WO1998049317A2 - Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase - Google Patents

Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase Download PDF

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Publication number
WO1998049317A2
WO1998049317A2 PCT/US1998/008439 US9808439W WO9849317A2 WO 1998049317 A2 WO1998049317 A2 WO 1998049317A2 US 9808439 W US9808439 W US 9808439W WO 9849317 A2 WO9849317 A2 WO 9849317A2
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Prior art keywords
seq
sad
alk
alp
ptp05
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PCT/US1998/008439
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English (en)
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WO1998049317A3 (fr
Inventor
Greg D. Plowman
Douglas Clary
Bahija Jallal
Elior Peles
Susan Onrust
Dave Markby
Sara A. Courtneidge
Harald App
Terence H. HUI
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Sugen, Inc.
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Application filed by Sugen, Inc. filed Critical Sugen, Inc.
Priority to EP98919919A priority Critical patent/EP0979288A2/fr
Priority to JP54724498A priority patent/JP2002513289A/ja
Priority to AU72600/98A priority patent/AU7260098A/en
Priority to CA002288221A priority patent/CA2288221A1/fr
Publication of WO1998049317A2 publication Critical patent/WO1998049317A2/fr
Publication of WO1998049317A3 publication Critical patent/WO1998049317A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to protein tyrosine phosphatases.
  • the invention concerns proteins we have named PTP04, SAD, PTP05, PTP10, ALP, and ALK-7, nucleotide sequences encoding these proteins, and various products and assay methods that can be used for identifying compounds useful for the diagnosis and treatment of various diseases and conditions related to these proteins, for example cell proliferative disorders.
  • Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regula- tion of the activity of mature proteins by altering their structure and function.
  • the best characterized protein kinases in eukaryotes phosphorylate proteins on the alcohol moiety of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serines and threonines, and those specific for phosphorylating tyrosines.
  • the phosphorylation state of a given substrate is also regulated by a class of proteins responsible for removal of the phosphate group added to a given substrate by a protein kinase.
  • the protein phosphatases can also be classified as being specific for either serine/threonine or tyrosine.
  • the known enzymes can be divided into two groups - receptor and non- receptor type proteins.
  • Most receptor-type protein tyrosine phosphatases (RPTPs) contain two conserved catalytic tyrosine phosphatase domains each of which encompasses a segment of 240 amino acid residues (Saito et al, Cell Growth and Diff. 2:59- 65, 1991) .
  • RPTPs can be subclassified further based upon the amino acid sequence diversity of their extracellular domains (Saito, et al, supra; Krueger, et al, Proc. Natl. Acad. Sci. USA 89:7417-7421, 1992). Alignment of primary peptide sequences of both types of known PTPases shows some sequence consensus in catalytic domains and has made it possible to identify cDNAs encoding proteins with tyrosine phosphate activity via the poiymerase chain reaction (PCR) .
  • PCR poiymerase chain reaction
  • kinases and phosphatases are involved in regulatory cascades wherein their substrates may include other kinases and phosphatases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway.
  • tyrosine kinases and/or tyrosine phosphatases plays a role in a variety of human disorders including cell proliferative disorders such as cancer, fibrotic disorders, disorders of the immune system and metabolic disorders such as diabetes.
  • the present invention concerns PTP04, SAD, PTP05, PTP10, Alp, and ALK-7 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to the polypeptides, assays utilizing the polypeptides, and methods relating to all of the foregoing.
  • a first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 polypeptide.
  • isolated in reference to nucleic acid is meant a polymer of 14, 17, 21 or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized.
  • the isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature.
  • Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment.
  • nucleic acid By the use of the term "enriched" in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2 - 5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two.
  • enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19.
  • This term distinguishes the sequence from naturally occurring enrichment events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity.
  • the claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA)..
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • cDNA synthetic substance
  • the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • the term is also chosen to distinguish clones already in existence which may encode PTP04, SAD, PTP05, PTP10, ALP, or Alk-7 but which have not been isolated from other clones in a library of clones.
