, or homologues thereof.

A preferred nucleic acid molecule of the present invention is capable of hybridizing under stringent conditions to a nucleic acid that encodes at least a portion of an MEKK protein, or a homologue thereof. Also preferred is an MEKK protein nucleic acid molecule that includes a nucleic acid sequence having at least about preferably at least about 75%, and more preferably at least about 85% homology with the corresponding region(s) of the nucleic acid sequence encoding the catalytic domain of an MEKK protein, or a homologue thereof. Also preferred is an MEKK protein nucleic acid molecule that includes a nucleic acid sequence having at least about 20%, preferably at least about 30%, and more preferably at least about homology with the corresponding region(s) of the nucleic acid sequence encoding the NH 2 -terminal regulatory domain of an MEKK protein, or a homologue thereof. A particularly preferred nucleic acid sequence is a nucleic acid sequence having at least about 50%, preferably at least about and more preferably at least about 85% homology with a nucleic acid sequence encoding the catalytic domain of an amino acid sequence represented by SEQ ID NO:2, SEQ ID WO 95/28421 PCT/US94/11690 NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10. Another particularly preferred nucleic acid sequence is a nucleic acid sequence having at least about 20%, preferably at least about 30%, and more preferably at least about homology with a nucleic acid sequence encoding the NH 2 terminal regulatory domain of an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID Such nucleic acid molecules can be a full-length gene and/or a nucleic acid molecule encoding a full-length protein, a hybrid protein, a fusion protein, a multivalent protein or a truncation fragment. More preferred nucleic acid molecules of the present invention comprise isolated nucleic acid molecules having a nucleic acid sequence as represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.

Knowing a nucleic acid molecule of an MEKK protein of the present invention allows one skilled in the art to make copies of that nucleic acid molecule as well as to obtain additional portions of MEKK protein-encoding genes nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or MEKK protein nucleic acid molecule homologues. Knowing a portion of an amino acid sequence of an MEKK protein of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such an MEKK protein.

WO 95/28421 PCT/US94/11690 -51- The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of an MEKK protein, or a homologue thereof. A preferred oligonucleotide is capable of hybridizing, under stringent conditions, with a nucleic acid molecule that is capable of encoding at least a portion of an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10, or homologues thereof. A more preferred oligonucleotide is capable of hybridizing to a nucleic acid molecule having a nucleic acid sequence as represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9, or complements thereof.

Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The size of the oligonucleotide must also be sufficient for the use of the oligonucleotide in accordance with the present invention. Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic WO 95/28421 PCTIUS94/11690 -52applications to inhibit, for example, expression of MEKK proteins by cells. Such therapeutic applications include the use of such oligonucleotides in, for example, antisense-, triplex formation-, ribozyme- and/or RNA drugbased technologies. The present invention, therefore, includes use of such oligonucleotides and methods to interfere with the production of MEKK proteins.

In one embodiment, an isolated MEKK protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell that is capable of expressing the MEKK protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention.

Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed recombinant) cell in such a manner that their ability to be expressed is retained.

WO 95/28421 PCT/US94/11690 -53- The present invention also includes a recombinant vector which includes at least one MEKK protein nucleic acid molecule of the present invention inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, for example nucleic acid sequences that are not naturally found adjacent to MEKK protein nucleic acid molecules of the present invention. The vector can be either RNA or DNA, and either prokaryotic or eukaryotic, and is typically a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of MEKK protein nucleic acid molecules of the present invention. One type of recombinant vector, herein referred to as a recombinant molecule and described in more detail below, can be used in the expression of nucleic acid molecules of the present invention. Preferred recombinant vectors are capable of replicating in the transformed cell.

Preferred nucleic acid molecules to insert into a recombinant vector includes a nucleic acid molecule that encodes at least a portion of an MEKK protein, or a homologue thereof. A more preferred nucleic acid molecule to insert into a recombinant vector includes a nucleic acid molecule encoding at least a portion of an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and/or SEQ ID NO:10, or homologues thereof. An even more preferred nucleic acid molecule to insert into a recombinant vector includes a nucleic acid WO 95/28421 PCT/US94/11690 -54molecule represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID SEQ ID NO:7 and/or SEQ ID NO:9, or complements thereof.

Suitable host cells for transforming a cell can include any cell capable of producing MEKK proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule.

Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells. Preferred host cells include bacterial, yeast, insect and mammalian cells, with mammalian cells being particularly preferred.

A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.

Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can WO 95/28421 PCT/US94/11690 be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, insect, animal, and/or plant cells. As such, nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. As used herein, a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.

Preferred transcription control sequences include those which function in bacterial, yeast, and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as XPL and ApR and fusions that include such promoters), bacteriophage T7, WO 95/28421 PCT/US94/11690 -56- T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha mating factor, baculovirus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences, as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding an MEKK protein.

Preferred nucleic acid molecules for insertion into an expression vector include nucleic acid molecules that encode at least a portion of an MEKK protein, or a homologue thereof. A more preferred nucleic acid molecule for insertion into an expression vector includes a nucleic acid molecule encoding at least a portion of an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and/or SEQ ID NO:10, or homologues thereof. An even more preferred nucleic acid molecule for insertion into an expression vector includes a nucleic acid molecule represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID SEQ ID NO:7 and/or SEQ ID NO:9, or complements thereof.

WO 95/28421 PCT/US94/11690 -57- Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins. Inclusion of a fusion sequence as part of an MEKK nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protein encoded by the nucleic acid molecule.

Furthermore, a fusion segment can function as a tool to simplify purification of an MEKK protein, such as to enable purification of the resultant fusion protein using affinity chromatography. A suitable fusion segment can be a domain of any size that has the desired function increased stability and/or purification tool). It is within the scope of the present invention to use one or more fusion segments. Fusion segments can be joined to amino and/or carboxyl termini of an MEKK protein. Linkages between fusion segments and MEKK proteins can be constructed to be susceptible to cleavage to enable straight-forward recovery of the MEKK proteins. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an MEKK protein.

A recombinant cell of the present invention includes any cells transformed with at least one of any nucleic acid molecule of the present invention. A preferred recombinant cell is a cell transformed with at least one nucleic acid molecule that encodes at least a portion of an MEKK WO 95/28421 PCTIUS94/11690 -58protein, or a homologue thereof. A more preferred recombinant cell is transformed with at least one nucleic acid molecule that is capable of encoding at least a portion of an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 SEQ ID NO:8 and/or SEQ ID or homologues thereof. An even more preferred recombinant cell is transformed with at least one nucleic acid molecule represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and/or SEQ ID NO:9, or complements thereof. Particularly preferred recombinant cells include mammalian cells involved in a disease transformed with at least one of the aforementioned nucleic acid molecules.

It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.

Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals promoters, operators, enhancers), WO 95/28421 PCT/US94/11690 -59substitutions or modifications of translational control signals ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant protein production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing the resultant protein.

As used herein, amplifying the copy number of a nucleic acid sequence in a cell can be accomplished either by increasing the copy number of the nucleic acid sequence in the cell's genome or by introducing additional copies of the nucleic acid sequence into the cell by transformation.

Copy number amplification is conducted in a manner such that greater amounts of enzyme are produced, leading to enhanced conversion of substrate to product. For example, recombinant molecules containing nucleic acids of the present invention can be transformed into cells to enhance enzyme synthesis. Transformation can be accomplished using any process by which nucleic acid sequences are inserted into a cell. Prior to transformation, the nucleic acid sequence on the recombinant molecule can be manipulated to encode an enzyme having a higher specific activity.

In accordance with the present invention, recombinant cells can be used to produce an MEKK protein of the present WO 95/28421 PCT/US94/11690 invention by culturing such cells under conditions effective to produce such a protein, and recovering the protein. Effective conditions to produce a protein include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing an MEKK protein. Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins. The medium may comprise complex nutrients or may be a defined minimal medium.

Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultant MEKK proteins may either remain within the recombinant cell or be secreted into the fermentation medium. The phrase "recovering the protein" refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps WO 95/28421 PCTUS94/11690 -61of separation or purification. MEKK proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing and differential solubilization.

In addition, an MEKK protein of the present invention can be produced by isolating the MEKK protein from cells expressing the MEKK protein recovered from an animal. For example, a cell type, such as T cells, can be isolated from the thymus of an animal. MEKK protein can then be isolated from the isolated T cells using standard techniques described herein.

The present invention also includes a method to identify compounds capable of regulating signals initiated from a receptor on the surface of a cell, such signal regulation involving in some respect, MEKK protein. Such a method comprises the steps of: contacting a cell containing an MEKK protein with a putative regulatory compound; contacting the cell with a ligand capable of binding to a receptor on the surface of the cell; and (c) assessing the ability of the putative regulatory compound to regulate cellular signals by determining activation of a member of an MEKK-dependent pathway of the present invention. A preferred method to perform step (c) comprises measuring the phosphorylation of a member of an WO 95/28421 PCT/US94/11690 -62- MEKK-dependent pathway. Such measurements can be performed using immunoassays having antibodies specific for phosphotyrosines, phosphoserines and/or phosphothreonines.

Another preferred method to perform step comprises measuring the ability of the MEKK protein to phosphorylate MEK protein and/or JEK protein using methods described herein.

In another embodiment, a method to identify compounds capable of regulating signal transduction in a cell can comprise the steps of: contacting a putative inhibitory compound with an MEKK protein to form a reaction mixture; contacting the reaction mixture with MEK protein; and assessing the ability of the putative inhibitory compound to inhibit phosphorylation of the MEK protein by the MEKK protein. The results obtained from step can be compared with the ability of a putative inhibitory compound to inhibit the ability of Raf protein to phosphorylate MEK protein, to determine if the compound can selectively regulate signal transduction involving MEKK protein independent of Raf protein. MEKK, MEK and Raf proteins used in the foregoing methods can be recombinant proteins or naturally-derived proteins.

Moreover, one can determine whether the site of inhibitory action along a particular signal transduction pathway involves both Raf and MEKK proteins by carrying out experiments set forth above see discussion on MEKKdependent pathways).

WO 95/28421 PCT/US94/11690 -63- Another aspect of the present invention includes a kit to identify compounds capable of regulating signals initiated from a receptor on the surface of a cell, such signals involving in some respect, MEKK protein. Such kits include: at least one cell containing MEKK protein; (b) a ligand capable of binding to a receptor on the surface of the cell; and a means for assessing the ability of a putative regulatory compound to alter phosphorylation of the MEKK protein. Such a means for detecting phosphorylation include methods and reagents known to those of skill in the art, for example, phosphorylation can be detected using antibodies specific for phosphorylated amino acid residues, such as tyrosine, serine and threonine.

Using such a kit, one is capable of determining, with a fair degree of specificity, the location along a signal transduction pathway of particular pathway constituents, as well as the identity of the constituents involved in such pathway, at or near the site of regulation.

In another embodiment, a kit of the present invention can includes: MEKK protein; MEK protein; and a means for assessing the ability of a putative inhibitory compound to inhibit phosphorylation of the MEK protein by the MEKK protein. A kit of the present invention can further comprise Raf protein and a means for detecting the ability of a putative inhibitory compound to inhibit the ability of Raf protein to phosphorylate the MEK protein.

Another aspect of the present invention relates to the treatment of an animal having a medical disorder that is WO 95/28421 PCT/US94/11690 -64subject to regulation or cure by manipulating a signal transduction pathway in a cell involved in the disorder.

Such medical disorders include disorders which result from abnormal cellular growth or abnormal production of secreted cellular products. In particular, such medical disorders include, but are not limited to, cancer, autoimmune disease, inflammatory responses, allergic responses and neuronal disorders, such as Parkinson's disease and Alzheimer's disease. Preferred cancers subject to treatment using a method of the present invention include, but are not limited to, small cell carcinomas, non-small cell lung carcinomas with overexpressed EGF receptors, breast cancers with overexpressed EGF or Neu receptors, tumors having overexpressed growth factor receptors of established autocrine loops and tumors having overexpressed growth factor receptors of established paracrine loops.

According to the present invention, the term treatment can refer to the regulation of the progression of a medical disorder or the complete removal of a medical disorder cure). Treatment of a medical disorder can comprise regulating the signal transduction activity of a cell in such a manner that a cell involved in the medical disorder no longer responds to extracellular stimuli growth factors or cytokines), or the killing of a cell involved in the medical disorder through cellular apoptosis.

One aspect of the present invention involves the recognition that an MEKK protein of the present invention is capable of regulating the homeostasis of a cell by WO 95/28421 PCT/US94/11690 regulating cellular activity such as cell growth cell death, and cell function secretion of cellular products). Such regulation, in most cases, is independent of Raf, however, as discussed above (and as shown in Fig.

some pathways capable of regulation by MEKK protein may be subject to upstream regulation by Raf protein.

Therefore, it is within the scope of the present invention to either stimulate or inhibit the activity of Raf protein and/or MEKK protein to achieve desired regulatory results.

Without being bound by theory, it is believed that the regulation of Raf protein and MEKK protein activity at the divergence point from Ras protein (see Fig. 2) can be controlled by a "2-hit" mechanism. For example, a first "hit" can comprise any means of stimulating Ras protein, thereby stimulating a Ras-dependent pathway, including, for example, contacting a cell with a growth factor which is capable of binding to a cell surface receptor in such a manner that Ras protein is activated. Following activation of Ras protein, a second "hit" can be delivered that is capable of increasing the activity of JNK activity compared with MAPK activity, or vice versa. A second "hit" can include, but is not limited to, regulation of JNK or MAPK activity by compounds capable of stimulating or inhibiting the activity of MEKK, JEK, Raf and/or MEK. For example, compounds such as protein kinase C or phospholipase C kinase, can provide the second "hit" needed to drive the divergent Ras-dependent pathway down the MEKK-dependent WO 95/28421 PCT/US94/11690 -66pathway in such a manner that JNK is preferentially activated over MAPK.

One embodiment of the present invention comprises a method for regulating the homeostasis of a cell comprising regulating the activity of an MEKK-dependent pathway relative to the activity of a Raf-dependent pathway in the cell. As used herein, the term "homeostasis" refers to the tendency of a cell to maintain a normal state using intracellular systems such as signal transduction pathways.

Regulation of the activity of an MEKK-dependent pathway includes increasing the activity of an MEKK-dependent pathway relative to the activity of a Raf-dependent pathway by regulating the activity of a member of an MEKK-dependent pathway, a member of a Raf-dependent pathway, and combinations thereof, to achieve desired regulation of phosphorylation along a given pathway, and thus effect apoptosis. Preferred regulated members of an MEKKdependent pathway or a Raf-dependent pathway to regulate include, but are not limited to, proteins including MEKK, Raf, JEK, MEK, MAPK, JNK, TCF, ATF-2, Jun and Myc, and combinations thereof.

In one embodiment, the activity of a member of an MEKK-dependent pathway, a member of a Raf-dependent pathway, and combinations thereof, are regulated by altering the concentration of such members in a cell. One preferred regulation scheme involves altering the concentration of proteins including MEKK, Raf, JEK, MEK, MAPK, JNK, TCF, Jun, ATF-2, and Myc, and combinations WO 95/28421 PCT/US94/11690 -67thereof. A more preferred regulation scheme involves increasing the concentration of proteins including MEKK, JEK, JNK, Jun, ATF-2, and Myc, and combinations thereof.

Another more preferred regulation scheme involves decreasing the concentration of proteins including Raf, MEK, MAPK, and TCF, and combinations thereof. It is also within the scope of the present invention that the regulation of protein concentrations in two or more of the foregoing regulation schemes can be combined to achieve an optimal apoptotic effect in a cell.

A preferred method for increasing the concentration of a protein in a regulation scheme of the present invention includes, but is not limited to, increasing the copy number of a nucleic acid sequence encoding such protein within a cell, improving the efficiency with which the nucleic acid sequence encoding such protein is transcribed within a cell, improving the efficiency with which a transcript is translated into such a protein, improving the efficiency of post-translational modification of such protein, contacting cells capable of producing such protein with anti-sense nucleic acid sequences, and combinations thereof.

In a preferred embodiment of the present invention, the homeostasis of a cell is controlled by regulating the apoptosis of a cell. A suitable method for regulating the apoptosis of a cell is to regulate the activity of an MEKKdependent pathway in which the MEKK protein regulates the pathway substantially independent of Raf. A particularly preferred method for regulating the apoptosis of a cell WO 95/28421 PCTIUS94/11690 -68comprises increasing the concentration of MEKK protein by contacting a cell with a nucleic acid molecule encoding an MEKK protein that possesses unregulated kinase activity.

A preferred nucleic acid molecule with which to contact a cell includes a nucleic acid molecule encoding an MEKK protein represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10, and combinations thereof. A more preferred nucleic acid molecule with which to contact a cell includes a nucleic acid molecule encoding a truncated MEKK protein having only the kinase catalytic domain no regulatory domain) of an MEKK protein represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and/or SEQ ID NO:10. An even more preferred nucleic acid molecule with which to contact a cell includes a nucleic acid molecule including MEKKl 352 6 7, MEKK2 352 -6 19 MEKK3 358 626 MEKK4 8 11 -1195 MEKK5863- 1247 and combinations thereof.

Again, suitable variations of MEKK proteins described herein comprise those proteins encoded by a nucleic acid molecule that are able to hybridize to any of the above sequences under stringent conditions.

It is within the scope of the invention that the foregoing method can further comprise the step of decreasing the activity of MEK protein in the cell by contacting the cell with a compound capable of inhibiting MEK activity. Such compounds can include: peptides capable of binding to the kinase domain of MEK in such a manner that phosphorylation of MAPK protein by the MEK protein is inhibited; and/or peptides capable of binding to a portion WO 95/28421 PCT/US94/11690 -69of a MAPK protein in such a manner that phosphorylation of the MAPK protein is inhibited.

In another embodiment, the activity of a member of an MEKK-dependent pathway, a member of a Raf-dependent pathway, and combinations thereof, can be regulated by directly altering the activity of such members in a cell.

A preferred method for altering the activity of a member of an MEKK-dependent pathway, includes, but is not limited to, contacting a cell with a compound capable of directly interacting with a protein including MEKK, JEK, JNK, Jun, ATF-2, and Myc, and combinations thereof, in such a manner that the proteins are activated; and/or contacting a cell with a compound capable of directly interacting with a protein including Raf, MEK, MAPK, TCF protein, and combinations thereof in such a manner that the activity of the proteins are inhibited. A preferred compound with which to contact a cell that is capable of regulating a member of an MEKK-dependent pathway includes a peptide capable of binding to the regulatory domain of proteins including MEKK, JEK, JNK, Jun, ATF-2, and Myc, in which the peptide inhibits the ability of the regulatory domain to regulate the activity of the kinase domains of such proteins. Another preferred compound with which to contact a cell includes TNFa, growth factors regulating tyrosine kinases, hormones regulating G protein-coupled receptors and FAS ligand.

A preferred compound with which to contact a cell that is capable of regulating a member of a Raf-dependent WO 95/28421 PCT/US94/11690 pathway includes a peptide capable of binding to the kinase catalytic domain of a protein selected from the group consisting of Raf, MEK-1, MEK-2, MAPK, and TCF, in which the peptide inhibits the ability of the protein to be phosphorylated or to phosphorylate a substrate.

One aspect of the present invention relates to the recognition that an MEKK protein is capable of activating MAPK. MAPK is known to be involved in various cellular pathways in mammalian systems. MAPK is known to be involved in cellular mitogenesis, DNA synthesis, cell division and differentiation. MAPK is also recognized as being involved in the activation of oncogenes, such as cjun and c-myc. While not bound by theory, the present inventor believes that MAPK is also intimately involved in various abnormalities having a genetic origin. MAPK is known to cross the nuclear membrane and is believed to be at least partially responsible for regulating the expression of various genes. As such, MAPK is believed to play a significant role in the instigation or progression of cancer, neuronal diseases, autoimmune diseases, allergic reactions, wound healing and inflammatory responses. The present inventor, by being first to identify nucleic acid sequences encoding MEKK, recognized that it is now possible to regulate the expression of MEKK, and thus regulate the activation of MAPK.

The present invention also includes a method for regulating the homeostasis of a cell comprising injecting an area of a subject's body with an effective amount of a WO 95/28421 PCT/US94/11690 -71naked plasmid DNA compound. A naked plasmid DNA compound comprises a nucleic acid molecule encoding an MEKK protein of the present invention, operatively linked to a naked plasmid DNA vector capable of being taken up by and expressed in a recipient cell located in the body area. A preferred naked plasmid DNA compound of the present invention comprises a nucleic acid molecule encoding a truncated MEKK protein having deregulated kinase activity.

Preferred naked plasmid DNA vectors of the present invention include those known in the art. When administered to a subject, a naked plasmid DNA compound of the present invention transforms cells within the subject and directs the production of at least a portion of an MEKK protein or RNA nucleic acid molecule that is capable of regulating the apoptosis of the cell.

A naked plasmid DNA compound of the present invention is capable of treating a subject suffering from a medical disorder including cancer, autoimmune disease, inflammatory responses, allergic responses and neuronal disorders, such as Parkinson's disease and Alzheimer's disease. For example, a naked plasmid DNA compound can be administered as an anti-tumor therapy by injecting an effective amount of the plasmid directly into a tumor so that the plasmid is taken up and expressed by a tumor cell, thereby killing the tumor cell. As used herein, an effective amount of a naked plasmid DNA to administer to a subject comprises an amount needed to regulate or cure a medical disorder the naked plasmid DNA is intended to treat, such mode of WO 95/28421 PCT/US94/11690 -72administration, number of doses and frequency of dose capable of being decided upon, in any given situation, by one of skill in the art without resorting to undue experimentation.

Therapeutic compounds for use with a treatment method of the present invention can further comprise suitable excipients. A therapeutic compound for use with a treatment method of the present invention can be formulated in an excipient that the subject to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.

Other useful excipients include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.

Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to WO 95/28421 PCT/US94/11690 -73which sterile water or saline can be added prior to administration.

In another embodiment, a therapeutic compound for use with a treatment method of the present invention can also comprise a carrier. Carriers are typically compounds that increase the half-life of a therapeutic compound in the treated animal. Suitable carriers include, but are not limited to, liposomes, micelles, cells, polymeric controlled release formulations, biodegradable implants, bacteria, viruses, oils, esters, and glycols. Preferred carriers include liposomes and micelles.

A therapeutic compound for use with a treatment method of the present invention can be administered to any subject having a medical disorder as herein described. Acceptable protocols by which to administer therapeutic compounds of the present invention in an effective manner can vary according to individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art without resorting to undue experimentation. An effective dose refers to a dose capable of treating a subject for a medical disorder as described herein. Effective doses can vary depending upon, for example, the therapeutic compound used, the medical disorder being treated, and the size and type of the recipient animal. Effective doses to treat a subject include doses administered over time that are capable of regulating the activity, including growth, of cells WO 95/28421 PCT[S94/11690 -74involved in a medical disorder. For example, a first dose of a naked plasmid DNA compound of the present invention can comprise an amount of that causes a tumor to decrease in size by about 10% over 7 days when administered to a subject having a tumor. A second dose can comprise at least the same the same therapeutic compound than the first dose.

Another aspect of the present invention includes a method for prescribing treatment for subjects having a medical disorder as described herein. A preferred method for prescribing treatment comprises: measuring the MEKK protein activity in a cell involved in the medical disorder to determine if the cell is susceptible to treatment using a method of the present invention; and prescribing treatment comprising regulating the activity of an MEKKdependent pathway relative to the activity of a Rafdependent pathway in the cell to induce the apoptosis of the cell. The step of measuring MEKK protein activity can comprise: removing a sample of cells from a subject; stimulating the cells with a TNFa; and detecting the state of phosphorylation of JEK protein using an immunoassay using antibodies specific for phosphothreonine and/or phosphoserine.

The present invention also includes antibodies capable of selectively binding to an MEKK protein of the present invention. Such an antibody is herein referred to as an anti-MEKK antibody. Polyclonal populations of anti-MEKK antibodies can be contained in an MEKK antiserum. MEKK WO 95/28421 PCT/US94/11690 antiserum can refer to affinity purified polyclonal antibodies, ammonium sulfate cut antiserum or whole antiserum. As used herein, the term "selectively binds to" refers to the ability of such an antibody to preferentially bind to MEKK proteins. Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, enzyme immunoassays ELISA), radioimmunoassays, immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.

Antibodies of the present invention can be either polyclonal or monoclonal antibodies and can be prepared using techniques standard in the art. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein used to obtain the antibodies. Preferably, antibodies are raised in response to proteins that are encoded, at least in part, by a MEKK nucleic acid molecule.

More preferably antibodies are raised in response to at least a portion of an MEKK protein, and even more preferably antibodies are raised in response to either the amino terminus or the carboxyl terminus of an MEKK protein.

Preferably, an antibody of the present invention has a WO 95/28421 PCT/US94/11690 -76single site binding affinity of from about 103M 1 to about 12

M

1 for an MEKK protein of the present invention.

A preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of an MEKK protein to produce the antibody and recovering the antibodies. Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used to identify unique MEKK proteins and recover MEKK proteins.

Another aspect of the present invention comprises a therapeutic compound capable of regulating the activity of an MEKK-dependent pathway in a cell identified by a process, comprising: contacting a cell with a putative regulatory molecule; and determining the ability of the putative regulatory compound to regulate the activity of an MEKK-dependent pathway in the cell by measuring the activation of at least one member of said MEKK-dependent pathway. Preferred methods to measure the activation of a member of an MEKK-dependent pathway include measuring the transcription regulation activity of c-Myc protein, measuring the phosphorylation of a protein selected from the group consisting of MEKK, JEK, JNK, Jun, ATF-2, Myc, and combinations thereof.

The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

EXAMPLES

WO 95/28421 PCT/US94/11690 -77- Example 1 This example describes the structural characterization of MEKK 1 protein.

A. MEKK 1 Nucleotide Sequence MEKK 1 protein was cloned by the following method.

Unique degenerate inosine oligodeoxynucleotides were designed to correspond to regions of sequence identity between the yeast Stell and Byr2 genes. With primers and cDNA templates derived from polyadenylated RNA from NIH 3T3 cells, a polymerase chain reaction (PCR) amplification product of 320 base pairs (bp) was isolated. This 320 bp cDNA was used as a probe to identify an MEKK 1 cDNA of 3260 bp from a mouse brain cDNA library using standard methods in the art. The MEKK 1 nucleotide sequence was determined by dideoxynucleotide sequencing of double-stranded DNA using standard methods in the art.

Referring to Table 6, based on the Kozak consensus sequence for initiation codons, the starting methionine can be predicted to occur at nucleotide 486. With this methionine at the start, the cDNA encodes a protein of 672 amino acids, corresponding to a molecular size of 73 kD.

There is another in-frame methionine at position 441, which does not follow the Kozak rule, but would yield a protein of 687 amino acid residues (74.6 kD). Also referring to Table 6, 20% of the NH 2 -terminal 400 amino acids are serine or threonine and there are only two tyrosines. Several potential sites of phosphorylation by protein kinase C are WO 95/28421 PCT/US94/11690 -78apparent in the NH 2 -terminal region. The kinase catalytic domain is located in the COOH-terminal half of the MEKK 1.

B. Southern Blot Analysis of MEKK 1 Transcript Equal amounts (20 Ag) of total RNA were loaded onto the gel as indicated by ethidium bromide staining. Blots were probed with either a 320-bp cDNA fragment encoding a portion of the MEKK kinase domain or an 858-bp fragment encoding a portion of the NH 2 terminal region of MEKK using standard methods in the art. Referring to Fig. 3A, a 7.8 kb mRNA was identified with probes derived from both the and 3' ends of the MEKK cDNA in several cell lines and mouse tissues. The MEKK mRNA was highly expressed in mouse heart and spleen, an in lower amounts in liver.

C. Southern Blot Analysis Mouse genomic DNA (10 jg) was digested with either Bam HI, Hind III or Eco RI and applied to gels using standard methods in the art. Blots were probed with a 320-bp fragment of the MEKK gene. Fig. 3B shows the appearance of one band in the Bam HI and Hind III digests which indicates that MEKK is encoded by one gene. The appearance of two bands in the Eco RI digest indicates the likely presence of an Eco RI site within an intron sequence spanned by the probe.

D. Immunoblots Using Anti-MEKK Antibodies Three polyclonal antisera were prepared using three different antigens. A first polyclonal antiserum was prepared using an antigen comprising a 15 amino acid peptide DRPPSRELLKHPVER derived from the COOH-terminus of WO 95/28421 PCTIS94/11690 -79- MEKK. NZW rabbits were immunized with the peptide and antisera was recovered using standard methods known in the art. This first polyclonal antiserum is hereinafter referred to as the DRPP antiserum.

A second polyclonal antiserum was produced using a DNA clone comprising an MEKK cDNA digested with EcoRl and PstI, thereby creating a 1270 bp fragment that encodes the amino terminus of MEKK. This fragment was cloned into pRSETC to form the recombinant molecule pMEKK 1369 comprising amino acid residues 1 to 369 of MEKK 1. The pMEKK1 1 369 recombinant molecule was expressed in E. coli and protein encoded by the recombinant molecule was recovered and purified using standard methods known in the art. NZW rabbits were immunized with the purified recombinant MEKK1 1 369 protein and antisera was recovered using standard methods known in the art. This second polyclonal antiserum is hereinafter referred to as the MEKK1 1 9 antiserum.

A third polyclonal antiserum was produced using a DNA clone comprising an MEKK cDNA digested with Pst I and Kpn 1, thereby creating a 1670 bp fragment that encodes the catalytic domain of MEKK. This fragment was cloned into pRSETC to form the recombinant molecule pMEKK 370 .38 comprising amino acid residues 370 to 738 of MEKK 1 (encoded by base pairs 1592-3260). The pMEKK1 370 -38 recombinant molecule was expressed in E. coli and protein encoded by the recombinant molecule was recovered and purified using standard methods known in the art. NZW rabbits were immunized with the purified recombinant WO 95/28421 PCT/US94/11690

MEKK

370 -73 8 protein and antisera was recovered using standard methods known in the art. This second polyclonal antiserum is hereinafter referred to as the MEKK1 370 -8 antiserum.

The DRPP antiserum was used to probe Western Blots of soluble cellular protein derived from several rodent cell lines. Soluble cellular protein (100 ug) or recombinant MEKK COOH-terminal fusion protein (30 ng) was loaded onto a 10% Tris Glycine SDS-PAGE gel and the protein transferred to a nylon filter using methods standard in the art. The nylon filter was immunoblotted with affinity purified DRPP antiserum (1:300 dilution). Referring to Fig. 3C, a 78 kD immunoreactive protein was identified in the samples comprising protein from Pheochromocytoma (PC12), Rat la, and NIH 3T3 cells. A prominent 50 kD immunoreactive band was also commonly present but varied in intensity from preparation to preparation indicating the band is a proteolytic fragment. Visualization of both the 78 kD and kD immunoreactive bands on immunoblots was inhibited by pre-incubation of the 15 amino acid peptide antigen with the affinity purified DRPP antiserum. The MEKK protein detected by immunoblotting is similar to the molecular size predicted from the open reading frame of the MEKK cDNA.

In a second immunoblot experiment, PC12 cells stimulated or not stimulated with EGF were lysed and resolved on 10% Tris Glycine SDS-PAGE gel as described above. MEKK proteins contained in the cell lysates were identified by immunoblot using affinity purified MEKK1 1369 antiserum (1:300) using methods standard in the art.

WO 95/28421 PCT/US94/11690 -81- Referring to Fig. 4, MEKK 1 and two higher molecular weight proteins having MEKK activity, MEKK a and MEKK B, were identified using the affinity purified MEKKl 1369 antiserum.

MEKK 1, and not MEKK a and MEKK B, were identified using the affinity purified MEKK 1 9 antiserum.

Using the same procedure described above, two MEKK immunoreactive species of approximately 98 kD and 82 kD present in PC12, Ratla, NIH3T3, and Swiss3T3 cell lysates were recognized by affinity purified MEKK1 13 antiserum as shown in Fig. 5. It should be noted that the 98 kD MEKK protein described herein was originally identified as a kD MEKK protein in the related PCT application (International application no. PCT/US94/04178). Subsequent Tris Glycine SDS-PAGE gel analysis has led to the determination that the modification in molecular weight.

Visualization of both of these proteins was inhibited by incubation of the affinity purified MEKK1 1 antiserum with purified recombinant MEKK1 1 9 fusion protein antigen. A single 98 kD MEKK protein was present in MEKK immunoprecipitates, but not in immunoprecipitates using preimmune serum. More of the 98 kD MEKK was expressed in PC12 cells relative to fibroblast cell lines.

Immunoblotting with antibodies that specifically recognize Raf-1 or Raf-B indicated that neither of these enzymes were present as contaminants of MEKK immunoprecipitates. 98 kD MEKK in MEKK immunoprecipitates did not comigrate with Raf- 1 or Raf-B in PC12 cell lysates and no cross-reactivity between MEKK and Raf antibodies was observed.

WO 95/28421 PCT/US94/11690 -82- Example 2 This example describes the isolation of nucleic acid sequences encoding MEKK 2, MEKK 3 and MEKK 4 protein.

PCR primers were designed based on the nucleotide sequence of MEKK 1. PCR amplification of fragments from DNA isolated from reverse transcriptase reactions of RNA isolated form PC12 and HL60 cells was conducted using standard techniques. The resultant PCR products were cloned into the pGEX cloning vector (Promega, Wisconsin) using standard procedures and submitted to DNA sequence analysis using standard techniques.

Example 3 This example describes the expression of MEKK 1 protein in COS-1 cells to define its function in regulating the signaling system that includes MAPK.

COS cells in 100-mm culture dishes were transfected with either the pCVMV5 expression vector alone (1 gg: control) or the pCVMV5 MEKK construct (1 gg: MEKK). After 48 hours, the cells were placed in serum-free medium containing bovine serum albumin (0.1 percent) for 16 to 18 hours to induce quiescence. Cells were then treated with human EGF (30 ng/ml) (+EGF) or buffer (control) for minutes, washed twice in cold phosphate buffered saline (PBS), and lysed in cell lysis buffer containing 50 mM 8glycerophosphate (pH 100 uM sodium vanadate, 2 mM MgC12, ImM EGTA Triton X-100 (0.5 percent), leupeptin (2 gg/ml), aprotinin (2Ag/ml), and 1 mM dithiothreitol (600 Al). After centrifugation for 10 minutes at maximum speed WO 95/28421 PCT/US94/11690 -83in a microfuge, COS cell lysates containing 0.5 to 1 mg of soluble protein were subjected to FPLC on a MONO Q column, and eluted fractions were assayed for MAPK activity according to the method described in Heasley et al., p.

545, 1992, Mol. Biol. Cell, Vol. 3.

Referring to Fig. 6A, when MEKK 1 was overexpressed in COS 1 cells, MAPK activity was four to five times greater than that in control cells transfected with plasmid lacking an MEKK 1 cDNA insert. The activation of MAPK occurred in COS cells deprived of serum and in the absence of any added growth factor. The activity of MAPK was similar to that observed after stimulation of control cells with EGF.

Stimulation of COS cells transiently overexpressing MEKK with EGF resulted in only a slight increase in MAPK activity compared to that observed with MEKK expression alone.

To ensure that MEKK protein was present in the samples tested for MAPK activity, protein from cell lysates of the transfected COS 1 cells were immunoblotted with MEKK specific antiserum. Equal amounts (100 jg) of soluble protein lysate from COS cells were placed on the gel for immunoblotting using the methods described in Example 1.

The filters were immunoblotted using the affinity purified DRPP antiserum (1:300) and affinity purified MEKK, 369 antiserum (1:300). Referring to Fig. 6B, the results indicate that expression of MEKK in cells transfected with vector encoding MEKK that were treated with or without EGF.

Only the 50 kD MEKK immunoreactive fragment was detected in WO 95/28421 PCT/US94/11690 -84lysates from control COS cells using the DRPP antiserum.

Transient expression of MEKK in COS cells yielded a predominant 82 kD band that was slightly larger than that observed in PC12, Rat la, or NIH 3T3 cells. Addition of the 15 amino acid DRPP peptide antigen to the antiserum during immunoblotting prevented detection of all of the immunoreactive bands; these bands were not detected in extracts of control COS cells, an indication that they were derived from the expressed MEKK protein.

Example 4 This Example describes the expression of MEKK 1 in COS cells to test the ability of MEKK protein to activate MEK protein.

Recombinant MAPK was used to assay MEK activity in COS cell lysates that had been fractionated by fast protein liquid chromatography (FPLC) on a Mono S column. A cDNA encoding p42 MAPK from Xenopus laevis was cloned into the pRSETB expression vector. This construct was used for expression in the LysS strain of Escherichia coli BL21(DE3) of a p42 MAPK fusion protein containing a polyhistidine sequence at the NH 2 -terminus. Cultures containing the expression plasmid were grown at 37 0 C to an optical density of 0.7 to 0.9 at 600 nM. Isopropyl-B-thiogalactopyranoside mM) was added to induce fusion protein synthesis and the cultures were incubated for 3 hours. The cells were then collected and lysed by freezing, thawing, and sonication. The lysate was centrifuged at 10,000g for minutes at 4°C. The supernatant was then passed over a Ni 2 minutes at 4 0 C. The supernatant was then passed over a Ni 2 WO 95/28421 PCT/US94/11690 charged Sepharose resin and the soluble recombinant MAPK was eluted in sodium phosphate buffer (pH The purified recombinant MAPK was more than 80 percent pure.

The purified recombinant MAPK served as a substrate for MEK and catalyzed the phosphorylation of a peptide consisting of residues 662 to 681 of the EGF receptor (EGFR 6 2681 Soluble cell lysates from COS cells transiently transfected with MEKK, mock-transfected (control), or mocktransfected and treated with EGF (30 ng/ml) were fractionated by FPLC on a Mono S column and endogenous MEK activity was measured. Endogenous MAPK eluted in fractions 2 to 4, whereas MEK was contained in fractions 9 to 13.

For assaying endogenous MEK activity, cells were washed twice in cold PBS and lysed in 650 pl of a solution containing 50 mM B-glycerophosphate, 10 mM 2-Nmorpholinoethane-sulfonic acid (pH 100 jM sodium vanadate, 2 mM MgCl 2 1 mM EGTA, Triton X-100 (0.5 percent), leupeptin (5 Mg/ml), aprotinin (2 Mg/ml), and 1 mM dithiothreitol. After centrifugation at maximum speed for 10 minutes in a microfuge, soluble cell lysates (1 to 2 mg of protein) were applied to a Mono S column equilibrated in elution buffer (50 mM B-glycerophosphate, 10 mM MES (pH 100 gM sodium vanadate, 2 mM MgCl, 1 mM EGTA, and 1 mM dithiothreitol. The column was washed with buffer (2 ml) and bound proteins were eluted with a 30ml linear gradient of 0 to 350 mM NaCl in elution buffer. A portion pl) of each fraction was assayed for MEK activity by mixing with buffer (25 mM B-glycerophosphate, 40 mM N-(2- WO 95/28421 PCT/US94/11690 -86hydroxyethyl)piperazine-N' (2-ethanolsulfonic acid) (pH 7.2) mM sodium vanadate, 10 mM MgCl 2 100 AM y- 32 P-ATP (3000 to 4000 cpm/pmol), inhibitor protein-20 TTYADFIASGRTGRRNAIHD; 25 Ag/ml), 0.5 mM EGTA, recombinant MAP kinase (7.5 Ag/ml), and 200 AM EGFR 662 681 in a final volume of 40 l1. After incubation at 30 0 C for 20 minutes, the incorporation of y- 32 P-ATP into EGFR 6 2 681 was measured.

In this assay, the ability of each column fraction to activate added recombinant MAPK was measured by the incorporation of y- 32 P-ATP into the MAPK substrate, a peptide derived from the EGF receptor (EGFR).

Referring to Fig. 7, the first peak of activity eluted represents endogenous activated MAPK, which directly phosphorylates the EGFR peptide substrate. The second peak of activity represents the endogenous MEK in COS cells.

The activity of endogenous MEK activity was characterized by fractionation of Mono S FPLC. COS cell lysates were fractionated by FPLC on a Mono Q column to partially purify the expressed MEKK. Purified recombinant MEK-1 was then used as a substrate for MEKK in the presence of y- 32 P-ATP to determine whether MEKK directly phosphorylates MEK-1.

A cDNA encoding MEK-1 was obtained from mouse B cell cDNA templates with the polymerase chain reaction and oligodeoxynucleotide primers corresponding to portions of the 5' coding region and 3' untranslated region of MEK-1.

The catalytically inactive MEK-1 was generated by sitedirected mutagenesis of Lys 343 to Met. The wild-type MEK-1 WO 95/28421 PCT/US94/11690 -87and catalytically inactive MEK-1 proteins were expressed in pRSETA as recombinant fusion proteins containing a polyhistidine sequence at their NH 2 -termini.

Lysates from COS cells transfected with MEKK or mocktransfected (control) were subjected to FPLC on a Mono Q column as described above. Portions (20 l) of fractions containing MEKK were mixed with buffer containing 50 mM 8glycerophosphate (pH 100 AM sodium vanadate, 2 mM MgCl 2 ImM EGTA, 50 AM ATP, IP-20 (50 xg/ml), and 10 Al y- 32 P-ATP in a reaction volume of 40 Al and incubated for minutes in the presence or absence of recombinant, catalytically inactive MEK-1 (150 ng)(kinase-MEK-1).

Reactions were stopped by the addition of 5 x SDS sample buffer (10 pl), 1 x SDS buffer contains 2 percent SDS, percent glycerol, 62.5 mM tris-HCl (pH 5 percent Bmercaptoethanol, and 0.001 percent bromophenol blue. The samples were boiled for 3 minutes and subjected to SDS-PAGE and autoradiography.

Referring to Fig. 8A, autophosphorylated recombinant wild-type MEK-1 (WT MEK-1) comigrated with phosphorylated catalytically inactive MEK-1. Thus, MEKK was capable of phosphorylating MEK-1. Corresponding fractions of lysates from control cells, however, were not able to phosphorylate MEK-1.

Example This example describes studies showing that the modified form of MEK-1 that was used in the phosphorylation WO 95/28421 PCT/US94/11690 -88assay of Example 4 did not autophosphorylate as does wildtype MEK-1.

Phosphorylation of catalytically inactive MEK-1 by MEKK was time dependent (Fig. 8B); MEKK was also phosphorylated. Fraction 22 from FPLC on a Mono Q column pl) was incubated with or without recombinant catalytically inactive MEK-1 (0.15 Mg) for the indicated times. Referring to Fig. 8B, phosphorylation of kinase MEK-1 and MEKK was visable after 5 minutes and maximal after about 20 minutes. The time-dependent increase in MEKK phosphorylation correlated with a decreased mobility of the MEKK protein during SDS-PAGE. Referring to Fig. 8C, immunoblotting demonstrated that the MEKK protein co-eluted (after FPLC on a Mono Q column) with the peak of activity (fraction 22) that phosphorylated MEK. The slowly migrating species of MEKK were also detected by immunoblotting. Thus, expression of MEKK appears to activate MAPK by activating MEK.

Example 6 This Example describes that the phosphorylation of MEK by overexpressed MEKK resulted in activation of MEK, recombinant wild-type MEK-1 and a modified form of MAPK that is catalytically inactive.

COS cell lysates were separated by Mono Q-FPLC and fractions containing MEKK were assayed for their ability to activate added wild-type MEK-1 such that it would phosphorylate catalytically inactive recombinant MAPK in the presence of y- 2 P-ATP. Lysates from COS cells WO 95/28421 PCT/US94/11690 -89transfected with MEKK or mock-transfected (control) were fractionated by FPLC on a Mono Q column and portions Al) of fractions containing MEKK were mixed with buffer.

Each fraction was incubated in the presence or absence of purified recombinant wild-type MEK-1 (150 ng) and in the presence of purified recombinant, catalytically inactive (kinase') MAPK (300 ng). Referring to Fig. 9A, fractions 20 to 24 from lysates of COS cells transfected with MEKK activated MEK-1. Thus, MEKK phosphorylated and activated MEK-1, leading to MAPK phosphorylation.

Example 7 This Example describes studies demonstrating that MEKK activated MEK directly, and not through the activation of one or more other kinases contained in the column fractions.

Overexpressed MEKK was immunoprecipitated from COS cell lysates with affintiy purified MEKK,69 antiserum.

Immunoprecipitated MEKK was resuspended in 10 to 15 p1 of PAN (10 mM piperazine-N, N'-bis-2-ethanesulfonic acid (Pipes) (pH 100 mM NaCl, and aprotinin (20 Cjg/ml) and incubated with or without catalytically inactive MEK-1 (150 ng) and 25 pCi of y- 32 P-ATP in 20 mM pipes (pH 10 mM MnCl 2 and aprotinin (20 /g/ml) in a final voume of 20 p1 for 15 minutes 30 0 C. Reactions were stopped by the addition of 5 x SDS sample buffer (5 The samples were boiled for 3 minutes and subjected to SDS-PAGE and autoradiography.

WO 95/28421 PCT/US94/11690 Referring to Fig. 9B, MEKK phosphorylated catalytically inactive MEK-1, which comigrated with wildtype MEK-1 on SDS-PAGE. Several phosphorylated bands of overexpressed MEKK were detected in the immunoprecipitates.

These bands probably resulted from autophosphorylation of MEKK and corresponded to the forms of MEKK identified by immunoblotting of lysates from COS cells transfected with MEKK. Immunoprecipitates obtained with pre-immune serum contained no MEKK and did not phosphorylate MEK-1. Thus, MEKK appears to directly phosphorylate MEK.

Taken together, the results from Examples 4 through 7 show that MEKK can phosphorylate and activate MEK, which in turn phosphorylates and activates MAPK.

Example 8 This Example demonstrates that Raf can also phosphorylate and activate MEK.

COS cells deprived of serum were stimulated with EGF, and Raf was immunoprecipitated with an antibody to the COOH-terminus of Raf-l. Cos cells were transiently transfected with vector alone (control) or with the MEKK construct (MEKK). Quiescent control cells were treated with or without human EGF (30 ng/ml) for 10 minutes and Raf was immunoprecipitatd from cell lysates with an antibody to a COOH-terminal peptide from Raf.

Immunoprecipitated Raf was incubated with catalytically inactive MEK-1 (150 ng) and 25 1l of y- 32 P-ATP. The immunoprecipitated Raf phosphorylated MEK-1 in the presence of y- 32 P-ATP (Fig. 10A). Little or no phosphorylation of WO 95/28421 PCT/US94/11690 -91- MEK-1 by Raf was observed in immunoprecipitates of Raf from COS cells overexpressing MEKK. Treatment of COS cells overexpressing MEKK with EGF resulted in a similar degree of phosphorylation of MEK-1 by immunoprecipitated Raf (Fig.

10B). Cells transfected with MEKK and deprived of serum were treated with EGF, and Raf was immunoprecipitated and incubated with catalytically inactive MEK-1. Equal amounts of Raf were immunoprecipitated in each sample as demonstrated by immunoblotting with antibodies to Raf. The slowest migrating band represents an immunoprecipitated phosphoprotein that is unrelated to Raf or MEK-1. The amount of Raf in the immunoprecipitates from control cells and cells transfected with MEKK was similar as shown by subsequent SDS-PAGE and immunoblotting with the antibody to Raf. Thus, both MEKK and Raf can independently activate

MEK.

Example 9 This Example describes the activation of a 98 kD MEKK protein isolated from PC12 cells in response to stimulation of cells containing MEKK protein by growth factors.

PC12 cells were deprived of serum by incubation in starvation media (DMEM, 0.1% BSA) for 18-20 hours and MEKK was immunoprecipitated from lysates containing equal amounts of protein from untreated controls or cells treated with EGF (30ng/ml) or NGF (100ng/ml) for 5 minutes with the above-described anti-MEKK antibodies speicific for the NH 4 terminal portion of MEKK. Immunoprecipitated MEKK was resuspended in 8pl of PAN (10mM piperazine-N,N'-bis-2- WO 95/28421 PCT/US94/11690 -92ethanesulfonic acid (Pipes) (pH 100mM NaCl, and aprotinin (20Ag/ml)) and incubated with catalytically inactive MEK-1 (150ng) and 40Ci of (y- 32 P)ATP in universal kinase buffer (20mM piperazine-N,N'-bis-2-ethanesulfonic acid (Pipes) (pH 10mM MnC12, and aprotinin in a final volume of 2 01 for 25 minutes at 30 0

C.

Reactions were stopped by the addition of 2X SDS sample buffer (20il). The samples were boiled for 3 minutes and subjected to SDS-PAGE and autoradiography. Raf-B was immunoprecipitated from the same untreated and treated PC12 cell lysates as above with an antiserum to a COOH-terminal peptide of Raf-B (Santa Cruz Biotechnology, Inc.) and assayed similarly. Raf-1 was immunoprecipitated with an antiserum to the 12 COOH-terminal amino acids of Raf-1 (Santa Cruz Biotechnology, Inc.). Epidermal growth factor (EGF) treatment of serum starved PC12 cells resulted in increased MEKK activity.