  • the term covers clones encoding PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 which are isolated from other non-PTP04, non-SAD, non-PTP05, non-PTPIO, non-ALP, or non-ALK-7 clones.
  • a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence.
  • the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, or SEQ ID NO: 8, a nucleic acid sequence that hybridizes to the nucleic acid sequence set forth in SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a functional derivative (as defined below) of either.
  • the nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester or other method or by using an automated DNA synthesizer.
  • hybridize refers to a method of interacting a nucleic acid sequence with a DNA or RNA molecule in solution or on a solid support, such as cellulose or nitrocellulose. If a nucleic acid sequence binds to the DNA or RNA molecule with high affinity, it is said to "hybridize” to the DNA or RNA molecule.
  • the strength of the interaction between the probing sequence and its target can be assessed by varying the stringency of the hybridization conditions. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations.
  • high stringency conditions hybridization at 50-65 °C, 5X SSPC, 50% formamide, wash at 50- 65 °C, 0.5X SSPC
  • Low stringency conditions hybridization at 35-37 °C, 5X SSPC, 40-45% formamide, wash at 42 °C SSPC
  • sequences having regions which are greater than 35-45% complementarity will hybridize to the probe.
  • nucleic acid is an isolated conserved or unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, or for the design of PCR probes to facilitate cloning of additional polypeptides.
  • conserved nucleic acid regions regions present on two or more nucleic acids encoding a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions.
  • lower stringency conditions suitable for screening for nucleic acids encoding PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptides are provided in Abe, et al. J. Biol. Chem. 19:13361 (1992).
  • conserved regions differ by no more than 5 out of 20 continguous nucleotides.
  • unique nucleic acid region it is meant a sequence present in a full length nucleic acid coding for a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide that is not present in a sequence coding for any other known naturally occurring polypeptide.
  • Such regions preferably comprise 14, 17, 21 or more contiguous nucleotides present in the full length nucleic acid encoding a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide.
  • a unique nucleic acid region is preferably of human origin.
  • the invention also features a nucleic acid probe for the detection of a nucleic acid encoding a PTP04, a SAD, a. PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide in a sample.
  • the nucleic acid probe contains nucleic acid that will hybridize specifically to a sequence of at least 14, preferably 17, 20 or 22, continguous nucleotides set forth in SEQ ID NO:l or a functional derivative thereof.
  • the probe is preferably at least 14, 17 or more bases in length and selected to hybridize specifically to a unique region of a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 endocing nucleic acid.
  • the nucleic acid probe hybridizes to nucleic acid encoding at least 14 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a functional derivative thereof.
  • Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under highly stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
  • Methods for using the probes include detecting the presence or amount of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 RNA.
  • the nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed. , 1992)). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
  • the invention also features recombinant nucleic acid, preferably in a cell or an organism.
  • the recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell.
  • the recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complimentary to an RNA sequence encoding a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide and a transcriptional termination region functional in a cell.
  • Another aspect of the invention features an isolated, enriched, or purified PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide.
  • PTP04 polypeptide it is meant an amino acid sequence substantially similar to the sequence shown in SEQ ID NO: 9, or fragments thereof.
  • SAD polypeptide it is meant an amino acid sequence substantially similar to the sequence shown in SEQ ID NO: 10, or fragments thereof.
  • PPP05 polypeptide or “PTPIO polypeptide” it is meant an amino acid sequence substantially similar to the sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or fragments thereof.
  • ALP polypeptide it is meant an amino acid sequence substantially similar to the sequence shown in SEQ ID NO: 15, or fragments thereof.
  • ALK-7 polypeptide an amino acid sequence substantially similar to the sequence shown in SEQ ID NO: 16, or fragments thereof. Two substantially similar sequences will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to each other.
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
  • isolated in reference to a polypeptide is meant a polymer of 6, 12, 18 or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized.
  • the isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature.
  • Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90 - 95% pure at least) of material naturally associated with it.
  • enriched in reference to a polypeptide it is meant that the specific amino acid sequence constitutes a significantly higher fraction (2 - 5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two.
  • enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more.