Referring to Fig. 11, the results were obtained by measuring the phosphorylation of purified MEK-1 (a kinase inactive form) by immunoprecipitates of MEKK in in vitro kinase assays. NGF stimulated a slight increase in MEKK activity compared to control immunoprecipitates from untreated cells. Stimulation of MEKK activity by NGF and EGF was similar to Raf-B activation by these agents, although Raf-B exhibited a high basal activity. Activation of c-Raf-1 by NGF and EGF was almost negligible in comparison to that of MEKK or Raf-B.

WO 95/28421 PCT/US94/11690 -93- A timecourse of MEKK stimulation by EGF was established by immunoprecipitating MEKK or Raf-B protein from lysates of PC12 cells treated with EGF (30ng/ml) for 0, 1, 3, 5, 10, or 20 minutes and incubating the protein with catalytically inactive MEK-1 (150ng) and (y- 32 P)ATP as described above. Data represent the relative magnitude of the response for each timepoint as quantitated by phosphorimager analysis of radioactive gels from a typical experiment. A timecourse of EGF treatment indicated that MEKK activation reached maximal levels following 5 minutes and persisted for at least 30 minutes (Fig. 12). Raf-B exhibited a similar timecourse; peak activity occurred within 3-5 minutes following EGF treatment and was persistent for up to 20 minutes.

To further dissociate EGF-stimulated MEKK activity from that of Raf-B, Raf-B was immunodepleted from cell lysates prior to MEKK immunoprecipitation. Raf-B was precleared from lysates of serum-starved PC12 cells which had been either treated or not treated with EGF (30ng/ml) for 5 minutes. Raf-B was pre-cleared two times using antisera to Raf-B or using preimmune IgG antisera as a control. The pre-cleared supernatant was then immunoprecipitated with either MEKK or Raf-B antisera and incubated with catalytically inactive MEK-1 and (y- 32 P)ATP as described in detail above. EGF-stimulated and unstimulated PC12 cell lysates were precleared with either IgG or Raf-B antisera and then subjected to immunoprecipitation with MEKK antiserum or Raf-B antibodies. The results shown in Fig.

WO 95/28421 PCT/US94/11690 -94- 13 indicate that pre-clearing with Raf-B resulted in a diminution of Raf-B activity as measured by phosphorimager analysis of Raf-B in vitro kinase assays. EGF-stimulated MEKK activity was unaffected by Raf-B depletion, suggesting that Raf-B is not a component of MEKK immunoprecipitates.

At least 40% of the Raf-B activity is resistant to preclearing with Raf-B antibodies. Recombinant wild type MEKK over-expressed in COS cells readily autophosphorylates on serine and threonine residues and the amino-terminus of MEKK is highly serine and threonine rich. MEKK contained in immunoprecipitates of PC12 cells were tested for selective phosphorylation of purified recombinant MEKK amino-terminal fusion protein in in vitro kinase assays.

Serum-starved PC12 cells were treated with EGF (30ng/ml) for 5 minutes and equal amounts of protein from the same cell lysates were immunoprecipitated with either MEKK, Raf-B, or preimmune antiserum as a control.

Immunoprecipitates were incubated with purified recombinant MEKK NH 2 -terminal fusion protein (400ng) and (y- 32 P)ATP as described above. The results shown in Fig. 14 indicate that MEKK immunoprecipitated from lysates of EGF-stimulated and unstimulated PC12 cells robustly phosphorylated the inert 50 kD MEKK NH 2 -fusion protein, while Raf-B or preimmune immunoprecipitates from EGF-stimulated or unstimulated cells did not use the MEKK NH 2 -fusion protein as a substrate. Thus, the EGF-stimulated MEKK activity contained in MEKK immunoprecipites is not due to contaminating Raf kinases.

WO 95/28421 PCT/US94/11690 Example This Example describes MEKK activity in FPLC Mono Q ino-exchnage column fractions of PC12 cell lysates.

Cell lysates were prepared from EGF-stimulated PC12 cells. Portions (900 pl) of 1 ml column fractions (1 to 525 mM NaCl gradient) were concentrated by precipitation with trichloroacetic acid and loaded on gels as described above. The gels were blotted and then immunoblotted with MEKK specific antibody. The results are shown in Fig. indicate that 98 kD MEKK immunoreactivity eluted in fractions 10 to 12. The peak of B-Raf immunoreactivity eluted in fraction 14, whereas Raf-1 was not detected in the eulates from the column. Portions (30 pl) of each fraction from the PC12 lysates of unstimulated control cells or EGF-treated cells were assayed as described above in buffer containing purified recombinant MEK-1 (150 ng) as a substrate. The results shown in Fig. 15B indicate that the peak of MEKK activity eluted in fractions 10 to 12 from EGF-stimulated PC12 cells phosphorylated MEK, whereas little MEK phosphorylation occurred in fractions from unstimulated cells.

Example 11 This Example describes studies demonstrating that the phosphorylation of both MEK-1 and the MEKK NH 2 -terminal fusion protein were due to the activity of the 98 kD PC12 cell MEKK.

Cell lysates prepared from EGF-stimulated and unstimulated cells were fractionated by FPLC on a Mono-Q WO 95/28421 PCT/US94/11690 -96column to partially purify the endogenous MEKK. Lysates from unstimulated control PC12 cells or cells treated with EGF (30ng/ml) for 5 minutes were fractionated by FPLC on a Mono Q column using a linear gradient of 0 to 525 mM NaCl.

A portion (301) of each even numbered fraction was mixed with buffer (20mM piperazine-N,N'-bis-2-ethanesulfonic acid (Pipes) (pH 10mM MnCl 2 aprotinin (20Ag/ml), 50mM Bglycerophosphate (pH ImM EGTA, IP-20 (50gg/ml), NaF, and 30Ci (y- 32 P)ATP) containing purified recombinant MEK-1 (150ng) as a substrate in a final volume of 40pl and incubated at 30 0 C for 25 minutes. Reactions were stopped by the addition of 2X SDS sample buffer (401), boiled and subjected to SDS-PAGE and autoradiography. The peak of MEKK activity eluted in fractions 10-12. Portions of each even numbered fraction from lysates of EGF-treated PC12 cells were mixed with buffer as described above except containing purified recombinant MEKK NH 2 -terminal fusion protein (400ng) as a substrate instead of MEK-1. Purified recombinant kinase inactive MEK-1 or the MEKK NH 2 -terminal fusion protein were then used as substrates in the presence of (y- 32 p)ATP to determine whether 98 kD MEKK directly phosphorylates either substrate. Fractions 10-14 of lysate from PC12 cells treated with EGF phosphorylated MEK-1 while little MEK-1 phosphorylation occurred in untreated control fractions. The MEKK NH 2 -terminal fusion protein was also phosphorylated in the same fractions as was MEK-1, although the peak of activity was slightly broader (fractions 8-16).

WO 95/28421 PCT/US94/11690 -97- Referring to Fig. 16, immunoblotting of column fractions demonstrated that the 98 kD MEKK protein coeluted with the peak of activity that phosphorylated either exogenously added kinase inactive MEK-1 or the 50 kD MEKK

NH

2 -terminal fusion protein. Portions (900pl) of even numbered column fractions were concentrated by precipitation with trichloroacetic acid and immunoblotted with MEKK antibody. The peak of immunoreactivity eluted in fractions 10-12.

Example 12 This Example describes the activation of MEK by a 98 kD MEKK.

98 kD MEKK was immunoprecipitated using the MEKK.1 369 antiserum described in Example 1 from untreated or EGFtreated PC12 cell lysates. The immunoprecipitates were incubated in the presence or absence of purified recombinant wild-type MEK (150 ng) and in the presence of purified recombinant catalytically inactive MAPK (300 ng) and (y- 32 P)ATP. The results shown in Fig. 17A indicate that immunoprecipitated MEKK from EGF-stimulated cells phosphorylated and activated MEK, leading to MAPK phosphorylation. No phosphorylation of MAPK occurred in the absence of added recombinant MEK. Immunoblotting demonstrated that there was no contaminating MAPK (Fig.

17B) or contaminating MEK (Fig. 17C) in the MEKK immunoprecipitates from the EGF-stimulated PC12 cells.

Thus, phosphorylation and activation of MEK is due to EGF WO 95/28421 PCT/US94/11690 -98stimulation of MEKK activity measured in the immunoprecipitates.

Example 13 This Example describes whether 98 kD PC12 cell MEKK and Raf-B require functional Ras proteins for growth factor mediated signalling.

Dominant negative Ha-ras(Asn 17) (N 17 Ras) was expressed in PC12 cells and EGF-stimulated MEKK or Raf-B activation was assayed in immunoprecipitates using kinase inactive MEK-1 as a substrate. PC12 cells stably expressing dexamethasone inducible N 17 Ras were serum starved for 18-20 hours in media containing 0.1% BSA with or without 1gM dexamethasone and then untreated or treated with EGF for 5 minutes. Equal amounts of soluble protein from cell lysates was immunoprecipitated with either MEKK or Raf-B antisera and incubated with purified recombinant catalytically inactive MEK-1 and (y- 32 P)ATP as described above. Expression of N 1 Ras was induced in PC12 clones stabley transfected with the N1'Ras gene by the addition of dexamethasone to the starvation media. N"Ras expression inhibited the activation of MEKK by EGF as measured by its ability to phosphorylate kinase inactive MEK. EGF-mediated activation of Raf-B was also greatly reduced in N 1 Ras expressing PC12 cells compared to uninduced N 1 'Ras transfectants. Addition of dexamethasone to wild type PC12 cells had no effect on the magnitude of MEKK or Raf-B activation elicited by EGF. PC12 cell clones stably transfected with the N 1 7Ras gene are less responsive to EGF- WO 95/28421 PCTfUS94/11690 -99mediated activation of MEKK activity than are wild type PC12 cells. These results indicate that functional Ras is required for growth factor stimulated activation of both Raf-B and MEKK in PC12 cells, suggesting that Ras may mediate its effects on cell growth and differentiation through the activation of multiple protein kinase effectors from both the Raf and MEKK families. Thus, EGF stimulated a peak of MEKK activity within 5 minutes which persisted for at least 30 minutes following treatment, and was similar to the timecourse of Raf-B activation. Nerve growth factor (NGF) and the phorbol ester TPA also activated MEKK, although to a lesser degree than EGF. MEKK activity in immunoprecipitates or column fractions was dissociable from that of EGF-stimulated c-Raf-1 and Raf-B activities. Forskolin pretreatment abolished both MEKK and Raf-B activation by EGF, NGF, and TPA (Fig. 18). Both MEKK and Raf-B activation in response to EGF was inhibited by stable expression of dominant negative N 17 Ras. These findings represent the first demonstration of Ras-dependent MEKK regulation by growth factors and suggest the emergence of a complex intracellular kinase network in which Ras may alternately couple between members of the Raf and MEKK families.

To determine whether the growth factor-mediated activation of 98 kD PC12 cell MEKK was inhibited by PKA, forskolin was used to elevate intracellular cAMP and activate PKA. Serum-starved PC12 cells were pretreated with or without forskolin (50AM) for 3 minutes to activate WO 95/28421 PCT/US94/11690 -100protein kinase A and then with EGF (30ng/ml), NGF (100ng/ml), or TPA (200nM) for 5 minutes and MEKK was immunoprecipitated from equal amounts of soluble protein from cell lysates and incubated with purified recombinant catalytically inactive MEK-1 and (y- 32 P)ATP as described above. Raf-B activity was also assayed from the same cell lysates to test whether its regulation differed from that of MEKK. Raf-B was immunoprecipitated from the same cell lysates as described above and assayed for its ability to phosphorylate MEK-1 as described above. Forskolin pretreatment abolished the activation of both MEKK and Raf- B by EGF, NGF, and TPA, as measured by their ability to phosphorylate kinase-inactive MEK-1 (Fig. 18). Forskolin treatment alone had no appreciable effect on either kinase.

These results demonstrate that in addition to Raf-1 and Raf-B, PKA activation inhibits growth factor stimulation of 98 kD PC12 cell MEKK, suggesting the existence of a common regulatory control point for PKA action which lies between or downstream of Ras and upstream or at the level of each of these three kinases.

Example 14 This Example describes the determination of whether a similar or distinct MEK activity is involved in activation of MAPK though G, protein coupled receptors by measuring MEK activity in cell lysates from thrombin stimulated Rat la cells.

Thrombin stimulated cells exhibited a MEK activity which co-fractionated with the major MEK peak detected in WO 95/28421 PCT/S94/11690 -101- EGF stimulated cells. The magnitude of MEK activity from thrombin challenged cells was generally two to three-fold less than that observed with EGF stimulation, which correlates with the smaller MAPK response the present inventors have observed in thrombin challenged cells.

Differential regulation of MEK in Rat la and NIH3T3 cells expressing gip2, v-src, v-ras, or v-raf led the present inventor to investigate the protein kinases that are putative regulators of MEK-1. Recently, it was shown that Raf-1 can phosphorylate and activate MEK. Raf activation was assayed in the following manner. Cells were serum starved and challenged in the presence or absence of the appropriate growth factors, as described above. Serum starved Rat la cells were challenged with buffer alone or with EGF and Raf was immunoprecipitated using an antibody recognizing the C terminus of Raf. Cells were lysed by scraping in ice cold RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1.0% Triton X 100, 10 mM sodium pyrophosphate, 25 mM sodium glycerophosphate, 2mM sodium vanadate, 2.1 gg/ml aprotinin) and were microfuged for 10 min to remove nuclei. The supernatants were normalized for protein content and precleared with protein A Sepharose prior to immunoprecipitation with rabbit antiserum to the C terminus of Raf-1 and protein A Sepharose for 2-3 h at 40C. The beads were washed twice with ice cold RIPA and twice with PAN (10 mM Pipes, pH 7.0, 100mM NaCl, 21gg/ml aprotinin).

A portion of the immunoprecipitate was diluted with SDS WO 95/28421 PCT/US94/11690 -102sample buffer and used for immunoblot analysis. The remainder was resuspended in kinase buffer (20 mM Pipes pH 10 mM MnCl 2 150 ng kinase-inactive MEK-1, 30 .Ci y- 32

P-

ATP and 20 pg/ml aprotinin) in a final volume of 50 Al for 30 min at 30 0 C. Wild type recombinant MEK-1 was autophosphorylated in parallel as a marker. Reactions were terminated by the addition of 12.5 pl 5X SDS sample buffer, boiled for 5 minutes and subjected to SDS-PAGE and autoradiography.

The immunoprecipitated Raf, in the presence of y- 32

P-

ATP, was able to phosphorylate MEK-1. The recombinant MEK- 1 used in this assay was kinase inactive to ensure it did not autophosphorylate as is observed with wild type MEK-1.

Little or no phosphorylation of MEK-1 by Raf was observed in immunoprecipitates from control cells. EGF challenge clearly stimulated Raf catalyzed phosphorylation of MEK-1; in contrast, thrombin challenge of Rat la cells did not measurably activate Raf even though endogenous MEK was clearly activated. EGF stimulated Raf phosphorylation of recombinant MEK-1 by approximately 2.6-fold over basal.

Little phosphorylation of MEK by Raf was observed in Raf immunoprecipitates from Gip2 or v-Src expressing Rat la cells. EGF stimulation was still capable of activating Raf catalyzed phosphorylation of MEK-1 in these cell lines by 1.8 and 1.4-fold, respectively. The blunting of the EFG response in Gip2 and v-Src expressing cells is likely a result of desensitization of the EFG receptor upon constitutive activation of MAPK. The amount of Raf in the

I

WO 95/28421 PCT/US94/11690 -103immunoprecipitates was shown to be similar by subsequent SDS-PAGE and immunoblotting using Raf antibody. Since thrombin stimulation of MEK is two to three-fold over basal, at least a 1.5-fold stimulation of MEK phosphorylation is expected if Raf significantly contributed to MEK activation by this growth factor. This level of activation was detectable in the EGF stimulated Gip2 and v-Src expressing cells lines. Thus, it is unlikely that the failure to detect thrombin activation of Raf is due to the sensitivity of the assay. Thrombin stimulation of MAPK is maximal at 3 minutes. Stimulation of Rat la cells for 1 or 5 minutes with thrombin did not increase Raf activity.

In NIH3T3 cells, as in Rat la cells, EGF activates Raf approximately 2.7-fold, while thrombin does not. V-Raf expressing NIH3T3 cells showed no increase in MEK-1 phosphorylation. This result was unexpected since MEK was clearly activated in v-Raf expressing NIH3T3 cells. Both the p90 and p75 gag-raf fusion proteins in addition to c- Raf-1 were immunoprecipitated from v-Raf NIH3T3 cells by the antisera. P75gag-raf has been shown to exhibit protein kinase activity, but it is possible that the NH2 terminal gag fusion protein sterically hinders Raf phosphorylation of recombinant MEK-1 in the in vitro assay system. Further studies will have to be done to measure v-Raf kinase activity. The results argue that activation of MEK cannot be accounted for exclusively by the activation of Raf.

Additional regulatory kinases for MEK must exist which WO 95/28421 PCT/US94/11690 -104contribute to MEK activation in thrombin stimulated, G protein coupled pathways and in gip2 and v-src transfected cells.

Example This Example demonstrates the ability of a PPPSS-trunc and Ncol-trunc of MEKK protein to activate MAPK activity compared with full-length MEKK protein and a negative control protein.

The results shown in Fig. 19 indicate that the truncated MEKK molecules were more active than the fulllength MEKK. Indeed, the truncated MEKK molecules were at least about 1.5 times more active than full-length MEKK protein. Thus, removal of the regulatory domain of MEKK deregulates the activity of the catalytic domain resulting in improved enzyme activity.

Example 16 This example describes the preferential activation of JNK by MEKK compared with Raf.

HeLa cells were transiently transfected with truncated

MEKK

370738 under control of an inducible mammary tumor virus promoter, together with epitope tagged JNK1 (described in detail in Derijard et al., p. 1028, 1994, Cell, Vol. 76).

Other HeLa cells were also transiently transfected with truncated BXB-Raf under control of an inducible mammary tumor virus promoter, together with epitope tagged JNK1 (Derijard et al., ibid.). The following day, MEKK 37 -73 8 expression and BXB-Raf expression were induced by administration of dexamethasone (10 gM) for 17 hours. Cell WO 95/28421 PCT/US94/11690 -105extracts were then prepared and assayed for JNK activity using an immune complex kinase assay (detailed in Derijard et al., ibid.). Phosphorylation was quantitated by phosphorimager analysis. The results shown in Fig. indicate that MEKK stimulated about 30-fold to about fold activation more JNK activity over unstimulated cells (basal) and about 15-fold to about 25-fold JNK activity over Raf stimulated cells.

Example 17 This example describes that the phosphorylation of c- Myc transactivation domain in response to MEKK expression activates MYC-GAL 4 transcriptional activity.

Two separate expression plasmids were constructed as follows. The expression plasmid pLNCX was ligated to a cDNA clone comprising c-myc (1-103) ligated to GAL4 (1-147) (Seth et al., pp. 23521-23524, 1993, J. Biol. Chem., Vol.

266) to form the recombinant molecule pMYC-GAL 4. The expression plasmid UASG-TK Luciferase (Sadowski et al., pp.

563-564, 1988, Nature, Vol. 335) was transfected with either pMYC-GAL 4 or pLU-GAL into Swiss 3T3 cells using standard methods in the art to form recombinant cells herein referred to as LU/GAL cells. Recombinant control cells were also produced by transfecting in pGAL4-Control plasmids containing GAL4 (1-147) alone in the absence of cmyc (1-103).

LU/Gal cells were transfected with either pMEKK 370 8 pMEKK (encoding full-length MEKK.1_7), BXB-Raf, pMyc-Gal4, pCREB-Gal4 (encoding CREB., 261 fused to Gal 41-.47; Hoeffler et WO 95/28421 PCT/US94/11690 -106al., pp. 868-880, 1989, Mol. Endocrinol., Vol. pGal4, or CREB fusion protein referred to as GAL4.

The transfected cells were incubated overnight and then lysed using methods standard in the art. The luciferase activity of each cell lysate was measure on a luminometer. The results shown in Fig. 21 indicate that MEKK is selectively capable of stimulating the phosphorylation of c-Myc transactivation domain in such a manner that the c-Myc domain is activated and induces transcription of the transfected luciferase gene. In addition, the results indicate that MEKK does not stimulate CREB activation. Also, activated Raf is unable to stimulate Myc activation. A schematic representation of the activation mechanism of c-Myc protein by MEKK is shown in Fig. 23.

Example 18 This Example describes the phosphorylation of p38 MAPK protein by MEKK 3 protein and not MEKK 1 protein.

COS cells were transfected with the expression plasmid pCVM5 ligated to cDNA clones encoding either MEKK 1 or MEKK 3 protein, or a control pCVM5 plasmid lacking MEKK cDNA inserts. Forty-eight hours after transfection, the COS cells were lysed and the lysate fractionated on a Mono Q FPLC column using conditions described in Example 4. The fractions were analyzed for tyrosine phosphorylation of MAP kinase-like enzymes using the kinase assay described in Example 4. Referring to Fig. 23, expression of MEKK 3 induces tyrosine phosphorylation of p38 MAPK and the p42 WO 95/28421 PCT/US94/11690 -107and p44 forms of MAPK. MEKK 1, however, only induces weak phosphorylation of p38 MAPK but does induce phosphorylation of p42 and p44 MAPK.

Example 19 This example describes MEKK-induced apoptosis.

Cells were prepared for the apoptosis studies as follows. Swiss 3T3 cells and REF52 cells were transfected with an expression plasmid encoding 8-Galactoctosidase (B- Gal) detection of injected cells. One set of B-Gal transfected cells were then microinjected with an expression vector encoding MEKK 370 p 8 protein. Another set of B-Gal transfected cells were then microinjected with an expression vector encoding truncated BXB-Raf protein.

A. Beauvericin-induced apoptosis A first group of transfected Swiss 3T3 cells and REF52 cells were treated with 50 pM beauvericin for 6 hours at 37 0 C. Beauvericin is a compound known to induce apoptosis in mammalian cells. A second group of cells were treated with a control buffer lacking beauvericin. The treated cells were then fixed in paraformaldehyde and permeabilized with saponin using protocols standard in the art. The permeabilized cells were then labelled by incubating the cells with a fluorescein-labelled anti-tubulin antibody (1:500; obtained from GIBCO, Gaithersburg, MD) to detect cytoplasmic shrinkage or 10 AM propidium iodide (obtained from Sigma, St. Louis, MO) to stain DNA to detect nuclear condensation. The labelled cells were then viewed by differential fluorescent imaging using a Nikon Diaphot WO 95/28421 PCT/US94/11690 -108fluorescent microscope. Fig. 24 shows two fields of Swiss 3T3 cells and REF52 cells, one field representing cells treated with the control buffer and a second field representing cells treated with beauvericin. The cells treated with beauvericin demonstrated cytoplasmic shrinkage (monitored by the anti-tubulin antibodies) and nuclear condensation (monitored by the propidium iodide) characteristic of apoptosis.

B. MEKK-induced apoptosis Swiss 3T3 cells and REF52 cells microinjected with a 8-galatoctosidase expression plasmid, and an MEKK encoding plasmid or a BXB-Raf encoding plasmid, were treated and viewed using the method described above in Section A. An anti-B-Gal antibody (1:500, obtained from GIBCO, Gaithersburg MD) was used to detect injected cells.

Referring to Fig. 25, microscopic analysis of REF52 cells indicated that the cells expressing MEKK protein underwent cytoplasmic shrinkage and nuclear condensation leading to apoptotic death. In contrast, cells expressing BXB-Raf protein displayed normal morphology and did not undergo apoptosis. Similarly, referring to Fig. 26, microscopic analysis of Swiss 3T3 cells indicated that the cells expressing MEKK protein underwent cytoplasmic shrinkage and nuclear condensation leading to apoptotic death. In contrast, cells expressing BXB-Raf protein displayed normal morphology and did not undergo apoptosis.

Fig. 27 shows 3 representative fields of RFE52 cells expressing MEKK protein which have undergone substantial WO 95/28421 PCT/US94/11690 -109cytoplasmic shrinkage and nuclear condensation compared with a control cell not expressing MEKK. Similarly, Fig.

28 shows 3 representative fields of Swiss 3T3 cells expressing MEKK protein which have undergone substantial cytoplasmic shrinkage and nuclear condensation compared with a control cell not expressing MEKK. Thus, MEKK and not Raf protein can induce apoptotic programmed cell death.

Example This Example describes regulation of MAPK activity by both MEKK and Raf protein.

COS cells were prepared using the method described in Example 3. In addition, COS cells were transfected with the pCVMV5 Raf construct (1 gg: Raf). FPLC MONO Q ionexchange column fractions were prepared as described in Example 3 and assayed for MAPK activity according to the method described in Heasley et al., ibid.

Referring to Fig. 29, both MEKK and Raf overexpression in COS 1 cells resulted in similar levels of stimulation of MAPK activity over basal levels.

The foregoing description of the invention has been presented for purposes of illustration and description.

Further, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge in the relevant art are within the scope of the present invention. The preferred embodiment described herein above is further intended to explain the best mode known of practicing the WO 95/28421 PCT/US94/11690 -110invention and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications required by their particular applications or uses of the invention. It is intended that the appended claims be construed to include alternate embodiments to the extent permitted by the prior art.

SEQUENCE LISTING INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3260 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: MEKK STRAIN: murine (vii) IMMEDIATE SOURCE: LIBRARY: mouse liver CLONE: MEKK cDNA (ix) FEATURES: (1) NAME/KEY: LOCATION: 1..485 (2) NAME/KEY: CDS LOCATION: 486..2501 35 (3) NAME/KEY: 3'UTR LOCATION: 2502..3260 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TACACTCCTT GCCACAGTCT GGCAGAAAGA ATCAAACTTC AGAGACTCCT CCGGCCAGTT

S

S.

GTAGACACTA TCCTTGTCAA CTACAGTGCT GGAACTCTGC TTAAAGCTGG GTCCATCGGG ACCAAGCTGA ATCAAACAAC TGCTGTTGGA ATTTCCTGCT CTGAGCCTGT TGAAATCAGG CCATTGACAA TTCCCACTCG CCAGG ATG GTG ACC GCA Met Val Thr Ala 1 GTGTGCAGAT CCAACAGCCG CACGAGTCAG CTGTCCATAT AAGGGCCAAG CAGGAGAGCT GGCGGTTGGG AGAGAAATAC GTTGGTGGTG TCGATTACGT CTTAAGTTGT ATCCTTGGAA TGGCAAGAAC TGCTGGGTCG CCTCTGTCTT ATAGACAGGT GAATTCTATC CTCATATTGT CAGTACTGAT GTCTCACAAG TACAAGAAGC TGCTCTCCCT CTTAACCTTT GCCTTGCAAT ATGGTTGGCA AGCTCTCTCG GAGGATATAT CTGAGCTCTG GTG CCC GCT GTG TTT TCC AAG CTG GTA ACC Val Pro Ala Val Phe Ser Lys Leu Val Thr 5 120 180 240 300 360 420 480 527 ATG CTT AAT GCT TCT GGC TCC ACC CAC TTC ACC AGG ATG CGC CGG CGT Met Leu Asn Ala Ser Gly Ser Thr His Phe Thr Arg Met Arg Arg Arg 20 25 Document2 CTG ATG GCT ATC GCG GAT GAG GTA GAA Leu Met Ala Ile Ala Asp Glu Val Glu GCC GAG GTC ATC Ala Glu Val Ile CAG CTG Gin Leu GGT GTG GAG Gly Val Glu GCC CCC ACC Ala Pro Thr

GAC

Asp ACT GTG GAT GGG Thr Val Asp Gly

CAT

His 55 CAG GAC AGC TTA Gin Asp Ser Leu CAG GCC GTG Gin Ala Val ACA GTC CAT Thr Val His AGC TGT CTA GAA Ser Cys Leu Glu AGC TCC CTT GAG Ser Ser Leu Glu

CAC

His 719 AGA GAG Arg Glu AAA ACT GGA AAA Lys Thr Gly Lys CTA AGT GCT ACG Leu Ser Ala Thr CTG AGT GCC AGC Leu Ser Ala Ser GAG GAC ATT TCT Glu Asp Ile Ser

GAC

Asp 100 AGA CTG GCC GGC Arg Leu Ala Gly

GTC

Val 105 TCT GTA GGA CTT Ser Val Gly Leu AGC TCA ACA ACA ACA GAA CAA CCA AAG Ser Ser Thr Thr Thr Glu Gin Pro Lys

CCA

Pro 120 GCG GTT CAA ACA Ala Val Gin Thr AAA GGC Lys Gly 125 AGA CCC CAC Arg Pro His TTA ATG TTC Leu Met Phe 145 CAG TGT TTG AAC Gin Cys Leu Asn TCC CCT TTG TCT Ser Pro Leu Ser CAT GCT CAA His Ala Gin 140 CCG TCT GTC Pro Ser Val CCA GCA CCA TCA Pro Ala Pro Ser

GCC

Ala 150 CCT TGT TCC TCT Pro Cys Ser Ser

GCC

Ala 155 CCA GAT Pro Asp 160 ATT TCT AAG CAC Ile Ser Lys His

AGA

Arg 165 CCC CAG GCA TTT Pro Gin Ala Phe CCC TGC AAA ATA Pro Cys Lys Ile ff fto o *f o o O e ft f ftf ft ft t f ft ft ft ftf ft f ft ft ft ft ftft ft ft ft ftf ft ft

CCT

Pro 175 TCC GCA TCT CCT Ser Ala Ser Pro

CAG

Gin 180 ACA CAG CGC AAG Thr Gin Arg Lys TCT CTA CAA TTC Ser Leu Gin Phe AGG AAC TGC TCT Arg Asn Cys Ser

GAA

Glu 195 CAC CGA GAC TCA His Arg Asp Ser

GAC

Asp 200 CAG CTC TCC CCA Gin Leu Ser Pro GTC TTC Val Phe 205 45 ACT CAG TCA Thr Gin Ser TCC CGA CCC 50 Ser Arg Pro 225

AGA

Arg 210 CCC CCA CCC TCC Pro Pro Pro Ser AAC ATA CAC AGG Asn Ile His Arg CCA AAG CCA Pro Lys Pro 220 GCC ACA AAA Ala Thr Lys GTT CCG GGC AGT Val Pro Gly Ser AGC AAA CTA GGG Ser Lys Leu Gly 1007 1055 1103 1151 1199 1247 1295 1343 1391 AGT AGC Ser Ser 240 ATG ACA CTT GAT Met Thr Leu Asp

CTG

Leu 245 GGC AGT GCT TCC Gly Ser Ala Ser

AGG

Arg 250 TGT GAC GAC AGC Cys Asp Asp Ser GGC GGC GGC GGC Gly Gly Gly Gly

AAC

Asn 260 AGT GGC AAC GCC Ser Gly Asn Ala

GTC

Val 265 ATA CCC AGC GAC Ile Pro Ser Asp

GAG

Glu 270 ACA GTG TTC ACG Thr Val Phe Thr GTG GAG GAC AAG Val Glu Asp Lys AGG TTA GAT GTG Arg Leu Asp Val AAC ACC Asn Thr 285 GAG CTC AAC TCC AGC ATC GAG GAC CTT CTT GAA GCA TCC ATG CCT TCA Document2 Glu Leu Asn Ser Ser Ile Glu Asp Leu Leu Glu Ala Ser Met Pro Ser

AGT

Ser

GAA

Glu

CAA

Gin 335

ATC

Ile

CAG

Gin

ACA

Thr

GAA

Glu

TCT

Ser 415

GTG

Val 40 GTG Val

CAT

45 His

TAC

Tyr 50

TTG

Leu 495

GAG

Glu

CAC

His

GAC

Asp

AAG

Lys 320

AAG

Lys

GCC

Ala

CTG

Leu

CCA

Pro

GAC

Asp 400

TCC

Ser

AAA

Lys

GTG

Val

CCA

Pro

AAC

Asn 480

AGT

Ser

CAG

Gin

AGA

Arg

ACG

Thr 305

GCC

Ala

TGC

Cys

ATG

Met

CAG

Gin

GAA

Glu 385

GCT

Ala

TGT

Cys

CAG

Gin

GAA

Glu

AAC

Asn 465

CTC

Leu

AAA

Lys

TTA

Leu

GAC

Asp 290

ACA

Thr

GAA

Glu

AAA

Lys

GCG

Ala

GTG

Val 370

ACT

Thr

GAG

Glu

TAC

Tyr

GTG

Val

GCG

Ala 450

ATC

Ile

TTC

Phe

TAC

Tyr

CTG

Leu

GTC

Val 530

GTC

Val

AAT

Asn

GAA

Glu

ATG

Met 355

GAA

Glu

CTT

Leu

TGG

Trp

CAA

Gin

ACG

Thr 435

TTG

Leu

ATC

Ile

ATT

Ile

GGA

Gly

CGT

Arg 515

AAA

Lys

ACT

Thr

GAC

Asp

AAG

Lys 340

TCA

Ser

AAT

Asn

CCA

Pro

CTG

Leu

GCA

Ala 420

TAC

Tyr

AGG

Arg

CGG

Arg

GAG

Glu

GCT

Ala 500

GGC

Gly

GGT

Gly

TTC

Phe

GAC

Asp 325

ATG

Met

GCG

Ala

GGA

Gly

GGA

Gly

AAA

Lys 405

CAG

Gin

GTC

Val

GAA

Glu

ATG

Met

TGG

Trp 485

TTC

Phe

CTT

Leu

GCC

Ala

AAG

Lys 310

ACC

Thr

GAA

Glu

TCT

Ser

GAA

Glu

CAT

His 390

GGC

Gly

GAT

Asp

AGA

Arg

GAG

Glu

CTG

Leu 470

ATG

Met

AAG

Lys

TCC

Ser

AAC

Asn 295

TCC

Ser

TAC

Tyr

GCT

Ala

CAG

Gin

GAT

Asp 375

ACC

Thr

CAG

Gin

GTG

Val

AAC

Asn

ATC

Ile 455

GGG

Gly

GCG

Ala

GAG

Glu

TAT

Tyr

CTG

Leu 535

GAA

Glu

AAA

Lys

GAA

Glu

GAT

Asp 360

ATT

Ile

AAA

Lys

CAG

Gin

GGG

Gly

ACA

Thr 440

CGG

Arg

GCC

Ala

GGA

Gly

TCA

Ser

CTC

Leu 520

CTC

Leu

GTC

Val

GAC

Asp

GAG

Glu 345

GCC

Ala

ATC

Ile

GCG

Ala

ATA

Ile

ACT

Thr 425

TCC

Ser

ATG

Met

ACG

Thr

GGA

Gly

GTC

Val 505

CAC

His

ATT

Ile

GCC

Ala

GAC

Asp 330

GAG

Glu

CTC

Leu

ATC

Ile

AAA

Lys

GGC

Gly 410

GGG

Gly

TCC

Ser

ATG

Met

TGC

Cys

TCT

Ser 490

GTC

Val

GAG

Glu

GAC

Asp

GTC

Val 315

GTC

Val

GAG

Glu

CCC

Pro

ATT

Ile

CAG

Gin 395

CTC

Leu

ACT

Thr

GAG

Glu

GGT

Gly

GAG

Glu 475

GTG

Val

ATT

Ile

AAC

Asn

AGC

Ser 300

CTC

Leu

AAT

Asn

GCT

Ala

ATC

Ile

CAG

Gin 380

CCT

Pro

GGA

Gly

TTA

Leu

CAG

Gin

CAC

His 460

AAG

Lys

GCT

Ala

AAC

Asn

CAG

Gin

ACC

Thr 540

TCT

Ser

CAT

His

TTA

Leu

GTC

Val 365

CAG

Gin

TAC

Tyr

GCA

Ala

ATG

Met

GAG

Glu 445

CTC

Leu

AGC

Ser

CAC

His

TAC

Tyr

ATC

Ile 525

GGT

Gly

CCG

Pro

AAT

Asn

GCG

Ala 350

CCT

Pro

GAC

Asp

AGA

Arg

TTT

Phe

GCT

Ala 430

GAG

Glu

AAC

Asn

AAC

Asn

CTC

Leu

ACT

Thr 510

ATT

Ile

CAG

Gin 1439 1487 1535 1583 1631 1679 1727 1775 1823 1871 1919 co g *o eoe'o 2015 2063 2111 2159 AGG CTG AGA ATT GCA GAC TTT GGA GCT GCT GCC AGG TTG GCA TCA AAA Arg Leu Arg Ile Ala Asp Phe Gly Ala Ala Ala Arg Leu Ala Ser Lys Document2 545

GGT

Gly 550

CAG

Gin 555

GGG

Giy GGA ACC Gly Thr 560 GCA GCA GAG Ala Gly Glu GGA GAG TTA Gly Gin Leu ACA ATT GCA Ihr Ile Ala

TC

Phe 575

GAT

Asp ATG GCC CCI GAG Met Ala Pro Gic

GIG

Vai 580

GGC

Gly CTA AGA GGT GAG Leu Arg Giy Gin

GAG

Gin 585 TAT GGT AGG AGC Tyr Giy Arg Ser GTA TGG ACT Vai Trp Ser

CT

Vai 595

CCA

Al a ICC CCC ATT Cys Aia ile

ATA

Ile 600 CAA AIC GC TGT Giu Met Aia Cys

GCA

Al a 605 GCA CCI TGG Pro Pro Trp AAC ATI CCT Lys Iie Ala 625 CCC CCI CTC Pro Civ Leu GAA AAA CAC Glu Lys His AAI CAT CIC CC Asn His Lou Ala TIC AlA III Leu Ile Phe 620 GAG GIG ICC His Leu Ser CCA ACT ACT Ala Thr Ihr

CCA

Al a 630 ICC ATG CCC Ser Ile Pro

ICA

Ser 635 CCC GAG GIG Arg Asp Val GIG CCC ICC TIA Val Arg Cys Leu

CAA

Glu 650 CIT GAG CCI GAG Leu Gin Pro Gin

GAG

Asp 655 CCC CCI CCC ICC Arg Pro Pro Ser GIG GIG AAA Leo Leu Lys CCC GIG TIC CCI Pro Val Phe Arg 2207 2255 2303 2351 2399 2447 2495 2551 2611 2671 2731 2791 2851 2911 2971 3031 3091 3151 3211 3260 AG ICC TAGTTAATTG TIGAGAICAG CTCTAATGGA GACACCATAT CGAACCGGGA Ihr Irp C C C

C

CAGAGAAAAG

AGCCAGAAAC

GTACCTAAC

GACACGCGG

GCATAAATTT

GCCGGCACA

III GGAGI IC

TAATATTCTT

IGICITITIA

CTICTACTA

AAATTCTACG

TAT IAGGAGG ACAAGT I C GCC CAGC C

ICCATAICCA

GCTGTGGCC

ATTICCAC

TTTCAAACIC

GCTCGCTG

AT I CITICG

ATITATTIC

CGIGAGCTGI

I CCTCI CA

TIACTAICTA

GGCGACCATC

GGGAACCGIA

GAGAIGIAGA

ACCACGAAGG

GACI 1111 A

ACACICTCCC

CTGCCTAAT IC CAT CAAAGCT

CTACTCGAAT

CCIATACATT

CIATCACTCC

AGCAAATGCT

CCCCTAACCC

CCTAAGCATG

CGCTCTCAG

CCAICGAGTT

GCTAATCAGT

AGAIGTCAAG

ICCICTTICG

CCACTCAAAA

TGIAAATAAC

TTTCII CIAC TAAGACT ICC

CACCCCTCAC

TGAITGACAA

GAACTGGAGA

IGGAIGACTA

AITACCAIT

AIGGACGCI

ITTTCCATGC

TTIGACIGIG

GIGIACGGG

CAAAGTT GAG

AGGGCTTAAG

GCCACIGAAC

AICAICACCI

CCTCCTTT

AAGAACAGAA

ACATCAACAI

TTTCAGT TTC AAATCT CAT C TAAT TATl I CII TAT ICCA ICTICAGAAI C

GGCTAACTCC

ACAAAAAAAA AAAAAAAAA INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENCTH: 672 amino acids TYPE: amino acid TOPOLOGY: linear Docunient2 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ TD NO:2: Met Val Thr Ala Val Pro Ala Val Phe Ser Lys Leu Val Thr Met Leu a..

a Asn Ala Glu Thr Lys Asp Thr His Pho 145 Ile Ala Cys Sor Pro 225 Met Gly Phe Asn Thr 305 Ala Ile Asp Ser Thr Ile Thr Ser 130 Pro Ser Sor Ser Arg 210 Val Thr Gly Thr Ser 290 Thr Ser Al a Thr Cys Gly Ser Thr 115 Gin Ala Lys Pro Glu 195 Pro Pro Lou Gly Pro 275 Ser Val Gly 20 Asp Val1 Leu Lys Asp 100 Glu Cys Pro His Gin 150 His Pro Gly Asp Asn 260 Val Ile Thr Ser Giu Asp Giu Gly Arg Gin Leu Ser Arg 165 Thr Arg Pro Ser Lou 245 Ser Glu Giu Phe Thr Val Gly Asn 70 Leu Leu Pro Aso Al a 150 Pro G In Asp Ser Thr 230 Gly Gly Asp Asp Lys 310 His Clu His 55 Ser Ser Al a Lys Ser 135 Pro Gin Ar g Ser Ser 215 Ser Ser Asn Lys Leu 295 S er Phe Ile 40 Gin Ser Ala Gly Pro 120 Ser Cys Al a Lys Asp 200 Asn Lys Al a Ala Cys 280 Leu Glua Thr 25 Al a Asp Leu Thr Val 105 Ala Pro Ser Phe Phe 185 Gin ile Lou Ser Val1 265 Arg Glu Val1 Ar g Glu Sor Glu Arg 90 Ser Val1 Leu Ser Val1 170 Ser Leu His Gly Arg 250 Ile Leu Al a Al a Met Val Leu His 75 Lou Val Gin Ser Ala 155 Pro Lou Ser Arg Asp 235 Cys Pro Asp Ser Val 315 Arg Ile Gin Thr Ser Gly Thr His 140 Pro Cys Gin Pro Pro 220 Al a Asp Ser Val Met 300 Lou Arg Gin Ala Val Ala Lou Lys 125 Al a Ser Lys Phe Val 205 Lys Thr Asp Asp Asn 285 Pro S or Arg Lou Val His Sor Pro 110 Gly Gin Val1 Ile Gin 190 Pho Pro Lys Ser Giu 270 Thr Ser Pro Lou Gly Al a Arg Sor Ser Arg Lou Pro Pro 175 Arg Thr Ser Ser Pho 255 Thr Glu Ser Glu Met Val Pro Glu Glu Sor Pro Met Asp 160 Ser As n Gin Arg Sor 240 Gly Val Lou Asp Lys 320 Docuument2 Ala Glu Asn Asp Asp Thr Tyr Lys Asp Asp Val Asn His Asn Gin Lys Cys Met Gin Glu 385 Ala Cys Gin Glu Asn 465 Leu Lys Leu Asp Arg 545 Gly Ala Trp Trp Ala 625 Leu Lys Ala Val 370 Thr Glu Tyr Val Ala 450 Ile Phe Tyr Leu Val 530 Ile Ala Pro Ser Asn 610 Ser Arg Glu Met 355 Glu Leu Trp Gin Thr 435 Leu Ile Ile Gly Arg 515 Lys Ala Gly Glu Val 595 Ala Ala Asp Lys 340 Ser Asn Pro Leu Ala 420 Tyr Arg Arg Glu Ala 500 Gly Gly Asp Glu Val 580 Gly Glu Thr Val 325 Met Ala Gly Gly Lys 405 Gin Val Glu Met Trp 485 Phe Leu Ala Phe Phe 565 Leu Cys Lys Thr Ala 645 335 Glu Ser Glu His 390 Gly Asp Arg Glu Leu 470 Met Lys Ser Asn Gly 550 Gin Arg Ala His Ala 630 Val Ala Gin Asp 375 Thr Gin Val Asn Ile 455 Gly Ala Glu Tyr Leu 535 Ala Gly Gly Ile Ser 615 Pro Arg Glu Asp 360 Ile Lys Gin Gly Thr 440 Arg Ala Gly Ser Leu 520 Leu Ala Gin Gin Ile 600 Asn Ser Cys Glu 345 Ala Ile Ala Ile Thr 425 Ser Met Thr Gly Val 505 His Ile Ala Leu Gin 585 Glu His Ile Leu Glu Leu Ile Lys Gly 410 Gly Ser Met Cys Ser 490 Val Glu Asp Arg Leu 570 Tyr Met Leu Pro Glu Glu Pro Ile Gin 395 Leu Thr Glu Gly Glu 475 Val Ile Asn Ser Leu 555 Gly Gly Ala Ala Ser 635 Leu Ala Ile Gin 380 Pro Gly Leu Gin His 460 Lys Ala Asn Gin Thr 540 Ala Thr Arg Cys Leu 620 His Gin Leu Val 365 Gin Tyr Ala Met Glu 445 Leu Ser His Tyr Ile 525 Gly Ser Ile Ser Ala 605 Ile Leu Pro Ala 350 Pro Asp Arg Phe Ala 430 Glu Asn Asn Leu Thr 510 Ile Gin Lys Ala Cys 590 Lys Phe Ser Gin Ile Gin Thr Glu Ser 415 Val Val His Tyr Leu 495 Glu His Arg Gly Phe 575 Asp Pro Lys Pro Asp Ala Leu Pro Asp 400 Ser Lys Val Pro Asn 480 Ser Gin Arg Leu Thr 560 Met Val Pro Ile Gly 640 Arg S S 650 655 Pro Pro Ser Arg Glu Leu Leu Lys His Pro Val Phe Arg Thr Thr Trp Document2 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 2503 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 466..2325 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGTGGCGGCC GCTCTAGAAC TAGTGGATCC CCCGGGCTGC GATCCAGCGG CAGAGTCGCC GCTTCCGCTT CGCTGCTTCT GGGGCTTCCT TTTCATCGGC CCAGCTTATT CCGCGGGCCC AGCGGCGAAG AGGCCCTGGG CTGCGCGCCC GCTGTCCCAT TCCCCGGCCC GTGCCCGGTT GTCTGCGGCC CTTCAGGCCT TGCTCCTGGG GGGCGCGGTG ACAGGCCGTG CGGGGGCGGA CTCTCGGCCC TCGCGTCCGC GATCCCGCCC AGCGGCCGGG 35 AGAACCATTT TCCTAATTTT CAAATTATTG AGCTGGTCGC AGGAATTCGG CACGAGGGAC CCGGTCGGCG ACGCGGGCCC CGGGGCTGCA GCTACCCAGA GTGAAGCAGG TTGGGCCTGG CAGGGACCCC CGCGAGGCGC GGGGCCAGCT CGGTGGCCTC CAATAAAGAA TGTTGATGGG GCATA ATG GAT GAT Met Asp Asp

S**

f o **o Q CAG CAA 40 Gin Gln 5 CCA GTC Pro Val GCT TTG AAT TCA Ala Leu Asn Ser

ATC

Ile 10

CTT

Leu ATG CAA GAT TTG Met Gin Asp Leu

GCT

Ala GTC CTT CAT AAG Val Leu His Lys GGC CAG CAT Gly Gin His TAC AAG AAA Tyr Lys Lys 45 20

CAT

His

CCA

Pro

TCA

Ser GGA AAG CAA AAC Gly Lys Gin Asn CAC CAA AAA His Gin Lys ATG ATG TTC Met Met Phe AAT TTG AAC Asn Leu Asn ATA GAG Ile Glu GAG GAA AAA Glu Glu Lys

AGG

Arg CTG CAG GTT Leu Gin Val

ACT

Thr 60 CCA GTT AAA Pro Val Lys CTA GAA GAC Leu Glu Asp GAT CTA CAC Asp Leu His 666 CTG AGA TCT Leu Arg Ser TAT ACC AAC Tyr Thr Asn GAC AAA GCT AAG TCT AAG ATC Lys Ser Lys Ile TTT GGG CAG TCT Phe Gly Gin Ser

ATG

Met AAT GAG TTG Asn Glu Leu GTG GAA CTG ATT CCG TTA ACT Ile Pro Leu Thr GAT CGC ACT ATT CAA GAT GAC TTG Gin Asp Asp Leu ATG AAG AGT CTC Asp 100 Lys Ala Val Glu Leu Leu Asp Arg Ser Ile His Met Lys Ser Docuinent2 AAG ATA TTA Lys Ile Leu CCA TCA CCG Pro Ser Pro AGG AAA AAG Arg Lys Lys 150 TCC CCT CCT Ser Pro Pro 165 CTT GTA Leu Val 120 GTA AAT GGG AGT Val Asn Gly Ser