  • the term also does not imply that there is no amino acid from other sources.
  • the other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • an amino acid sequence be in purified form.
  • purified in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e . g. , in terms of mg/mL).
  • Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • the substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
  • the invention features an isolated, enriched, or purified PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide fragment.
  • a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide fragment it is meant an amino acid sequence that is less than the full-length PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 amino acid sequence shown in SEQ ID NO: 2.
  • fragments include PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 domains, PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 mutants and PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific epitopes.
  • a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 domain it is meant a portion of the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide having homology to amino acid sequences from one or more known proteins wherein the sequence predicts some common function, interaction or activity.
  • domains are the SH2 (Src Homology 2) domain (Sadowski, et al, Mol. Cell. Biol. 6:4396, 1986; Pawson and Schlessinger, Curr. Biol. 3:434, 1993), the SH3 domain (Mayer, et al, Nature 332:272, 1988; Pawson and Schlessinger, Curr.
  • a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 mutant it is meant a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide which differs from the native sequence in that one or more amino acids have been changed, added or deleted. Changes in amino acids may be conservative or non- conservative. By “conservative” it is meant the substitution of an amino acid for one with similar properties such as charge, hydrophobicity, structure, etc.
  • polypeptides encompassed by this term include, but are not limited to, (1) chimeric proteins which comprise a portion of a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide sequence fused to a non-PTP04, a non-SAD, a non-PTP05, a non-PTPIO, a non-ALP, or a non-ALK-7 polypeptide sequence, for example a polypeptide sequence of hemagglutinin (HA), (2) PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 proteins lacking a specific domain, for example the catalytic domain, and (3) PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 proteins having a point mutation.
  • HA hemagglutinin
  • a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 mutant will retain some useful function such as, for example, binding to a natural binding partner, catalytic activity, or the ability to bind to a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 specific antibody (as defined below) .
  • PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific epitope it is. meant a sequence of amino acids that is both antigenic and unique to PTP04, SAD, PTP05, PTP10, ALP, or ALK- 7.
  • PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific epitope can be used to produce PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific antibodies, as more fully described below. Particularly preferred epitopes are shown in Examples below.
  • recombinant PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide it is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature) , purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide or polypeptide fragment.
  • an antibody e.g., a monoclonal or polyclonal antibody having specific binding affinity to a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide or polypeptide fragment.
  • specific binding affinity is meant that the antibody binds to target polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
  • Antibodies or antibody fragments are polypeptides which contain regions that can bind other polypeptides.
  • binding affinity describes an antibody that binds to a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
  • polyclonal refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • “Monoclonal antibodies” are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al.. Na ture 256:495-497 (1975), and U.S. Patent No. 4,376,110.
  • antibody fragment refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule.
  • a hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
  • Antibodies or antibody fragments having specific binding affinity to a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide may be used in methods for detecting the presence and/or amount of a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide in a sample by probing the sample with the antibody under conditions suitable for formation of an immunocomplex between the antibody and the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide and detecting the presence and/or amount of the antibody conjugated to the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
  • An antibody or antibody fragment with specific binding affinity to a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above .
  • hybridoma which produces an antibody having specific binding affinity to a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide.
  • hybridoma an immortalized cell line which is capable of secreting an antibody, for example a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 antibody.
  • the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 antibody comprises a sequence of amino acids that is able to specifically bind a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide.
  • the invention encompasses a recombinant cell or tissue containing a purified nucleic acid coding for a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK- 7 polypeptide.
  • the nucleic acid may be under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter.
  • exogenous it is meant a promoter that is not normally coupled transcriptionally to the coding sequence for the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide in its native state.
  • the invention features a method for identifying human cells containing a PTP04, a SAD, a PTP05, a PTPIO, an ALP, or an ALK-7 polypeptide or a related sequence.
  • the method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
  • the invention also features methods of screening cells for natural binding partners of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptides.
  • Natural binding partner refers to molecules, or portions of these molecules, that bind to the protein of interest in cells. Natural binding partners may be polypeptides or lipids, but do not include glutathione. Natural binding partners can play a role in propagating a signal in a protein signal transduction process. A change in the interaction between a protein and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of the protein/natural binding partner complex.
  • a protein's natural binding partner can bind to a protein's intracellular region with high affinity. High affinity represents an equilibrium binding constant on the order of IO "6 M or less.
  • a natural binding partner can also transiently interact with a protein's intracellular region and chemically modify it.
  • Natural binding partners of protein are chosen from a group that includes, but is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases or protein phosphatases. Methods of determining changes in interactions between proteins and their natural binding partners are readily available in the art.
  • the invention provides an assay to identify substances capable of modulating the activity of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7.
  • assays may be performed in vitro or in vivo can be obtained by modifying existing assays, such as the assays described in WO 96/40276, published December 19, 1996 and WO 96/14433, published May 17, 1996.
  • Other possibilities include testing for phosphatase activity on standard substrates such as Src kinase or synthetic amino acid substrates.
  • the substances so identified may be enhances or inhibitors of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity and can be peptides, natural products (such as those isolated from fungal strains, for example) or small molecular weight chemical compounds.
  • a preferred substance will be a compound with a molecular weight of less than 5,000, more preferably less than 1,000, most preferably less than 500.
  • the invention provides a method for treating or preventing an abnormal condition by administering a compound which is a modulator of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 function in vi tro.
  • the abnormal condition preferably involves abnormality in PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 signal transduction pathway, and most preferably is cancer.
  • Such compounds preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (such as the assays described in examples 5, 10, 15, 20, and 21 below) . Examples of substances that can be screened for favorable activity are provided in section XIV below.
  • Substances identified as modulators of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity can be used to study the effects of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 modulation in animal models of cell proliferative disorders.
  • inhibitors of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity can be tested as treatments for cell proliferative disorders such as leukemia or lymphoma using subcutaneous xenograph models in mice.
  • the invention provides a method for identifying modulators of protein activity.
  • the method involves the steps of: a) forming a captured protein by contacting the protein with a natural binding partner; b) contacting the captured protein with a test compound; and c) measuring the protein activity.
  • the method also includes the step of comparing the protein activity with the activity of a control protein, which has the same amino acid sequence as the protein in step (a) without the natural binding partner, to determine the extent of modulation.
  • modulator refers to a compound which has the ability of altering the activity of a protein.
  • a modulator may activate the activity of the protein, may activate or inhibit the activity of the protein depending on the concentration of the compound exposed to the protein, or may inhibit the activity of the protein.
  • modulator also refers to a compound that alters the function of a protein by increasing or decreasing the probability that a complex forms between a protein and a natural binding partner.
  • a modulator preferably increases the probability that such a complex forms between the protein and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protein and the natural binding partner depending on the concentration of the compound exposed to the protein, and most preferably decreases the probability that a complex forms between the protein and the natural binding partner.
  • activity of a protein in the context of the invention, defines the natural function of a protein in a cell. Examples of protein function include, but are not limited to, catalytic activity and binding a natural binding partner.
  • the term "activates” refers to increasing the natural function of a protein.
  • the protein function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
  • the term “inhibit” refers to decreasing the cellular function of a protein.
  • the protein function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
  • catalytic activity in the context of the invention, defines the rate at which a protein reacts with a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. When the protein is a protein kinase or a protein phosphatase, then the reaction with a substrate is the phosphorylation or dephosphorylation of the substrate, respectively.
  • Phosphorylation or dephosphorylation of a substrate occurs at the active-site of a protein kinase or a protein phosphatase.
  • the active-site is normally a cavity in which the substrate binds to the protein kinase or protein phosphatase and is phosphorylated.
  • substrate refers to a molecule which is acted upon by an enzyme. If the enzyme is a protein kinase then the substrate is phosphorylated by the protein kinase. If the enzyme is a protein phosphatase then the substrate is dephosphorylated by the protein phosphatase.
  • the term "compound” refers to a molecule which has at least two types of atoms in its composition.
  • the molecule may be a small organic molecule.
  • organic molecule refers