ACA

Thr 125 CAG GCT ACT AAT Gin Ala Thr Asn TTA GAA Leu Glu 130

TCA

Ser 135 CCA GAA GAT TTG Pro Glu Asp Leu AAT ACA CCA CTT Asn Thr Pro Leu GGT GCA GAG Gly Ala Glu 145 GAT AGA AGT Asp Arg Ser CGG CTA TCT GTA Arg Leu Ser Val

GTA

Val 155 GGT CCC CCT AAT Gly Pro Pro Asn

AGG

Arg 160 CCA GGA TAC Pro Gly Tyr CCA GAC ATA CTA Pro Asp Ile Leu CAG ATT GCC CGG Gln Ile Ala Arg

AAT

Asn 180 GGG TCA TTC ACT Gly Ser Phe Thr

AGC

Ser 185 ATC AAC AGT GAA Ile Asn Ser Glu GAG TTC ATT CCA Glu Phe Ile Pro AGC ATG GAC CAA Ser Met Asp Gln CTG GAT CCA TTG Leu Asp Pro Leu TTA AGC AGC CCT Leu Ser Ser Pro GAA AAT Glu Asn 210 TCT GGC TCA Ser Gly Ser AGC TGT CCG TCA Ser Cys Pro Ser GAT AGT CCT TTG Asp Ser Pro Leu GAT GGA GAA Asp Gly Glu 225 CCA GAT AAT Pro Asp Asn AGC TAC CCA Ser Tyr Pro 230 CAT CAG GAG 35 His Gln Glu 245 AAA TCA CGG ATG Lys Ser Arg Met

CCT

Pro 235 AGG GCA CAG AGC Arg Ala Gln Ser

TAC

Tyr 240 o TTT ACA GAC Phe Thr Asp GAT AAC CCC ATT Asp Asn Pro Ile

TTT

Phe 255 GAG AAA TTT GGA Glu Lys Phe Gly

AAA

Lys 40 260 GGA GGA ACA TAT Gly Gly Thr Tyr AGA AGG TAC CAC Arg Arg Tyr His TCC TAT CAT CAC Ser Tyr His His

CAG

Gln 275 1002 1050 1098 1146 1194 1242 1290 1338 1386 1434 1482 1530 1578 GAG TAT AAT GAC Glu Tyr Asn Asp

GGT

Gly 280 CGG AAG ACT TTT Arg Lys Thr Phe

CCA

Pro 285 AGA GCT AGA AGG Arg Ala Arg Arg ACC CAG Thr Gln 290 GGC ACC AGT Gly Thr Ser 50 TTA AGC ACT Leu Ser Thr 310 AGT CGA ATA Ser Arg Ile 325

TTC

Phe 295 CGG TCT CCT GTG Arg Ser Pro Val

AGC

Ser 300 TTC AGT CCT ACT Phe Ser Pro Thr GAT CAC TCC Asp His Ser 305 TAT GAC GAC Tyr Asp Asp AGT AGT GGA AGC Ser Ser Gly Ser AGA AGA CGG GGG Arg Arg Arg Gly 330 GTC TTT ACC CCA Val Phe Thr Pro

GAG

Glu 320 AGT GAC ATA GAC Ser Asp Ile Asp CCT ACT TTG ACT Pro Thr Leu Thr

GTC

Val 340 ACA GAC ATC AGC CCA CCC AGC CGT TCA CCT CGA GCT CCG ACC Thr Asp Ile Ser Pro Pro Ser Arg Ser Pro Arg Ala Pro Thr TGG AGA CTG GGC Trp Arg Leu Gly

AAG

Lys 360 CTG CTT GGC CAA Leu Leu Gly Gln

GGA

Gly 365 GCT TTT GGT AGG Ala Phe Gly Arg GTC TAC Val Tyr 370 Document2 CTC TGC TAT Leu Cys Tyr CAG TTT AAC Gin Phe Asn 390 GAG TGT GAA Glu Cys Glu 405

GAT

Asp 375 GTT GAT ACC GGA Val Asp Thr Gly

AGA

Arg 380 GAG CTG GCT GTT Glu Leu Ala Val AAG CAA GTT Lys Gin Val 385 AAT GCA CTT Asn Ala Leu CCT GAG AGC CCA Pro Glu Ser Pro

GAG

Glu 395 ACC AGC AAG GAA Thr Ser Lys Glu

GTA

Val 400 1626 1674 1722 ATT CAG TTG lie Gin Leu

TTG

Leu 410 AAA AAC TTG TTG Lys Asn Leu Leu GAG CGA ATT GTT Glu Arg Ile Val

CAG

Gln 420 TAT TAT GGC TGT Tyr Tyr Gly Cys

TTG

Leu 425 AGG GAT CCT CAG Arg Asp Pro Gin

GAG

Glu 430 AAA ACA CTT TCC Lys Thr Leu Ser ATC 1770 Ile 435 TTT ATG GAG CTC TCG CCA GGG GGT TCA Phe Met Glu Leu Ser Pro Gly Gly Ser 440 AAG GAC CAA CTA Lys Asp Gln Leu AAA GCC Lys Ala 450 TAC GGA GCT Tyr Gly Ala

CTT

Leu 455 ACT GAG AAC GTG Thr Glu Asn Val

ACG

Thr 460 AGG AAG TAC ACC Arg Lys Tyr Thr CGT CAG ATT Arg Gln Ile 465 CAT AGA GAT His Arg Asp CTG GAG GGG Leu Glu Gly 470 ATC AAA GGA Ile Lys Gly 485 GTC CAT TAT TTG Val His Tyr Leu AGT AAT ATG ATT Ser Asn Met Ile

GTC

Val 480 GCA AAT ATC Ala Asn Ile AGG GAT TCC ACA Arg Asp Ser Thr AAT ATC AAG TTA Asn Ile Lys Leu

GGA

Gly 500 GAC TTT GGG GCT Asp Phe Gly Ala

AGT

Ser 505 AAA CGG CTT CAG Lys Arg Leu Gin

ACC

Thr 510 ATC TGT CTC TCA lie Cys Leu Ser 9* a ACA GGA ATG AAG Thr Gly Met Lys GTC ACA GGC ACG Val Thr Gly Thr

CCA

Pro 525 TAC TGG ATG AGT Tyr Trp Met Ser CCT GAG Pro Glu 530 GTC ATC AGT Val Ile Ser GAA GGC TAT GGA Glu Gly Tyr Gly AAA GCA GAC ATC Lys Ala Asp Ile TGG AGT GTA Trp Ser Val 545 TGG GCT GAA Trp Ala Glu 1818 1866 1914 1962 2010 2058 2106 2154 2202 2250 2298 2352 2412 GCA TGT ACT Ala Cys Thr 550 TTT GAA GCA Phe Glu Ala 565 GTG GTA GAA ATG Val Val Glu Met ACT GAA AAG CCA Thr Glu Lys Pro ATG GCT GCC Met Ala Ala ATC TTT Ile Phe 570 AAG ATC GCC Lys Ile Ala

ACT

Thr 575 CAG CCA ACG AAC Gln Pro Thr Asn

CCA

Pro 580 AAG CTG CCA CCT Lys Leu Pro Pro GTC TCA GAC TAT Val Ser Asp Tyr CGG GAC TTC CTC Arg Asp Phe Leu

AAA

Lys 595 CGG ATT TTT GTA Arg Ile Phe Val GCC AAA CTT CGA Ala Lys Leu Arg TCA GCG GAG GAG Ser Ala Glu Glu CTC TTG Leu Leu 610 CGG CAC ATG Arg His Met

TTT

Phe 615 GTG CAT TAT CAC Val His Tyr His TAGCAGCGGC GGCTTCGGTC CTCCACCAGC 620 TCCATCCTCG CGGCCACCTT CTCTCTTACT GCACTTTCCT TTTTTATAAA AAAGAGAGAT Document2 120 GGGGAGAAAA AGACAAGAGG GAAAATATTT CTCTTGATTC TTGGTTAAAT TTGTTTAATA ATAATAGTAA ACTAAAAAAA AAAAAAAAAA A INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: 0 LENGTH: 619 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 2472 2503 Met Asp Asp Gln Gln Ala Leu Asn Ser Ile Met Gln Asp Leu Ala Val zu Leu Gln Asn Leu Asp Asp Lys Asn Gly 145 Asp Ile Ile Pro Asp 225 His Asn Ile Glu Leu Asp Ser Leu 130 Ala Arg Ala Pro Glu 210 Gly Lys Leu 35 Glu Asp His Leu Leu 115 Glu Glu Ser Arg Glu 195 Asn Glu Pro His Glu Leu Tyr Asp 100 Lys Pro Arg Ser Asn 180 Ser Ser Ser Val His Glu Arg Thr Lys Ile Ser Lys Pro 165 Gly Met Gly Tyr Gly Gln Lys Ser 70 Asn Ala Leu Pro Lys 150 Pro Ser Asp Ser Pro 230 Gln Lys Arg 55 Lys Asn Val Leu Ser 135 Arg Pro Phe Gln Gly 215 Lys His Asn 40 Ile Ser Glu Glu Val 120 Pro Leu Gly Thr Met 200 Ser Ser Tyr 25 Arg Leu Lys Leu Leu 105 Val Glu Ser Tyr Ser 185 Leu Cys Arg Leu Met Gln Ile Val 90 Leu Asn Asp Val Ile 170 Ile Asp Pro Met Tyr 250 Tyr Lys Met Phe Val Thr Ala Phe 75 Ile Pro Asp Arg Gly Ser Leu Asn 140 Val Gly 155 Pro Asp Asn Ser Pro Leu Ser Leu 220 Pro Arg 235 Lys Glu Arg Gly Leu Ser Thr 125 Asn Pro Ile Glu Ser 205 Asp Ala Pro Ser Pro Gln Thr Ile 110 Gln Thr Pro Leu Gly 190 Leu Ser Gin Gly Asn Val Ser Thr His Ala Pro Asn His 175 Glu Ser Pro Ser Lys Leu Lys Met Gln Met Thr Leu Arg 160 Gin Phe Ser Leu Tyr 240 Pro Asp Asn His Gln Glu Phe Thr Asp 245 Asp Asn Pro Ile Phe Glu 255 Document2 Lys Phe Gly His Arg Asp 305 Tyr Thr Pro Arg Lys 385 As n Arg Leu 35 Leu Arg 465 His Ile Leu Her Trp 545 T rp Pro His Thr 290 His Asp Lou Thr Val1 370 Gin Ala Ile Ser Lys 450 Gin Arg Lys Her Pro 530 Ser Ala Thr Gin 275 Gin Per Asp Thr As n 355 T yr Vali Lou Vai Ile 435 Aia le Asp Leu Gly 515 Giu Vai Giu Asn Lys 260 Giu Gly Leu Per Val 340 T rp Lou Gin Giu Gin 420 Phe Tyr Lou Ile Giy 500 Thr Val Aila Phe Pro 580 Gly Gly Thr Tyr Pro Arg Arg Tyr His Vai Ser Tyr T yr Thr Ser Arg 325 Thr Arg Cys Phe Gys 405 Tyr Met Giy Giu Lys 485 Asp Giy Ile Cys Giu 565 Lys Asn S or Thr 310 le Asp Leu T yr As n 390 Giu T yr Glu Aila Giy 470 Giy Phe Met Ser Thr 550 Al a Lou Asp Phe 295 Ser Arg Ile Giy Asp 375 Pro Ile Gly Leu Leu 455 Vai Al a Gly Lys Giy 535 Vai Met Pro Gly 280 Ar g Ser Arg Ser Lys 360 Vai Giu Gin Cys Ser 440 Thr His Asn Aia Ser 520 Giu Vai Ala Pro 265 Arg Per Giy Arg Pro 345 Leu Asp Ser Leu Leu 425 Pro Giu T yr Ile Ser 505 Vai Giy Giu Al a His 585 Lys Pro Per Gi y 330 Pro Leu Thr Pro Lou 41i0 Arg Ci y Asn Lou Leu 490 Lys Thr T yr Met le 570 Vali Phe Per 300 Val1 Asp Ar g Gin Arg 380 Thr Asn Pro Per Thr 460 Per Asp Leu Thr Arg 540 Thr Lys Asp 270 Arg Per Thr Asp Pro 350 Al a Leu Lys Lou Giu 430 Lys Lys Met Thr Thr 510 Tyr Al a Lys Al a Thr 590 Ala Pro Pro As n 335 Arg Phe Al a Glu His 415 Lys Asp Tyr Ile Gly 495 Ile I rp Asp Pro Thr 575 Arg Arg Thr Giu 320 Pro Al a Gly Val Val1 400 Glu Thr Gin Thr Val 480 Asn Gys Met Ile Pro 560 Gin Asp a a Phe Lou Lys Arg le Phe Val Giu Aia Lys Lou Arg Pro Per Ala Glu Documrent2 Glu Leu Leu Arg His Met Phe Val His Tyr His 610 615 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 3089 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 400..2280 (xi) SEQUENCE DESCRIPTION: SEQ ID AGGGAACAAA AGCTGGAGCT CCACCGCGGT GGCGGCCGCT CTAGAACTAG GGGCTGCAGG AATTCGGCAC GAGGAACAGT GGCCGGTCGG AGCGTCTTCT ACTCGCAGGC GGCCCGGTCG AGTGGCGCCG CCGAGGCCGG GTTGGGCCGA GCCGGGGATG TAGCGGGCCA ACCTGCTCAT GCCACAGCGC CCGGCCGCGG GCCTGGGGAG GCGGCGGGGG CCCCGAGCGC AGCCCACGGC CCCCGCGCGG GCTGCCGTCC CCGCCGCCCG CTCCCCCGGC ATGCAGCCCC GGCTGCGGAG 35 TGGGCTGTAG TCGCCACCGC CGCCTCCGCC ATCGCCACC ATG GAT GAA

TGGATCCCCC

GGACTTCAGG

GCCTGGGAGC

CCGAGCCGGA

AGCCAGGCCC

GTGACACTTC

CAA GAG 4*

S

*5 a 55 a.

a

S

Met Asp Glu Gin Glu GCA TTA GAC TCG 40 Ala Leu Asp Ser ATC ATG lie Met 10 AAG GAC CTG Lys Asp Leu GTG GCC Val Ala 15 CTC CAG ATG Leu Gin Met AGC CGA Ser Arg CGA ACC CGG Arg Thr Arg CAC CCA AAC His Pro Asn 40 GAG AGA CGA Glu Arg Arg TCT GGA TAT GAG Ser Gly Tyr Glu

ACC

Thr 30 ATG AAG AAT AAG Met Lys Asn Lys CAG AGT GAC Gin Ser Asp AGA ATC AAG Arg Ile Lys TTT GAA Phe Glu AGA TAC Arg Tyr GAC ACA GGT Asp Thr Gly CAC AAT GGG His Asn Gly GAA GAT GTG Glu Asp Val 510 ATT ATA GCA Ile Ile Ala AGC CGG CCT GTG Ser Arg Pro Val 55 GAG CAC Glu His AAG GTG ACA Lys Val Thr GTC TTT GGG Val Phe Gly CAG CCT Gin Pro 80 AAA AAC Lys Asn 95 CTT GAT TTG CAT Leu Asp Leu His

TAT

Tyr ATG AAT AAT GAG Met Asn Asn Glu

CTC

Leu TCC ATC CTG TTG Ser Ile Leu Leu CAA GAT Gin Asp GAT CTC GAT Asp Leu Asp 100 AGC CTT AGG Ser Leu Arg 115 AAA GCC ATT GAC ATT TTG GAT AGA AGC TCA AGT ATG AAA Lys Ala lie Asp Ile 105 Leu Asp Arg Ser Ser Met Lys Document2 ATA CTA CTG Ile Leu Leu 120 TTA TCC CAA GAC Leu Ser Gin Asp AAC CAT ACT AGT Asn His Thr Ser

TCC

Ser 130 TCT CCC CAC Ser Pro His TCT GGA Ser Gly 135 GTG TCC AGG CAG Val Ser Arg Gin CGG ATC AAG CCT Arg Ile Lys Pro

TCC

Ser 145 CAG TCT GCA GGG Gin Ser Ala Gly 846

GAT

Asp 150 ATA AAT ACC ATC Ile Asn Thr Ile

TAC

Tyr 155 CAA GCT CCT GAG Gin Ala Pro Glu

CCC

Pro 160 AGA AGC AGG CAC Arg Ser Arg His TCT GTC AGC TCC Ser Val Ser Ser AAC CCT GGC CGA Asn Pro Gly Arg TCT CCT CCC CCG Ser Pro Pro Pro GGA TAT Gly Tyr 180 GTA CCT GAG Val Pro Glu ATC AAC AGC Ile Asn Ser 200

CGA

Arg 185 CAA CAG CAC ATT Gin Gin His lle

GCC

Ala 190 CGG CAA GGA TCC Arg Gin Gly Ser TAT ACG AGC Tyr Thr Ser 195 CAG TGT ATG Gin Cys Met GAA GGT GAA TTC Glu Gly Glu Phe CCA GAG ACC AGC Pro Glu Thr Ser

GAA

Glu 210 CTA GAT Leu Asp 215 CCC CTC AGC AGT Pro Leu Ser Ser GAA AAT TCC TTG Glu Asn Ser Leu GGA AGC TGC CAA Gly Ser Cys Gin

TCC

Ser 230 TTG GAC AGG TCA GCA GAC AGC CCA TCC Leu Asp Arg Ser Ala Asp Ser Pro Ser 235

TTC

Phe 240 AGG AAA TCA CAA Arg Lys Ser Gin TCC CGA GCC CGG 35 Ser Arg Ala Arg

AGC

Ser 250 TTC CCA GAC AAC Phe Pro Asp Asn

AGA

Arg 255 AAG GAA TGC TCA Lys Glu Cys Ser GAT CGG Asp Arg 260 *0 090 000 *00000 *090 0 0000 0 0 00 00 00 00 0S 0 0) 00eS0~ GAG ACC CAG Glu Thr Gin CGC TAC CAT Arg Tyr His 280 TAT GAT AAA GGT Tyr Asp Lys Gly AAA GGT GGA ACC Lys Gly Gly Thr TAT CCC AGG Tyr Pro Arg 275 GGC AGA AGA Gly Arg Arg GTG TCT GTG CAT Val Ser Val His

CAC

His 285 AAA GAC TAC AAT Lys Asp Tyr Asn 1038 1086 1134 1182 1230 1278 1326 1374 1422 1470 1518 ACA TTT Thr Phe 295 CCC CGA ATA CGA Pro Arg Ile Arg

CGG

Arg 300 CAT CAA GGC AAC His Gin Gly Asn

CTA

Leu 305 TTC ACT CTG GTG Phe Thr Leu Val 50 CCC Pro 310 TCA AGT CGC TCC Ser Ser Arg Ser AGC ACA AAT GGC Ser Thr Asn Gly AAC ATG GGT GTA Asn Met Gly Val

GCT

Ala 325 GTG CAA TAC CTG Val Gin Tyr Leu CCC CGT GGG CGC Pro Arg Gly Arg CGG AGT GCA GAC Arg Ser Ala Asp AGT GAG Ser Glu 340 AAT GCC CTC ACT GTG CAG GAA AGG Asn Ala Leu Thr Val Gin Glu Arg 345

AAT

Asn 350 GTG CCA ACC AAA Val Pro Thr Lys TCT CCT AGT Ser Pro Ser 355 GGT GCC TTC Gly Ala Phe GCT CCC ATC Ala Pro Ile 360 AAT TGG CGT CGG Asn Trp Arg Arg

GGG

Gly 365 AAG CTC CTG GGT Lys Leu Leu Gly

CAA

Gin 370 Document2 GGC AGG Gly Arg 375 GTC TAC TTG TGC Val Tyr Leu Cys GAT GTG GAC ACA Asp Val Asp Thr

GGA

Gly 385 CGT GAA CTT GCT Arg Glu Leu Ala

TCT

Ser 390 AAG CAG GTC CAG Lys Gin Val Gin

TTT

Phe 395 GAC CCA GAT AGT Asp Pro Asp Ser GAG ACA AGC AAG Glu Thr Ser Lys GTG AGT GCT CTG Val Ser Ala Leu TGT GAG ATC CAG Cys Glu Ile Gin

TTG

Leu 415 CTG AAG AAC CTG Leu Lys Asn Leu CAG CAT Gin His 420 GAG CGC ATT Glu Arg Ile ATC CTC ACC lie Leu Thr 440

GTG

Val 425 CAG TAC TAC GGC Gin Tyr Tyr Gly CTG CGG GAC CGT Leu Arg Asp Arg GCT GAG AAG Ala Glu Lys 435 GTA AAA GAC Val Lys Asp ATC TTT ATG GAG Ile Phe Met Glu

TAT

Tyr 445 ATG CCA GGG GGC Met Pro Gly Gly CAG TTG Gin Leu 455 AAG GCC TAC GGA Lys Ala Tyr Gly CTG ACA GAG AGT Leu Thr Glu Ser

GTG

Val 465 ACC CGC AAG TAC Thr Arg Lys Tyr

ACC

Thr 470 CGG CAG ATT CTG Arg Gin Ile Leu GGC ATG TCA TAC Gly Met Ser Tyr CAC AGC AAC ATG His Ser Asn Met GTG CAT CGG GAC Val His Arg Asp

ATC

Ile 490 AAG GGA GCC AAT Lys Gly Ala Asn

ATC

Ile 495 CTC CGA GAC TCA Leu Arg Asp Ser GCT GGG Ala Gly 500

£*S

0 0* 0

S

0 0

OSGO

*5 0 0 0O S *5 0) S S 00 50 00 00 *0 0S 09 5 0 *0500*

S

0*

S

S.

AAT GTG AAG Asn Val Lys TGC ATG TCA Cys Met Ser 520 GGG GAT TTT GGG Gly Asp Phe Gly AGC AAA CGC CTA Ser Lys Arg Leu CAG ACC ATC Gin Thr Tle 515 CCC TAC TGG Pro Tyr Trp 1566 1614 1662 1710 1758 1806 1854 1902 1950 1998 2046 2094 2142 2190 2238 2287 2347 GGG ACA GGC ATT Gly Thr Gly Ile TCT GTC ACT GGC Ser Val Thr Gly ATG AGT Met Ser 535 CCT GAA GTC ATC Pro Glu Val lie

AGT

Ser 540 GGC GAG GGC TAT Gly Glu Gly Tyr

GGA

Gly 545 AGA AAG GCA GAC Arg Lys Ala Asp

GTG

Val 550 TGG AGC CTG GGC Trp Ser Leu Gly ACT GTG GTG GAA Thr Val Val Glu CTG ACA GAG AAA Leu Thr Glu Lys

CCA

Pro 565 CCT TGG GCA GAG 50 Pro Trp Ala Glu GAA GCT ATG GCT Glu Ala Met Ala ATT TTC AAG ATT Ile Phe Lys Ile GCC ACC Ala Thr 580 CAG CCT ACC Gln Pro Thr GAC TTC CTG Asp Phe Leu 600

AAT

Asn 585 CCT CAG CTG CCC Pro Gln Leu Pro CAC ATC TCA GAA His Ile Ser Glu CAC GGC AGG His Gly Arg 595 CCC TCA GCT Pro Ser Ala AGG CGC ATA TTT Arg Arg Ile Phe

GTG

Val 605 GAA GCT CGT CAG Glu Ala Arg Gln

AGA

Arg 610 GAG GAG Glu Glu 615 CTG CTC ACA CAC CAC TTT GCA CAG CTA GTG TAC TGAGCTCTCA Leu Leu Thr His His Phe Ala Gln Leu Val Tyr AGGCTATCAG GCTGCCAGCT GCCACCTGCT GAGCAGGCAA GGGGCTGCTG TCAGGCTCAG Document2

TGAAGTTGCT

TTGTCTGTGC

GACTGGGAGC

GAACGGCTGG

AACACTCCAG

GAATAGTGTT

AGCCTGTCTC

AGAGGTTTGG

AGATGAATGA

CACGGCCTGA

TTGTCTCTCT

CTTTGCCCTC

CATCGCCTCT

GCTTCTTCCA

CC CAT CT GC C

TCCAGCCTGT

AAACAGTATG

ACAGCACTGA

ACTTCATTCA

TGGGCTGCAA

GCTACCCCAC

AGCAAATGTT

GAGATGGAGC

TTTACAAGTT

TGAGCTCTGG

GCCCCTGGCC

GGCAAGGCTA

ACTGGGACTC

AAGACCCAAG

CAAGACTGCC

AGTCAAGAGG

GAGTGTTACT

GCCTGAGGTA

TATACCTTCC

ACACTGCCTT

TGCCTCCAGA

GGAGTCACTC

AGACCCATGT

TG

TGACCAGTGG

AAACCCACGA

AGCTTTAGCA

ATGGGTCCTG

GACTGGGGCA

TTGTTTCTCT

AAGCCCAGCA

AGGTATTCGG

ATTCTGGGAA

AACTGCGGAG

TTATGCTGTT

GGGCTTAGGC

AGCATCGGTC

TGGGATAGCT

CCTTAAGCTC

CCTACCCTCA

CAGGAGGTCC

CCCAATGTTT

T CCC CCACCC

TGTCAGTCCT

GGAGGAGCTA

ACCCAGTCTT

CCCAGTTTTA

TTGGACTGGA

CAGCCAT TGT

CTGGCATCAA

AGTATGGCGG

GATCTGTCCT

TCAAGGGTAT

GGAGACCACC

AACACAAGGT

GTCTTACCAA

CTCGGATAAG

GTCAATCCAA

AAACTCCACA

TGGAAGAGCT

2407 2467 2527 2587 2647 2707 2767 2827 2887 2947 3007 3067 3089 INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: LENGTH: 626 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:6: (i i) (xi) Met 1 Asp Gin Gin Giu Ala Leu Asp Ser Ile 10 Ser Met Lys Asp Len Val Ala Met Lys Leu Gln Met Aso Lys Asp Phe Gin His Ser Thr Arg Arg Thr Arg Leu 25 Cly Tyr Gin Thr Giy His Pro Arg Gin Ser Asp Arg Ile Lys Arg Pro Val Asn Gly Gin Arg Arg Ile Ile Ala Arg 65 Tyr Gin Asp Val His Lys Val Thr Thr '75 Val Phe Gly Gin Pro 55 Len Asp Len His Met Asn Asn Gin Ser Ile Len Len Lys Asn Gin Asp Asp Met Lys Her 115 Len 100 Len Asp Lys Ala le Asp 105 Len Asp Arg Ser Her Her 110 Asn His Thr Arq Ile Len Len Her Gin Asp Arg 125 Ser Ser 130 Her Pro His Her Gly Val 135 Ser Arg Gln Arg Ile Lys Pro DocumenQt Ser Gin 145 Arg Ser Pro Pro Gly Ser Ser Glu 210 Ser Gly 225 Arg Lys Glu Cys Gly Thr Asn Asp 290 Leu Phe 305 Asn Met Ser Ala Thr Lys Gly Gln 45 370 Gly Arg 385 Glu Thr Lys Asn Asp Arg Gly Ser 450 Val Thr Ser Ala Arg His Pro Gly 180 Tyr Thr 195 Gln Cys Ser Cys Ser Gln Ser Asp 260 Tyr Pro 275 Gly Arg Thr Leu Gly Val Asp Ser 340 Ser Pro 355 Gly Ala Glu Leu Ser Lys Leu Gln 420 Ala Glu 435 Val Lys Arg Lys Gly Leu 165 Tyr Ser Met Gln Met 245 Arg Arg Arg Val Ala 325 Glu Ser Phe Ala Glu 405 His Lys Asp Tyr Asp 150 Ser Val Ile Leu Ser 230 Ser Glu Arg Thr Pro 310 Val Asn Ala Gly Ser 390 Val Glu Ile Gin Thr lie Val Pro Asn Asp 215 Leu Arg Thr Tyr Phe 295 Ser Gln Ala Pro Arg 375 Lys Ser Arg Leu Leu 455 Arg Asn Ser Glu Ser 200 Pro Asp Ala Gln His 280 Pro Ser Tyr Leu Ile 360 Val Gln Ala Ile Thr 440 Lys Gln Thr Ser Arg 185 Glu Leu Arg Arg Leu 265 Val Arg Arg Leu Thr 345 Asn Tyr Val Leu Val 425 Ile Ala Ile Ile Tyr 155 Gln Asn 170 Gln Gln Gly Glu Ser Ser Ser Ala 235 Ser Phe 250 Tyr Asp Ser Val Ile Arg Ser Leu 315 Asp Pro 330 Val Gln Trp Arg Leu Cys Gin Phe 395 Glu Cys 410 Gln Tyr Phe Met Tyr Gly Leu Glu Gin Ala Pro Gly His Ile Phe Ile 205 Ala Glu 220 Asp Ser Pro Asp Lys Gly His His 285 Arg His 300 Ser Thr Arg Gly Glu Arg Arg Gly 365 Tyr Asp 380 Asp Pro Glu Ile Tyr Gly Glu Tyr 445 Ala Leu 460 Gly Met Pro Arg Ala 190 Pro Asn Pro Asn Val 270 Lys Gin Asn Arg Asn 350 Lys Val Asp Gin Cys 430 Met Thr Ser Glu Ser 175 Arg Glu Ser Ser Arg 255 Lys Asp Gly Gly Leu 335 Val Leu Asp Ser Leu 415 Leu Pro Glu Tyr Pro 160 Ser Gln Thr Leu Phe 240 Lys Gly Tyr Asn Glu 320 Arg Pro Leu Thr Pro 400 Leu Arg Gly Ser Leu •t *o o o*ooo* *o oto 465 470 475 480 His Ser Asn Met Ile Val His Arg Asp Ile Lys Gly Ala Asn Ile Leu Docunient2 490 Gly Arg Asp Ser Arg Leu Gln 515 Ala 500 Thr Asn Val Lys Asp Phe Gly 495 Ala Ser Lys 510 Ser Val Thr Glu Gly Tyr Ile Cys Met Ser 520 Pro Thr Gly Ile Arg 525 Gly Gly Thr 530 Gly Arg 545 Pro Tyr Trp Met Glu Val lie Lys Ala Asp Val 550 Ser Leu Gly Cys 555 Val Val Glu Leu Thr Glu Lys Pro Trp Ala Glu Tyr 570 Pro Glu Ala Met Ala Ala Ile 575 His Ile Phe Lys Ile Ser Glu His 595 Gln Arg Pro 610 Val Tyr 625 Gin Pro Thr Gln Leu Pro Gly Arg Asp Phe Leu 600 Arg Arg Ile Phe Val 605 Glu Ala Arq Ser Ala Glu Leu Leu Thr His Phe Ala Gin Leu INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 3913 base pairs ln) l-IfT: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (1) NAME/KEY: CDS LOCATION: 747..3417 *44 4 4 4.

4* NAME/KEY: N G,A,C or T LOCATION: 1094 NAME/KEY: Xaa Any amino acid LOCATION: 116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AATTCGGCAC GAGAACCTAT CAGACATTGG CTGGCCAGTG TTTGAAATCC CCTCCCCTCG GCCGTCCAAG GGCTACGAGC CAGAGGACGA GGTCGAGGAC ACGGAGGTTG AGCTGAGGGA GCTGGAGAGC GGGACGGAGG AGAGTGACGA GGAGCCAACC CCCAGTCCGA GGGTGCCAGA GCTCAGGCTG TCCACAGACA CCATCTTGGA CAGTCGCTCC CAGGGCTGCG TCTCCAGGAA S GCTGGAGAGG CTCGAGTCAG AGGAAGATTC CATAGGCTGG GGGACAGCGG ACTGTGGCCC Document2

TGAAGCCAGC

GCAAATCGG

AAGAGCTCGT

CC CAT CT CC

TGTAGCGCTG

TTCCTGCTGC

GAACAGAGC

AAGAGCTCCT

AGGCATTGTT

CTAACAAAGT

C TAC C TC TGC

CCTCCCATTA

TCTCCTGGCA

TCTCTCCCCT

TCCGGCGAG

AAAGGGCGGG

TGACTTCTAT

TAATTTTACG

TGAAGGACGA

TCTCCACTTG

AGAACTGAGA

CCTGCTCAAC

CCTTCCCTCT

CTCCTG ATG CTATAGACCA TTCGTCCACA AACCACTCAA ACTTCATAAG CTTATCAATC CCTCCTTGCA CCGTCACTCC ACTTCTCTCA CTTTCCACCT TCCCCAACAC CTCCTTCCTC AGAGCACAAC GCCATGGACC TGCCTTCCTT TGACCCCGCC GTGATCCACC ACTCCCTCAA CCTCCCCTG TCAGTATCAA ACAGCTAGTG CGAGAGTCTA AAG CAG TAT TAC CAG TTC ATG CTG Met Lys Cmn Tyr Tyr Cmn Phe Met Leu

CAC

Cmn

TTT

Phe CAC CTC CTC GC Clu Val Leu Gly CTG CAC AAC ACC Leu Clu Lys Thr TCC AAC ATC CAT Cys Asn Met Asp CAC CAC CAC Clu Clu Asp

CTC

Leu 30 CAC AAC ATC CTC Gin Lys Met Leu

ATC

Met 35 CTC TAT TTT CAT Val Tyr Phe Asp

TAC

T yr ACA ACC TCC Arg Ser Trp TTA AAA AAC Leu Lys Asn CAA ATC CTA CAC Cmn Met Leu Cmn TTA CCT CAC GCT Leu Pro Cmn Ala TCC CAT AC Per His Ser CAA ATA ACC Clu Ile Thr CTA CAA CAC Leu Glu Glu TCC AAT TTC ACC Trp Asn Phe Thr

AAA

Lys His Tyr Ile Arg Cly Cly Clu Ala Gin Ala Cly Leu Phe Cys Asp a a p a p

ATC

40 Ile

CTC

Leu

CCA

Al a GGG ATG CTC Cly Met Lou AAA TCC ACA CCC Lys Per Thr Cly CTC CAA TCC Lou Ciu Ser CAC CAC AC Cmn Ciu Ser

TCT

Cys 110

CTA

Leu CCT CAC CTC TCC Ala Giu Leu Trp

ACC

Thr 115 AT C Tie GCC CAC CAC Ala Asp Asp AAC CCT Asn Cly 120 CCT CCC CAC Ala Ala Asp AAC CAC CTC Lys Ciu Leu 140 TTT CCT AAA Phe Ala Lys AGO ACA TCT Arg Arg Per

GTC

Val 130

CAA

Giu CAC ATC AC Ciu Ile Ser CAC CAA GCC His Ciu Ala ACA CCC TCC Arg Ala Per

AAC

Lys 150

CCA

Al a CCA CCA CTC Arg Ala Leu 135 CCC CTC GC Ala Leu Cly CAC TTC CTG Clu Phe Val 1613J~ 1061 1109 1157 1205 1253 1301 1349 ATG CTC ACC Met Lou Arg 155 CTA TCT 60 Lou Ser

AAC

Lys 160

GAG

Clu CTA CAA ATA Lou Ciu Ile

CCA

Al a 165

CTC

Leu CCA TCA GCC Ala Ser Ala 170

TAT

CCA

Arg 175 AT T CTC CTC CAC Leu Leu Asp

CCT

Ala 180

AAT

AAA GCA AAG Lys Ala Lys

CAC

Gin 185 CTC CTT AAC CTA CAC CCC CCC TTA CAC TTC CAC CTC TTT Tyr Val Lys Val Cn Ile Pro Gly Lou Ciu Asn Lou His Val Phe Val Docu'llln2

CCC

Pro

GCT

Al a

CAT

Asp

GAA

Glu 250

CAG

Gin

CG

Leu

TTC

Phe

TCT

Her

GCA

33~ A-Lct 330

CAC

His 40

GICC

Ala

TTT

Phe

TTC

Phe

ATC

55 Met 410

AGA

Arg 60

GAG

Asp

CC

Ala

GCC

Ala 235

CAT

Asp

GIG

Val

CTG

Leu

CAG

Gin

ACC

Her 315

CT

Leu

ATG

Met

ACG

Thr

CCC

Gly

AGG

Arg 395

AAT

Asn

ICC

I rp

AGC

Her

ACA

Thr 220

TTC

Phe

GC

Cly

GAG

Ciu

CII

Val1

GAG

Gin 300

CAG

Gin

GAG

GlIL

TIC

Phe

CG

Leu

TTT

Phe 380

GAG

Gin

TAG

Tyr

GCC

Al a

GIG

Leu 205

GGA

Gly

CTC

Leu

TGG

T rp

ACT

Thr

GIG

Val1 285

TGG

Ser

CCC

Pro

CTA

L e LI

ACG

Thr

GAG

Gin 365

GAG

Glu

AAG

Lys

CTC

Val1

ACG

Ihr

CI

Al a

AAG

Lys

GIG

Lou

GCC

Ci y

GIG

Val 270

ATC

Met AT T Ile

ATG

Ile

IC

cyb

CTG

Leu 350

GAG

Gin

TAT

Tyr

ATA

Ile

GTG

Lou

CAA

Gin 430

GAG

Giu

GAG

Asp

CG

Lou

ACA

Thr 255

GAG

Asp

GAG

Glu

GAG

Giu

ATG

Ile

AAC

I-"jli 335

GAG

Ciu

TAG

T yr

CAT

His

GGA

Gly

ACC

Thr 415

GGA

Cly

GAG

Giu

IC

Gys

ACC

Thr 240

TG

T rp

ACC

Thr

TCT

Ser

CCC

Gly

CCC

Al a 320

ACA

AMuG

TIC

Phe

TAG

Tyr

AAA

Lys

GAG

Asp 400

AAA

Lys

TTT

Phe

AAG

Lys

TGA

Ser 225

AAC

Lys

GAA

Ciu

GTG

Lou

CI

Al a

GTG

Lou 305

AAA

Lys AT C 13-u

CAT

Asp

CCA

Arg

GAA

Ciu 385

AAA

Lys

IC

Cys

CAT

Asp

AA

Lys 210

AAG

Lys

CAT

His

CI

Al a

AGA

Arg

GAG

His 290

ATG

Met

CCI

Cly

AGG

CI

Ala

CA

Giu 370

CT

Vai

TAT

Tyr

GAG

Ciu

TC

Phe ATT ATI TIGCGAG CIA GIG PAT le Tie Lou Gin Lou Lou Asn

CAT

Asp

CCC

Cly

CCC

Arg

AGG

Ser 275

GIG

Lou

ACT

Ihr

TIC

Leu

CAT

TIP

GAG

Clu 355

CC

Ala

CII

Val1 AlA Tle

AGC

Her

CIA

Lou 435

CCA

Pro

GAG

Asp

C

Aia 260

AIG

Met

CIA

Val

CIA

Vai

GAG

Cmn

CC

I-lld 340

GC

Vai

AIC

Met

CCI

Arg

AG

Ihr

C

Gly 420

CA

Gin

GAG

Asp

GA

Arg 245

GC

Val

GAG

Gin

CII

Lou

CC

Arg

GAG

Cmn 325

AIC

!It--

GAC

Ciu All Ile

TTG

Lou

TC

Phe 405

ACA

Arg

GCC

Al a

GC

Asp 230

CC

Al a

AA

Lys

GIG

Val

GAG

Cmn

CAT

His 310

GIG

Lou

CAC

A bp

GAC

Clu

CAC

Cin AT C Met 390

GCC

Ala

GCC

Gly AT I TIe 215

GIG

Vali

CCI

Ar g All le

GAG

Asp

ACA

Ar g 295

GAG

Giu

PAG

Lys

CCI

-L

IGI

Her

GC

Cly 375

ICI

Her

GAG

Gin

ACA

Ihr

CA

Clu

TC

Phe

GC

Asp

CIC

Val1

AG

Asn 280

AA

Lys

GAG

Gin

AG

Asn

CIC

V, 1

GAG

Clu 360

TAG

Tyr

CCC

Ci y

AC

Lys

AGA

Arg

CCI

Pro 440

AIC

Met I CA Her

CCI

Pro 265

CII

Lou

CC

Al a

ACA

Ihr

CAT

Asp

GC

I bp 345

ICC

Her

PAC

As n

CA

Clu

ICC

Irp

CCC

Pro 425

GCC

Al a 1397 1445 1493 1541 1589 1637 1685 1733 1781 1829 1877 1925 1973 2021 2069 2117 a a a a a a.

a a a III ATT ICA CI TIA GGA CPA CAT GC TIC TIC ACT TIC CPA CCC CG Phe le Her Ala Lou Pro Gbu Asp 445 Asp 450 Phe Lou Her Lou Cmn Ala Lou 455 Docurnent2 ATG AAT GAG TGC ATC GGG CAC GTC ATA GGA AAG CCA CAC AGC CCT GTC Met

ACA

Thr

CAC

His 490

TTC

Phe

GCA

Ala

GAG

Glu

CGG

Arg

GCC

Ala 570

TTT

Phe

CAA

Gln

CTG

Leu 45 GGA Gly

CTG

50 Leu 650

ATC

Ile 55

CCC

Pro

TAC

Tyr Asn

GCT

Ala 475

AGT

Ser

AGG

Arg

GAA

Glu

CGA

Arg

AGA

Arg 555

TCT

Ser

GAA

Glu

GTG

Val

AGG

Arg

CAG

Gln 635

ATG

Met

AAG

Lys

AAC

Asn

ATC

Ile Glu 460

ATC

Ile

GAC

Asp

GGT

Gly

CTG

Leu

GAC

Asp 540

AGC

Ser

AAG

Lys

GAG

Glu

TGC

Cys

AAG

Lys 620

TAT

Tyr

GCC

Ala

GAG

Glu

CTG

Leu

TTC

Phe Cys

CAT

His

CCT

Pro

TCC

Ser

CAG

Gln 525

GAG

Glu

TGG

Trp

CAG

Gln

AGG

Arg

GAT

Asp 605

GTG

Val

GGA

Gly

ATG

Met

ACT

Thr

GTC

Val 685

ATG

Met Ile Gly CGG AAC Arg Asn CCT AAC Pro Asn 495 AGT GTC Ser Val 510 TTC AGG Phe Arg CCA GCG Pro Ala GAA CTT Glu Leu GGG CCC Gly Pro 575 AGG TAT Arg Tyr ACC CCT Thr Pro ACA TTT Thr Phe AAA GTA Lys Val AAG GAG Lys Glu 655 GCA GAC Ala Asp 670 CGG TAT Arg Tyr GAG TAC Glu Tyr His

AGC

Ser 480

CCT

Pro

CCT

Pro

TCT

Ser

TAT

Tyr

CGA

Arg 560

ATA

Ile

CGA

Arg

AAG

Lys

AAG

Lys

TAC

Tyr 640

ATT

Ile

GAG

Glu

TTT

Phe

TGT

Cys Val 465

CCC

Pro

CAC

His

GAA

Glu

CTG

Leu

CCT

Pro 545

ACA

Thr

GAA

Glu

GAG

Glu

TCC

Ser

TGG

Trp 625

ACC

Thr

CGA

Arg

TTG

Leu

GGC

Gly

GAT

Asp 705 Ile

CGC

Arg

CTC

Leu

AAC

Asn

AGT

Ser 530

CGG

Arg

CTC

Leu

GCT

Ala

ATG

Met

TAT

Tyr 610

CAA

Gln

TGC

Cys

TTT

Phe

AAA

Lys

GTG

Val 690

GAG

Glu Gly Lys CCT GTG Pro Val ATC ATC Ile Ile 500 GAC CGC Asp Arg 515 CGG CAC Arg His AGT GAC Ser Asp ATC AGC Ile Ser ATC CAG Ile Gln 580 AGG AGA Arg Arg 095 GAT AAC Asp Asn AGA GGA Arg Gly ATC AGT Ile Ser CAG CCT Gln Pro 660 ATA TTT Ile Phe 675 GAG CTT Glu Leu GGT ACA Gly Thr Pro

AAG

Lys 485

CCG

Pro

TTG

Leu

TCA

Ser

TCA

Ser

CAG

Gln 565

AAG

Lys

AAG

Lys

GTC

Val

AAC

Asn

GTT

Val 645

AAC

Asn

GAA

Glu

CAC

His

CTA

Leu His 470

GTG

Val

ACT

Thr

GCC

Ala

AGC

Ser

AGT

Ser 550

ACC

Thr

TCA

Ser

AAT

Asn

ATG

Met

AAA

Lys 630

GAC

Asp

GAC

Asp

GGC

Gly

AGG

Arg

GAG

Glu Ser

CCC

Pro

CCA

Pro

TCC

Ser

CCC

Pro 535

GGA

Gly

AAA

Lys

GTC

Val

ATC

Ile

CAT

His 615

ATT

Ile

ACA

Thr

CAC

His

ATC

Ile

GAA

Glu 695

GAG

Glu Pro

CGA

Arg

GAG

Glu

ATA

Ile 520

ACG

Thr

TCA

Ser

GAC

Asp

CGA

Arg

ATC

Ile 600

GTT

Val

GGA

Gly

GGG

Gly

AAG

Lys

AAG

Lys 680

GAG

Glu

GTG

Val Val

TGC

Cys

GGA

Gly 505

GCT

Ala

GAA

Glu

ACT

Thr

TCG

Ser

CTG

Leu 585

GGC

Gly

GGA

Gly

GAA

Glu

GAG

Glu

ACT

Thr 665

CAC

His

ATG

Met

TCA

Ser 2165 2213 2261 2309 2357 2405 2453 2501 2549 2597 2645 2693 2741 2789 2837 2885 2933 S e fr o a a a *o o *o **o 700 710 CGA CTG GGC CTG CAG GAG CAC GTC ATC AGG TTA TAT ACC AAG CAG ATC Documen2 Arg Leu Gly Leu Gin Giu His Val Ile Arg Leu Tyr Thr Lys Gin Ile

ACT

Thr AT C Ile

GGA

Giy

CCC

Pro

GAA

Gin AT C Ile 810

CCT

Pro

ATG

Met

GCC

Ala 40 CCC

GCC

Aila

GGT

Giy TT T Phe

GAG

Gin 780 AT T Ile

AGT

Ser

CAT

His

GAG

His

CTC

Len 860

GAG

AAG

As n

AAT

As n 750

TGG

Gys

AAC

Asn

GGA

Arg

GG

Giy

TAT

T yr 830

CGA

Pro

GAG

His

GIG

720

CTC

Len

TTC

Phe

GTA

Vai

ACC

Thr

AAA

Lys 800

GTC

Vai

GAG

His AT C Ile

CGG

Len

GAG

CAT

His

CT

Len

AAA

Lys

GTA

Len 785

CCA

Giy

GTC

Vai

AAG

Asn

CCC

Pro

GAA

Gin 065

GCT

GAG

His

TCA

Her 755

AAA

Lys

ACA

Thr

GCC

Giy

GAG

Gin

GAG

Gin 835

AGG

Ar g

GAG

Asp

GTC

GC

Gly 740

TCT

Ser

AAG

As n

GCA

Aia

GAG

His AT C Met 820

ATT

Ile

CIA

Len

CCC

Pro

AAG

725 AT C Ile

GGA

Giy

AAG

As n

GGT

Ala

GCA

Gly 805

GTG

Vai

ATG

Met

AGG

Ser

AAG

Lys

GTT

CT

Val

CIA

Len

GCC

Al a

TAG

T yr 790

GT

Ar g

ACT

Thr

TAG

Tyr

CCT

Pro

ATA

le 870

TGG,

GAG

His

ATG

Ile

GAG

Gin 775

ATG

Met

GC

Al a

GC

Giy

AAG

Lys

GAA

Gin 855

CGC

Arg

ACA

CGA

Arg

AAG

Lys 760

ACC

Ihr

GCC

Aila

GCA

Ala

AAC

Lys

CTC

Vai 840

GA

Cly

TGG

T rp,

CAT

GAG

Asp 745

CTG

Len

ATC

Met

CCT

Pro

CAT

Asp

CCC

Arg 825

CCC

Giy

AAG

Lys

ACA

Thr

CAA

2981 3029 3077 3125 3173 3221 3269 3317 3365 3413 3467 3527 3587 3647 3707 3767 3827 3887 3913 9 9* 9 9 .9 .9 9 9 .9 9* 9* Ala Ser Gin Len Len Asp His Ala Phe Val Lys Val Gys Ihr Asp Gin 875 880 GAG T GAAGTGAACC ACTCCGTGGC CTACIACICT CTGGAGAGAA ICCGGGC 45 Gin 890 ACTACTGTAI CTAATATTTA GATAAAGACT GACTCAACAC TAGAGCCCTG AGAACTCA GACTCCIGAG GITAAAGGAG CCCCACGTTA GTTGTCGCG TGCGCGGG TCTGTGAG TGACCTCCIC GCCTTCAGAA GAACATCTAG ACCTCCTCCT CCTTCGCTCT GCGTTGAG TGCAAAGGGT CGTAAGTGAA GCTCAACAAA CCAAAGCAA AAAAAAAAAA AAAAAA INFORMATION FOR SEQ ID NO:8:

CCACCCAGG

CITCTCCTC

AGTGGGATIA

GAACCAACCG

IGITCCGGG

CTCIGGIICT

ACOT TCT T TT CGGCCITCCT AACCTCCCAC ICCTGTGGCC TCCTCACTGA GTACGTCA CGCCACGGC IGCATCACC ACAGITTIT TTCGGGGC GITTATTIGA GGAAAGGTGG ACCTTCTTA TGAATAAATC GTTTATTITA Docurnent2 SEQUENCE CHARACTERISTICS: LENGTH: 890 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Xaa Any amino acid LOCATION: 116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Lys Gln Tyr Tyr Gln Phe Met Leu Gln Glu Val Leu Gly Gly Leu 1 5 10 Glu Lys Thr Asp Cys Asn Met Asp Ala Phe Glu Glu Asp Leu Gln Lys 25 Met Leu Met Val Tyr Phe Asp Tyr Met Arg Ser Trp Ile Gln Met Leu 40 Gin Gln Leu Pro Gln Ala Ser His Ser Leu Lys Asn Leu Leu Glu Glu 50 55 Glu Trp Asn Phe Thr Lys Glu Ile Thr His Tyr Ile Arg Gly Gly Glu 70 75 Ala Gln Ala Gly Lys Leu Phe Cys Asp Ile Ala Gly Met Leu Leu Lys 90 Ser Thr Gly Ser Phe Leu Glu Ser Gly Leu Gln Glu Ser Cys Ala Glu 100 105 110 Leu Trp Thr Xaa Ala Asp Asp Asn Gly Ala Ala Asp Glu Leu Arg Arg 115 120 125 Ser Val Ile Glu Ile Ser Arg Ala Leu Lys Glu Leu Phe His Glu Ala 130 135 140 Arg Glu Arg Ala Ser Lys Ala Leu Gly Phe Ala Lys Met Leu Arg Lys 145 150 155 160 45 Asp Leu Glu Ile Ala Ala Glu Phe Val Leu Ser Ala Ser Ala Arg Glu 165 170 175 L Leu Asp Ala Leu Lys Ala Lys Gln Tyr Val Lys Val Gln Ile Pro 180 185 190 Gly Leu Glu Asn Leu His Val Phe Val Pro Asp Ser Leu Ala Glu Glu :195 200 205 SLys Lys Ile Ile Leu Gln Leu Leu Asn Ala Ala Thr Gly Lys Asp Cys 55 210 215 220 Ser Lys Asp Pro Asp Asp Val Phe Met Asp Ala Phe Leu Leu Leu Thr 225 230 235 240 60 Lys His Gly Asp Arg Ala Arg Asp Ser Glu Asp Gly Trp Gly Thr Trp 245 250 255 Glu Ala Arg Ala Val Lys Ile Val Pro Gln Val Glu Thr Val Asp Thr 260 265 270 Document2 Leu Arg Ala His 290 Leu Met 305 Lys Gly Ile Ser Asp Ala Arg Glu 370 Glu Val 385 Lys Tyr Cys Glu Asp Phe Asp Asp 450 Val lie 465 40 Pro Arg His Leu Glu Asn Leu Ser 50 530 Pro Arg 545 55 Thr Leu Glu Ala Glu Met Ser Met 275 Leu Val Thr Val Leu Gin Asp Ala 340 Glu Val 355 Ala Met Val Arg Ile Thr Ser Gly 420 Leu Gin 435 Phe Leu Gly Lys Pro Val Ile Ile 500 Asp Arg 515 Arg His Ser Asp Ile Ser Ile Gin 580 Arg Arg 595 Gin Leu Arg Gin 325 Ile Glu Ile Leu Phe 405 Arg Ala Ser Pro Lys 485 Pro Leu Ser Ser Gin 565 Lys Lys Val Gin His 310 Leu Asp Glu Gin Met 390 Ala Gly Ile Leu His 470 Val Thr Ala Ser Ser 550 Thr Ser Asn Asp Arg 295 Glu Lys Arg Ser Gly 375 Ser Gin Thr Glu Gin 455 Ser Pro Pro Ser Pro 535 Gly Lys Val Ile Asn Leu 280 Lys Ala Gin Thr Asn Asp Val Asp 345 Glu Ser 360 Tyr Asn Gly Glu Lys Trp Arg Pro 425 Pro Ala 440 Ala Leu Pro Val Arg Cys Glu Gly 505 Ile Ala 520 Thr Glu Ser Thr Asp Ser Arg Leu 585 Ile Gly Leu Phe Ser Ala 330 His Ala Phe Phe Met 410 Arg Phe Met Thr His 490 Phe Ala Glu Arg Ala 570 Phe Gin Leu Gin Ser 315 Leu Met Thr Gly Arg 395 Asn Trp Ile Asn Ala 475 Ser Arg Glu Arg Arg 555 Ser Glu Val Val Gin 300 Gin Glu Phe Leu Phe 380 Gin Tyr Ala Ser Glu 460 Ile Asp Gly Leu Asp 540 Ser Lys Glu Cys Lys 620 Val Met 285 Ser Ile Pro Ile Leu Cys Thr Leu 350 Gin Gin 365 Glu Tyr Lys Ile Val Leu Thr Gin 430 Ala Leu 445 Cys Ile His Arg Pro Pro Ser Ser 510 Gin Phe 525 Glu Pro Trp Glu Gin Gly Arg Arg 590 Asp Thr 605 Glu Glu lie Asn 335 Glu Tyr His Gly Thr 415 Gly Pro Gly Asn Asn 495 Val Arg Ala Leu Pro 575 Tyr Pro Ser Gly Ala 320 Arg Phe Tyr Lys Asp 400 Lys Phe Glu His Ser 480 Pro Pro Ser Tyr Arg 560 Ile Arg Lys a. a a a a a Ser Tyr 610 Asp Asn Val Met His Val Gly Leu Arg 615 Val Thr Phe Lys Document2 Trp Gln Arg Gly Asn 625 Thr Arg Leu Gly Asp 705 Val His Leu Lys Leu 785 Gly Val Asn Pro Glu 865 50 Ala Cys Phe Lys Val 690 Glu Ile Glu Thr Leu 770 Gly Glu Ile Phe Glu 850 Ser Phe lie Gin Ile 675 Glu Gly Arg His Ser 755 Lys Thr Gly Glu Gln 835 Arg Asp Val Ser Pro 660 Phe Leu Thr Leu Gly 740 Ser Asn Ala His Met 820 Ile Leu Pro Lys Val 645 Asn Glu His Leu Tyr 725 Ile Gly Asn Ala Gly 805 Val Met Ser Lys Val 885 Lys lie Gly Glu Gly 630 Asp Thr Gly Glu Leu 650 Asp His Lys Thr Ile 665 Gly Ile Lys His Pro 680 Arg Glu Glu Met Tyr 695 Glu Glu Val Ser Arg 710 Thr Lys Gln Ile Thr 730 Val His Arg Asp Ile 745 Leu Ile Lys Leu Gly 760 Ala Gln Thr Met Pro 775 Tyr Met Ala Pro Glu 790 Arg Ala Ala Asp Ile 810 Thr Gly Lys Arg Pro 825 Tyr Lys Val Gly Met 840 Pro Glu Gly Lys Ala 855 Ile Arg Trp Thr Ala 870 Cys Thr Asp Glu Glu 890 Gln 635 Met Lys Asn Ile Leu 715 Val Lys Asp Gly Val 795 Trp Trp Gly Phe Ser 875 Tyr Ala Glu Leu Phe 700 Gly Ala Gly Phe Glu 780 Ile Ser His His Leu 860 Gln Gly Met Thr Val 685 Met Leu Ile Ala Gly 765 Val Thr Leu Glu Lys 845 Ser Leu Lys Lys Ala 670 Arg Glu Gin Asn Asn 750 Cys Asn Arg Gly Tyr 830 Pro His Leu Val Glu 655 Asp Tyr Tyr Glu Val 735 Ile Ser Ser Ala Cys 815 Glu Pro Cys Asp Tyr 640 Ile Glu Phe Cys His 720 Leu Phe Val Thr Lys 800 Val His Ile Leu His Rsn 0 00.

INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 4574 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS Document2 LOCATION: 355. .4095 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AAGAAGAAGG ACAGGGAGCA GAGGGGACAA GAAAACACGG TCGAACGAAC TGATCTGGTT AGAACTOCAG GCCTGGCACG CAGGACCTCT TTCTCTACAC AGCCCGCCAG GCCATCCCAG ACCTTCAAAG TTAACTACOG GAGCATTOCC TTCTCCAOCA COOTTOOTAC AAGGCCAOTO CAGAACCCCT CAGGAOACAA TACCACOAC ACCTCCAOCO CCAOAGOOTC TCGTTTOAOC CT OCTTITCTG

CGGOCCOCAC

ACATCATCAA

ATOOAOCCOG

ACCO TOT OG

GTTCAACCGA

CAT CAATOAC

TGAOATCCTC

TTTCAACGGG

CT OCT CATCO 120 180 240 300 AGGTGAAGCG GATA ATO Met 1 GAG CTO CTO GAO TAC ATO GAG GCA Olu Leu Leu Olu Tyr Met Olu Ala TAC CCA TCC TTG Tyr Pro Ser Leu CAC AAG GAO Gin Lys Asp CAG 000 CTC Gin Ala Leu TAT O1V\ CGG TAO Tyr Olu Arg Tyr

GCC

Ala

AAC

Asn AAG GAC TIT Lys Asp Phe CAC OCT OTG Gin Ala Leu GAC AGA OTO Asp Arg Val AAT CAG AAO Asn Gin Lys TOO OTO TOO Cys Leu Trp ATO ACO AAA Tle Thr Lys 35 OTO CG Leu Arg

OAT

Asp

ITO

Phe ATO ATO 000 Ile Met Oly OTO 000 ATO Leu Gly Ile OTA TOA GAO Leu Ser Asp 000 Gly AT T Ile TOO OCA OTO Trp Pro Val

AAA

Lys

GAG

Glu ATO CCC TOO Ile Pro Ser

OCT

Pro 75

GAG

Glu COO TOO AAO Pro Ser Lys 000 TAO Oly Tyr a..

a a a.

S

a *5 a

S

a.

S

a a a a.

a a a a

S

a.

GAO OCA GAO Olu Pro Glu GAO AGO 000 45 Glu Ser Oly 100 OTO OCA GAG Vol Pro Glu 115

GAO

Asp 85

ACO

Thr OTO GAO GAO Val Glu Asp

AG

Thr 90

GAG

Olu OTT GAG OTO Val Olu Leu GAO GAO AGT Olu Glu Ser GAG OCA ACC Olu Pro Thr

CCC

Pro 110

GAO

Asp AGO GAG 010 Arg 0Th Leu AOT COG AGO Ser Pro Arg AOT COO TOO Ser Arg Ser 693 OTO AGO OTO Leu Arg Leu

TOO

Ser 120 GAO ACC ATO Asp Thr Ile

ITO

Leu 125

CAG

Gbn 55 130

TOO

Ser 000 TOO OTO TOO Gly Cys Vol Ser

AGO

Arg 135

ACA

Thr AAG 010 GAG AGO Lys Lou Olu Arg

OTO

Leu 140

CT

Pro GAO TOA GAG GAA Glu Ser Glu Olu ATA 000 TG Ile Gly Trp 000 Oly 150 000 GAO TOT Ala Asp Cys 000 Gly 155

OTO

Val GAA 000 AGO Olu Ala Ser AGO OAT Arg His 160 AAG CAA Lys Gbn TOT TO ACT Cys Leu Thr ATO TAT AGA OCA le Tyr Arq Pro GAO AAA OCA Asp Lys Ala

OTO

Leu 175 ATO 000 CTA AGA AAO TTA ATT TTA OGA OTT CAT AAO OTT ATO AAI 000 933 D..ument2 Met Gly Leu 180 Arg Lys Leu Ile Arg Leu His Lys Leu 190 Met Asn Gly TCC TTG Ser Leu 195 CAA AGA GCT CGT Gin Arg Ala Arg

GTA

Val 200 GCT CTG GTG AAG Ala Leu Val Lys

GAC

Asp 205 GAC CGT CCA GTG Asp Arg Pro Val

GAG

Glu 210 TTC TCT GAC TTT Phe Ser Asp Phe

CCA

Pro 215 GGT CCC ATG TGG Gly Pro Met Trp

GGC

Gly 220 TCG GAT TAT GTG Ser Asp Tyr Val

CAG

Gln 225 TTG TCG GGA ACA Leu Ser Gly Thr CCT TCC TCA GAG Pro Ser Ser Glu AAG TGT AGC GCT Lys Cys Ser Ala GTG TCC Val Ser 240 TGG GAA GAA Trp Glu Glu

CTG

Leu 245 AGA GCC ATG GAC Arg Ala Met Asp

CTG

Leu 250 CCT TCC TTT GAG Pro Ser Phe Glu CCC GCC TTC Pro Ala Phe 255 TGC CTG AAG Cys Leu Lys CTG GTG CTC Leu Val Leu 260 CTG CGG CTG Leu Arg Leu 275 TGT CGG GTC CTG Cys Arg Val Leu

CTG

Leu 265 AAC GTG ATC CAC Asn Val Ile His

GAG

Glu 270 GAA CAG AGG Glu Gin Arg GCC GGG GAG CCT Ala Gly Glu Pro CTC TTG AGT ATC Leu Leu Ser Ile

AAA

Lys 290 CAG CTA GTG CGA Gln Leu Val Arg TGT AAA GAG GTC Cys Lys Glu Val

CTA

Leu 300 AAG GGC GGG CTC Lys Gly Gly Leu ATG AAG CAG TAT Met Lys Gln Tyr

TAC

Tyr 310 CAG TTC ATG CTG Gin Phe Met Leu

CAG

Gin 315 GAG GTC CTG GGC Glu Val Leu Gly GGA CTG Gly Leu 320 GAG AAG ACC Glu Lys Thr TGC AAC ATG GAT Cys Asn Met Asp TTT GAG GAG GAC Phe Glu Glu Asp CTG CAG AAG Leu Gln Lys 335 CAA ATG CTA Gln Met Leu 1029 1077 1125 1173 1221 1269 1317 1365 1413 1461 1509 1557 1605 1653 1701 So. e: *to.

at ATG CTG ATG Met Leu Met 340 CAG CAG TTA 45 Gln Gln Leu 355 GTG TAT TTT GAT Val Tyr Phe Asp ATG AGA AGC TGG Met Arg Ser Trp CCT CAG GCT Pro Gln Ala CAT AGC TTA AAA His Ser Leu Lys

AAC

Asn 365 CTG CTA GAA GAG Leu Leu Glu Glu

GAA

Glu 370 TGG AAT TTC ACC Trp Asn Phe Thr

AAA

Lys 375 GAA ATA ACC CAT Glu Ile Thr His

TAT

Tyr 380 ATC CGT GGC GGA Ile Arg Gly Gly GCG CAG GCT GGA Ala Gln Ala Gly CTT TTC TGT GAC Leu Phe Cys Asp GCA GGG ATG CTG Ala Gly Met Leu CTG AAA Leu Lys 400 TCC ACA GGG Ser Thr Gly TTT CTG GAA TCC GGC CTG CAG GAG AGC Phe Leu Glu Ser Gly Leu Gln Glu Ser TGT GCT GAG Cys Ala Glu 415 CTA AGG AGA Leu Arg Arg CTG TGG ACC Leu Trp Thr 420 AGC GCC GAC GAC Ser Ala Asp Asp GGT GCT GCC GAC Gly Ala Ala Asp TCT GTC ATC GAG ATC AGC CGA GCA CTC AAG GAG CTC TTC CAC GAA GCC Ser Val Ile Glu Ile Ser Arg Ala Leu Lys Glu Leu Phe His Glu Ala Document2 445 AAA ATG CTG AGG Lys Met Leu Arg

AGG

Arg 450 GAA AGA GCC TCC Glu Arg Ala Ser

AAG

Lys 455 GCC CTG GGC TTT Ala Leu Gly Phe

GCT

Ala 460 GAC CTA GAA ATA Asp Leu Glu Ile GCA GAG TTC GTG Ala Glu Phe Val TCT GCA TCA GCC Ser Ala Ser Ala CGA GAG Arg Glu 480 CTC CTG GAC Leu Leu Asp

GCT

Ala 485 CTG AAA GCA AAG Leu Lys Ala Lys

CAG

Gln 490 TAT GTT AAG GTA Tyr Val Lys Val CAG ATT CCC Gin Ile Pro 495 GCT GAG GAG Ala Glu Glu GGG TTA GAG Gly Leu Glu 500 AAG AAA ATT Lys Lys Ile 515 AAT TTG CAC GTG Asn Leu His Val GTC CCC GAC AGC Val Pro Asp Ser

CTC

Leu 510 ATT TTG CAG Ile Leu Gin CTC AAT GCT GCC Leu Asn Ala Ala GGA AAG GAC TGC Gly Lys Asp Cys

TCA

Ser 530 AAG GAT CCA GAC Lys Asp Pro Asp

GAC

Asp 535 GTC TTC ATG GAT Val Phe Met Asp

GCC

Ala 540 TTC CTG CTC CTG Phe Leu Leu Leu AAG CAT GGG GAC Lys His Gly Asp CGA GCC CGT GAC TCA GAA Arg Ala Arg Asp Ser Glu 550 555 GAT GGC TGG GGC Asp Gly Trp Gly ACA TGG Thr Trp 560 GAA GCT CGG Glu Ala Arg GTC AAA ATT GTG Val Lys Ile Val CAG GTG GAG ACT Gin Val Glu Thr GTG GAC ACC Val Asp Thr 575 ATG GAG TCT Met Glu Ser 1749 1797 1845 1893 1941 1989 2037 2085 2133 2181 2229 2277 2325 2373 2421 2469 CTG AGA AGC Leu Arg Ser 580 GCT CAC CTC 40 Ala His Leu 595 ATG CAG GTG GAC Met Gin Val Asp

AAC

Asn 585 CTT CTG CTG GTT Leu Leu Leu Val

GTC

Val 590 GTA CTT CAG Val Leu Gin

S.

S..

S.

V..

V

4 Sq*

S

V

V.

S

.4

S

V S

S*

S

.9 4.

*4

V.

9*

S

.9

V

*9

AGA

Arg 600 AAA GCC TTC CAG Lys Ala Phe Gin

CAG

Gin 605 TCC ATT GAG GGG Ser Ile Glu Gly

CTG

Leu 45 610 ATG ACT GTA CGC Met Thr Val Arg GAG CAG ACA TCT Glu Gin Thr Ser CAG CCC ATC ATC Gin Pro Ile Ile

GCC

Ala 625 AAA GGT TTG CAG Lys Gly Leu Gin CTC AAG AAC GAT Leu Lys Asn Asp CTT GAG CTA TGC Leu Glu Leu Cys AAC AGA Asn Arg 640 ATC AGC GAT Ile Ser Asp 55 GAT GCT GAG Asp Ala Glu 660 CGA GAA GCC 60 Arg Glu Ala 675

GCC

Ala 645 ATC GAC CGT GTG Ile Asp Arg Val

GAC

Asp 650 CAC ATG TTC ACC CTG GAG TTC His Met Phe Thr Leu Glu Phe GTC GAG GAG TCT Val Glu Glu Ser

GAG

Glu 665 TCG GCC ACG CTG Ser Ala Thr Leu

CAG

Gin 670 CAG TAC TAC Gin Tyr Tyr ATG ATT CAG Met Ile Gln

GGC

Gly 680 TAC AAC TTT GGG Tyr Asn Phe Gly GAG TAT CAT AAA Glu Tyr His Lys

GAA

Glu 690 GTT GTT CGT TTG Val Val Arg Leu TCT GGG GAA TTC Ser Gly Glu Phe CAG AAG ATA GGA Gin Lys Ile Gly

GAC

Asp 705 Document2

AAA

Lys

TGC

Cys

GAT

Asp

GAT

Asp

GTG

Val1 770 Gee Pro

CAC

His

ICC

Ser

CGT

Arg

GAC

Asp 40 850

TCC

Ser

GAG

Gin

GAG

Glu

TGG

Trp

GAG

Gin 930

TAT

Tyr

GAG

Glu

TTC

Phe

GAG

Asp 755

ATA

Ilie

CG

Ar g

CTG

Leu

GAG

Asp

GGG

Al a 835

TGT

Ser

AGT

Ser

TTG

Phe

GGA

Pro

GAA

Glu 915

GGG

Giy

ATA

Ile

AGG

Ser

GTA

Leu 740

TTG

Phe

GGA

Gly

GGT

Pro

ATG

Ile

GGI

Aia 820

GCC

Ala

GIG

Val

GIG

Vai

AGG

Arg

GG

Al a 900

GTT

Leu

GGG

Pro

AGG

Ser

GGG

Giy 725

GAA

Gin

TTG

Leu

AAG

Lys

GTG

Val

ATG

Tie 805

CGG

Arg

GGG

Aia

GGA

Pro

GGT

Pro

TGT

Ser 885

TAT

Tyr

GGA

Arg

ATA

Ile TTG GGG Phe Ala 710 AGA GGG Arg Gly GGG ATT Aia Ile AGT TTG Per Leu GGA GAG Pro His 775 AAG GTG Lys Val 790 GGG AGT Pro Thr AGG GAT Thr His AGG AGT Thr Thr GGG AAA Ala Lys 855 GAA AAG Glu Asn 870 GTG AGT Leo Ser GGT GG Pro Arg AGA GIG Thr Leu GAA GGT Glu Aia 935

GAG

Gin

AGA

Thr

GAA

Gio

GAA

Gin 760

AGG

Ser

GGG

Pro

GGA

Pro

GGG

Gly

GGT

Al a 840

CT

Pro

GAG

Asp

CGG

Arg

AGT

Ser AT G Ile 920

ATG

Tie AAG TGG Lys Trp AGA GGG Arg Pro 730 GGT GGG Pro Ala 745 GGG CG Ala Leo GGT GIG Pro Val GGA TGG Arg Gys GAG GGA Gbu Gly 810 AAG IGI Aso Ser 825 GGT GGG Ala Gly GIG AAG Vai Aso GG TIG Arg Leo GAG IGA His Ser 890 GAG IGA Asp Ser 905 AGG GAG Per Gin GAG AAG Gin Lys AT G Met 715

AGA

Arg

TTI

Phe

AIG

Met

AGA

Ihr

GAG

His 795

TTG

Phe

GTT

Val1

GG

Arg

AGT

lhr

GGG

Aia 875

AGG

Per

AGT

Ser

AGG

Ihr

IGA

Ser AAI TAG GIG GIG AGG AAA Asn Tyr Val Leo Ihr Lys 720

TGG

I rp All ile

AAI

Asn GG I Al a 780

AGI

Ser

AGC

Ser

GGI

Aila

GGI

Pro

GGG

Aila 860

TGG

Ser

GGG

Pro

GGA

Giy

AAA

Lys

GIG

Val1 940 GGG AGG Ala Ihr IGA GGT Per Ala 750 GAG IGG Gbu Gys 765 AIG CAT Ile His GAG GGI Asp Pro AGG CGG Thr Arg GGI GGI Ala Ala 830 GGG GGA Gly Pro 845 GGI GAT Pro Asp AlA GGI le Aia AGG GAA lhr Gbu IGA AGT Ser Thr 910 GAG IG Asp Per 925 GGA CG Arg Leo

GAA

Gin 735

TIA

Leo AT G le

CGG

Arg

GGT

Pro

AGG

Per 815

GGI

Al a

GGI

Gly

ACC

Thr

GGA

Al a

GAG

Glu 895

CGG

Ar g

GGG

Al a

III

Phe

GGA

Gly

GGA

Pro

GGG

Gly

AAG

Asn

AAG

Asn 800

GIG

Vai

GGT

Ala

GGI

Gly

AGG

Arg

GAA

Glu 880

GGA

Arq

AGA

Arg

TGI

Per

GAA

Glu

III

Phe GAlA Glu

GAG

His

AGG

Per 785

GGT

Pro

GGT

Pro GTl Val

GGT

Gly

GGT

Gly 865

CG

Leo

GAG

Asp

AGG

Per

AAG

Lys

GAG

Gbu 945 2517 2565 2613 2661 2709 2757 2805 2853 2901 2949 2997 3045 3093 3141 3189 3237 fee 0.9.

0..0 AGG AGG TAT GGA GAG ATG AGG AGA AAG AAT AIG ATG GGG GAlA GIG IGG Arg Arg Iyr Arg Glu Met Arg Arq Lys Asn Ile ile Gly Gin Vai Gys C950 955 960 Docurnent2 139 GAT ACC CCT AAG TCC TAT GAT AAC GTC ATG CAT GTT GGA CTG AGG AAG 3285 Asp Thr Pro Lys Ser Tyr Asp Asn Val Met His Val Gly Leu Arg Lys 965 970 975 GTG ACA TTT AAG TGG CAA AGA GGA AAC AAA ATT GGA GAA GGA CAG TAT 3333 Val Thr Phe Lys Trp Gin Arg Gly Asn Lys Ile Gly Glu Gly Gin Tyr 980 985 990 GGA AAA GTA TAC ACC TGC ATC AGT GTT GAC ACA GGG GAG CTG ATG GCC 3381 Gly Lys Val Tyr Thr Cys Ile Ser Val Asp Thr Gly Glu Leu Met Ala 995 1000 1005 ATG AAG GAG ATT CGA TTT CAG CCT AAC GAC CAC AAG ACT ATC AAG GAG 3429 Met Lys Glu Ile Arg Phe Gin Pro Asn Asp His Lys Thr Ile Lys Glu 1010 1015 1020 1025 ACT GCA GAC GAG TTG AAA ATA TTT GAA GGC ATC AAG CAC CCC AAC CTG 3477 Thr Ala Asp Glu Leu Lys Ile Phe Glu Gly Ile Lys His Pro Asn Leu 1030 1035 1040 GTC CGG TAT TTT GGC GTG GAG CTT CAC AGG GAA GAG ATG TAC ATC TTC 3525 Val Arg Tyr Phe Gly Val Glu Leu His Arg Glu Glu Met Tyr Ile Phe 1045 1050 1055 ATG GAG TAC TGT GAT GAG GGT ACA CTA GAG GAG GTG TCA CGA CTG GGC 3573 Met Glu Tyr Cys Asp Glu Gly Thr Leu Glu Glu Val Ser Arg Leu Gly 1060 1065 1070 CTG CAG GAG CAC GTC ATC AGG TTA TAT ACC AAG CAG ATC ACT GTC GCC 3621 Leu Gin Glu His Val Ile Arg Leu Tyr Thr Lys Gin Ile Thr Val Ala 1075 1080 1085 ATC AAC GTC CTC CAT GAG CAC GGC ATC GTT CAC CGA GAC ATC AAA GGT 3669 Ile Asn Val Leu His Glu His Gly Ile Val His Arg Asp Ile Lys Gly 1090 1095 1100 1105 GCC AAT ATC TTC CTT ACG TCA TCT GGA CTA ATC AAG CTG GGA GAT TTT 3717 Ala Asn Ile Phe Leu Thr Ser Ser Gly Leu Ile Lys Leu Gly Asp Phe 1110 1115 1120 GGA TGC TCT GTA AAA CTT AAA AAC AAC GCC CAG ACC ATG CCC GGA GAG 3765 Gly Cys Ser Val Lys Leu Lys Asn Asn Ala Gin Thr Met Pro Gly Glu 1125 1130 1135 45 GTG AAC AGC ACC CTA GGG ACA GCA GCT TAC ATG GCC CCT GAA GTT ATT 3813 Val Asn Ser Thr Leu Gly Thr Ala Ala Tyr Met Ala Pro Glu Val Ile 1140 1145 1150 ACC CGA GCC AAA GGA GAA GGC CAC GGA CGT GCG GCA GAT ATC TGG AGT 3861 *o 50 Thr Arg Ala Lys Gly Glu Gly His Gly Arg Ala Ala Asp Ile Trp Ser G* 1155 1160 1165 o. CTG GGG TGC GTC GTC ATA GAG ATG GTG ACT GGC AAG CGG CCT TGG CAT 3909 Leu Gly Cys Val Val Ile Glu Met Val Thr Gly Lys Arg Pro Trp His 55 1170 1175 1180 1185 GAG TAT GAA CAC AAC TTT CAG ATT ATG TAC AAG GTG GGG ATG GGA CAC 3957 Glu Tyr Glu His Asn Phe Gin Ile Met Tyr Lys Val Gly Met Gly His 1190 1195 1200 AAG CCA CCA ATC CCG GAA AGG CTA AGC CCT GAA GGA AAG GCC TTT CTC 4005 Lys Pro Pro Ile Pro Glu Arg Leu Ser Pro Glu Gly Lys Ala Phe Leu S1205 1210 1215 6RAC z 65 TCG CAC TGC CTG GAA AGT GAC CCG AAG ATA CGG TGG ACA GCC AGC CAG 4053 Docunment2 Ser His Cys Leu Glu Ser Asp Pro Lys Ile Arg 1220 1225 CTC CTC GAC CAC GCT TTT GTC AAG GTT TGC ACA Leu Leu Asp His Ala Phe Val Lys Val Cys Thr Trp Thr Ala Ser Gin 1230 GAT GAA GAG Asp Glu Giu 1245 1235 1240

TGAACTCAAC

TGTAATATTT

CTACAGGGGT

GGTTAAAGGA

CT CCGACCCT

GGGGTTCAGA

TCGTTGCCTC

CAGTCCGTGG

ACATAAAGAC

GACAAGCCTC

GCCGCACGTT

CTCGTGACTG

AGAACATGTA

TGACTGTGGA

AGCTCAAGAA

CC TAG TAGT G

TGCAGCGCAG

ACT TCTGCTG

AAGTGCCATT

ACAACCAACC

CT CT TCCC CC

GCCTCCTTGT

AAGGTTCTTT

TGTGGACAGA

GCGGCCTTCC

CT CCTOT CC

ACTACTGTAC

GTGTCATCAG

CTGTC COCOA

TCGAAAGCTG

TTCAATAAAT

ATCCCGTGAT

TAACCTCCCA

CTGCTOAGTG

ACOGCCACCG

CACAGTGTTT

COTTTATTTC

CAGGTTTOTT

GGTTTATTTT

CACTACTGTA

GGACTGAAGA

ACAGTCCTGA

CCTCTOTCCC

TTGAGCTCCT

AACCTCCTGG

ATGCAAAGGC

AGGAAAGCGA

4095 4155 4215 4275 4335 4395 4455 4515 4575 4592

TCGTAACTGA

AAAAAAAAAA AAAAAAA INFORMATION FOR SEQ ID NO:l0: SEQUENCE CHARACTERISTICS: LENGTH: 1247 amino acids TYPE: amino acid TOPOLOGY: linear (ii) (xi) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:i0: a a a a a a.

Met 1 Giu Leu Leu Olu Tyr Met Glu Ala Tyr Pro Ser Leu Gin Ala Leu Gin Lys Val Gin Ala 35 Asp 20 Tyr Olu Arg Tyr Ala Lys Asp Phe Oiu Asp Arg Leu Asn Gin Leu Cys Leu Trp Leu Asn Ile Thr Lys Lys Leu Arg Ile Met Gly Val Leu Oly Ile Phe Leu Ser Asp Ile Tyr Gly Trp Pro Val Lys 70 Ile Pro Ser Pro 75 Arg Pro Ser Lys Oly Glu Oiu Pro Olu Giu Val Oiu Asp Olu Val Oiu Lou Arg Leu Oiu Ser Arg Val Pro 115 Ser Gin Gly 130 Oly 100 Oiu Giu Ser Oiu Pro Thr Giu Leu Arg Leu Ser 120 Lys Thr Asp Thr le Leu 125 Giu Pro Ser Pro 110 Asp Ser Arg Ser Giu Oiu Cys Val Ser Arg 135 Leu Oiu Arg Asp Ser Ile Oly Trp Oly Thr Ala Asp Cys Oly Pro Oiu Ala Ser Arg DOCLnMent2 a 4 141 145 150 155 160 His Cys Leu Thr Ser Ile Tyr Arg Pro Phe Val Asp Lys Ala Leu Lys 165 170 175 Gin Met Gly Leu Arg Lys Leu Ile Leu Arg Leu His Lys Leu Met Asn 180 185 190 Gly Ser Leu Gln Arg Ala Arg Val Ala Leu Val Lys Asp Asp Arg Pro 195 200 205 Val Glu Phe Ser Asp Phe Pro Gly Pro Met Trp Gly Ser Asp Tyr Val 210 215 220 Gln Leu Ser Gly Thr Pro Pro Ser Ser Glu Gln Lys Cys Ser Ala Val 225 230 235 240 Ser Trp Glu Glu Leu Arg Ala Met Asp Leu Pro Ser Phe Glu Pro Ala 245 250 255 Phe Leu Val Leu Cys Arg Val Leu Leu Asn Val Ile His Glu Cys Leu 260 265 270 Lys Leu Arg Leu Glu Gln Arg Pro Ala Gly Glu Pro Ser Leu Leu Ser 275 280 285 Ile Lys Gln Leu Val Arg Glu Cys Lys Glu Val Leu Lys Gly Gly Leu 290 295 300 Leu Met Lys Gln Tyr Tyr Gln Phe Met Leu Gln Glu Val Leu Gly Gly 305 310 315 320 Leu Glu Lys Thr Asp Cys Asn Met Asp Ala Phe Glu Glu Asp Leu Gln 325 330 335 Lys Met Leu Met Val Tyr Phe Asp Tyr Met Arg Ser Trp Ile Gln Met 340 345 350 Leu Gin Gln Leu Pro Gln Ala Ser His Ser Leu Lys Asn Leu Leu Glu 40 355 360 365 Glu Glu Trp Asn Phe Thr Lys Glu Ile Thr His Tyr Ile Arg Gly Gly 370 375 380 45 Glu Ala Gln Ala Gly Lys Leu Phe Cys Asp Ile Ala Gly Met Leu Leu 385 390 395 400 Lys Ser Thr Gly Ser Phe Leu Glu Ser Gly Leu Gln Glu Ser Cys Ala 405 410 415 Glu Leu Trp Thr Ser Ala Asp Asp Asn Gly Ala Ala Asp Glu Leu Arg 420 425 430 Arg Ser Val Ile Glu Ile Ser Arg Ala Leu Lys Glu Leu Phe His Glu 55 435 440 445 S* Ala Arg Glu Arg Ala Ser Lys Ala Leu Gly Phe Ala Lys Met Leu Arg 450 455 460 Lys Asp Leu Glu Ile Ala Ala Glu Phe Val Leu Ser Ala Ser Ala Arg 465 470 475 480 Glu Leu Leu Asp Ala Leu Lys Ala Lys Gln Tyr Val Lys Val Gln Ile 485 490 495 j q Pro Gly Glu Lys Cys Ser 530 Thr Lys 545 Trp Glu Thr Leu Ser Ala Gly Leu 610 Ala Lys 625 Arg Ile Phe Asp Tyr Arg Lys Glu 690 Asp Lys 705 Lys Cys Phe Asp Glu Asp His Val 770 Ser Pro 785 Pro His Pro Ser Leu Glu Asn Leu His Val Phe Val Pro Asp Ser Leu Ala Glu 500 Lys Ile 515 Lys Asp His Gly Ala Arg Arg Ser 580 His Leu 595 Met Thr Gly Leu Ser Asp Ala Glu 660 Glu Ala 675 Val Val Tyr Tle Glu Ser Phe Leu 740 Asp Phe 755 Ile Gly Arg Pro Leu Ile Asp Ala Ile Leu Pro Asp Asp Arg 550 Ala Val 565 Met Gin Val Leu Val Arg Gin Gin 630 Ala Ile 645 Val Glu Met Ile Arg Leu Ser Phe 710 Gly Arg 725 Gin Ala Leu Ser Lys Pro Val Lys 790 Ile Pro 805 Arg Thr Gin Asp 535 Ala Lys Val Gin His 615 Leu Asp Glu Gin Met 695 Ala Gly Ile Leu His 775 Val Thr His Leu 520 Val Arg Ile Asp Arg 600 Glu Lys Arg Ser Gly 680 Ser Gin Thr Glu Gin 760 Ser Pro Pro Gly 505 Leu Phe Asp Val Asn 585 Lys Gin Asn Val Glu 665 Tyr Gly Lys Arg Pro 745 Ala Pro Arg Glu Asn Asn Ala Met Asp Ser Glu 555 Pro Gin 570 Leu Leu Ala Phe Thr Ser Asp Ala 635 Asp His 650 Ser Ala Asn Phe Glu Phe Trp Met 715 Pro Arg 730 Ala Phe Leu Met Val Thr Cys His 795 Gly Phe 810 Ser Val Ala Ala 540 Asp Val Leu Gin Ser 620 Leu Met Thr Gly Arg 700 Asn Trp Ile Asn Ala 780 Ser Ser Al a Thr 525 Phe Gly Glu Val Gin 605 Gin Glu Phe Leu Phe 685 Gin Tyr Ala Ser Glu 765 Ile Asp Thr Ala 510 Gly Leu Trp Thr Val 590 Ser Pro Leu Thr Gin 670 Glu Lys Val Thr Ala 750 Cys His Pro Arg Ala Lys Leu Gly Val 575 Met Ile Ile Cys Leu 655 Gin Tyr Ile Leu Gin 735 Leu Ile Arg Pro Ser 815 Ala Asp Leu Thr 560 Asp Glu Glu Ile Asn 640 Glu Tyr His Gly Thr 720 Gly Pro Gly Asn Asn 800 Val Ala 0.

0 0 00 0 00 820 825 830 Val Arg Ala Ala Ala Thr Thr Ala Ala Gly Arg Pro Gly Pro Gly Gly Document2 143 835 840 845 Gly Asp Ser Val Pro Ala Lys Pro Val Asn Thr Ala Pro Asp Thr Arg 850 855 860 Gly Ser Ser Val Pro Glu Asn Asp Arg Leu Ala Ser Ile Ala Ala Glu 865 870 875 880 Leu Gln Phe Arg Ser Leu Ser Arg His Ser Ser Pro Thr Glu Glu Arg 885 890 895 Asp Glu Pro Ala Tyr Pro Arg Ser Asp Ser Ser Gly Ser Thr Arg Arg 900 905 910 Ser Trp Glu Leu Arg Thr Leu Ile Ser Gln Thr Lys Asp Ser Ala Ser 915 920 925 Lys Gln Gly Pro Ile Glu Ala Ile Gln Lys Ser Val Arg Leu Phe Glu 930 935 940 Glu Arg Arg Tyr Arg Glu Met Arg Arg Lys Asn Ile Ile Gly Gln Val 945 950 955 960 Cys Asp Thr Pro Lys Ser Tyr Asp Asn Val Met His Val Gly Leu Arg 965 970 975 Lys Val Thr Phe Lys Trp Gln Arg Gly Asn Lys Ile Gly Glu Gly Gin 980 985 990 Tyr Gly Lys Val Tyr Thr Cys Ile Ser Val Asp Thr Gly Glu Leu Met 995 1000 1005 Ala Met Lys Glu Ile Arg Phe Gin Pro Asn Asp His Lys Thr Ile Lys 1010 1015 1020 Glu Thr Ala Asp Glu Leu Lys Ile Phe Glu Gly Ile Lys His Pro Asn 1025 1030 1035 1040 Leu Val Arg Tyr Phe Gly Val Glu Leu His Arg Glu Glu Met Tyr lie 40 1045 1050 1055 Phe Met Glu Tyr Cys Asp Glu Gly Thr Leu Glu Glu Val Ser Arg Leu 1060 1065 1070 45 Gly Leu Gln Glu His Val Ile Arg Leu Tyr Thr Lys Gln Ile Thr Val 1075 1080 1085 Ala Ile Asn Val Leu His Glu His Gly Ile Val His Arg Asp Ile Lys 1090 1095 1100 Gly Ala Asn Ile Phe Leu Thr Ser Ser Gly Leu Ile Lys Leu Gly Asp 1105 1110 1115 1120 Phe Gly Cys Ser Val Lys Leu Lys Asn Asn Ala Gln Thr Met Pro Gly 1125 1130 1135 Glu Val Asn Ser Thr Leu Gly Thr Ala Ala Tyr Met Ala Pro Glu Val 1140 1145 1150 Ile Thr Arg Ala Lys Gly Glu Gly His Gly Arg Ala Ala Asp Ile Trp 1155 1160 1165 Ser Leu Gly Cys Val Val Ile Glu Met Val Thr Gly Lys Arg Pro Trp 1170 1175 1180 Document2 4 f 144 His Glu Tyr Glu His Asn Phe Gin Ile Met Tyr Lys Val Gly Met Gly 1185 1190 1195 1200 His Lys Pro Pro Ile Pro Glu Arg Leu Ser Pro Glu Gly Lys Ala Phe 1205 1210 1215 Leu Ser His Cys Leu Glu Ser Asp Pro Lys Ile Arg Trp Thr Ala Ser 1220 1225 1230 Gln Leu Leu Asp His Ala Phe Val Lys Val Cys Thr Asp Glu Glu 1235 1240 1245 e

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Document2

Claims

1. An isolated and/or recombinant protein, or at least a portion thereof, comprising an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with the complementary strand of a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID

2. An isolated nucleic acid molecule capable of hybridizing under stringent conditions with the complementary strand of a nucleic acid sequence selected from the ~group consisting of SEQ ID NO: 7, and SEQ ID NO: 9.

3. A recombinant molecule, comprising a nucleic acid molecule capable of hybridizing under stringent conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, or the complementary sequence thereof, said nucleic acid molecule being operatively linked to an expression vector.

4. A recombinant molecule, comprising a nucleic acid molecule capable of hybridizing under stringent conditions with the nucleic acid sequence of SEQ ID NO: 1, or the complementary sequence thereof, said nucleic acid molecule being operatively linked to an expression vector. A recombinant cell transformed with the recombinant molecule of either of claims 3 or 4.

6. An isolated nucleic acid molecule which selectively hybridizes under stringent conditions to an MEKK nucleic acid sequence selected from the group consisting of i 146 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, which nucleic acid molecule can specifically detect the MEKK nucleic acid sequence.

7. An isolated nucleic acid molecule which selectively hybridizes under stringent conditions to the MEKK nucleic acid sequence of SEQ ID NO: 1, which nucleic acid molecule can specifically detect the MEKK nucleic acid sequence.

8. An isolated and/or recombinant protein comprising an amino acid sequence encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions with the complementary strand of a nucleic acid sequence selected from the group consisting of SEQ ID NO:7, and SEQ ID NO: 9, which protein modulates 10 activation of map kinase kinases.

9. The protein of claim 8, which protein modulates activation of a map kinase.

10. The protein of claim 9, which protein includes a kinase domain. S11. The protein of claim 10, which protein phosphorylates a map kinase kinase. o. 12. A fusion protein comprising all or a portion of a MEKK protein, which fusion 15 protein further comprises a second polypeptide sequence having an amino acid sequence distinct from an MEKK amino acid sequence.

13. An isolated and/or recombinant protein comprising an amino acid sequence at least identical to a MEKK sequence of SEQ ID NO: 8 or SEQ ID NO:

14. The protein of claim 13, comprising an amino acid sequence at least 85% identical to a MEKK sequence of SEQ ID NO: 8 or SEQ ID NO: The protein of claim 14, comprising an amino acid sequence at least 90% identical to a MEKK sequence of SEQ ID NO: 8 or SEQ ID NO: 147

16. An isolated and/or recombinant protein encoded by a nucleic acid sequence having at least 80% homology with the nucleic aicd sequence of SEQ ID NO: 7 or SEQ ID NO: 9.

17. The protein of claim 16, which is encoded by a nucleic acid sequence having at least 85% homology with the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9.

18. The protein of claim 17, which is encoded by a nucleic acid sequence having at least 90% homology with the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO:9.

19. The nucleic acid molecule of claim 2, which nucleic acid molecule has a nucleotide sequence represented in the MEKK coding sequences selected from the group 10 consisting of SEQ ID NO: 7 and SEQ ID NO: 9. A nucleic acid vector comprising a coding sequence for a recombinant protein, S: which coding sequence includes a nucleotide sequence which hybridizes under stringent S•conditions to the complementary strand of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9.

21. A nucleic acid vector comprising a coding sequence for a recombinant protein, which coding sequence includes a nucleotide sequence which hybridizes under stringent conditions to the complementary strand of the nucleic acid sequence SEQ ID NO: 1.

22. An isolated and/or recombinant protein comprising the amino acid sequence, as set forth in SEQ ID NO: 8 or SEQ ID NO: 10, or a functional fragment thereof.

23. The protein or functional fragment of claim 22, wherein said fragment comprises a kinase catalytic domain.

24. The protein or functional fragment of claim 22, wherein said fragment comprises a regulatory domain.

148- A method for regulating homeostasis of a cell comprising introducing into said cell a nucleic acid molecule according to claim 2 or a recombinant molecule according to claim 3, which encodes a MEKK protein such that homeostasis of said cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein. 26. A method for regulating homeostasis of a cell comprising introducing into said cell a nucleic acid molecule capable of hybridizing under stringent conditions with the complementary strand of the nucleic acid of SEQ ID NO: 1 or a recombinant molecule according to claim 4, which encodes a MEKK protein such that homeostasis of said cell 10 is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein. 27. A method for regulating apoptosis of a cell comprising introducing into said cell a nucleic acid molecule according to claim 2 or a recombinant molecule according to claim 3, which encodes an MEKK protein such that apoptosis of the cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein. 28. A method for regulating apoptosis of a cell comprising introducing into said cell a nucleic acid molecule capable of hybridizing under stringent conditions with the complementary strand of the nucleic acid of SEQ ID NO: 1 or a recombinant molecule according to claim 4, which encodes an MEKK protein such that apoptosis of the cell is regulated, wherein said MEKK protein regulates the activity of a MAPK signal transduction protein. 149 29. A method for regulating mitogen activated protein kinase (MAPK) activity in a cell, comprising contacting the cell with a nucleic acid molecule according to claim 2 or a recombinant molecule according to claim 3, which encodes a MEKK protein such that MAPK activity is regulated in the cell. 30. A method for regulating mitogen activated protein kinase (MAPK) activity in a cell, comprising contacting the cell with a nucleic acid molecule capable of hybridizing under stringent conditions with the complementary strand of the nucleic acid of SEQ ID e* NO: 1 or a recombinant molecule according to claim 4, which encodes a MEKK protein S..1 such that MAPK activity is regulated in the cell. 10 31. The method of any of claims 25 to 30, wherein said MEKK protein comprises a kinase catalytic domain having an amino acid sequence at least 80% identical with the kinase catalytic domain of a MEKK protein selected from the group consisting of SEQ S• ID NO: 8, and SEQ ID NO: 32. The method of any of claims 25 to 30, wherein said MEKK protein comprises a kinase catalytic domain having an amino acid sequence at least 80% identical with the kinase catalytic domain of a MEKK protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. 33. The method of claim 31, wherein said nucleic acid molecule encodes an MEKK protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 34. The method of claim 32, wherein said nucleic acid molecule encodes an MEKK protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. I

150- The method of any of claims 25 to 30, wherein said nucleic acid molecule is capable of hybridizing under stringent conditions with a nucleic acid molecule selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. 36. The method of any of claims 25 to 30, wherein said nucleic acid molecule is capable of hybridizing under stringent conditions with a nucleic acid sequence of SEQ ID NO: 1. 37. The method of claim 35, wherein said nucleic acid molecule comprises a 10 nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 10 5, SEQ ID NO: 7, and SEQ ID NO: 9. 38. The method of claim 36, wherein said nucleic acid molecule has a nucleotide sequence of SEQ ID NO: 1. 39. The method of any of claims 25 to 32, wherein the nucleic acid molecule encodes a MEKK protein having the amino acid sequence of SEQ ID NO:2. 15 40. A method for regulating mitogen ERK kinase kinase (MEKK) protein activity in a cell comprising transforming said cell with an antisense nucleic acid molecule such that MEKK protein activity is regulated in said cell. 41. The method of any one of claims 25 to 30 or 40, wherein said cell is a mammalian cell. 42. An isolated and/or recombinant protein according to any one of claims 1, 8 to 11, to 20 or 24 to 26, and substantially as herein described with reference to any one of the examples. 151 43. An isolated nucleic acid molecule according to any one of claims 2, 6, 7, or 19 to 23 and substantially as herein described with reference to any one of the examples. 44. A recombinant molecule according to any one of claims 3 to 5 and substantially as herein described with reference to any one of the examples. 45. A nucleic acid vector according to claim 20 or claim 21 and substantially as herein described with reference to any one of the examples. 46. A fusion protein according to claim 12 and substantially as herein described with reference to any one of the examples. 47. A method of regulating homeostasis of a cell, substantially as herein described 10 with reference to any one of the examples. 48. A method of regulating apoptosis of a cell, substantially as herein described with S" reference to any one of the examples. 49. A method of regulating mitogen activated protein kinase (MAPK) activity in a cell, substantially as herein described with reference to any one of the examples. a DATED this 12th day of January 1999 NATIONAL JEWISH CENTER FOR IMMUNOLOGY AND RESPIRATORY MEDICINE Attorney: PAUL G. HARRISON Fellow Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS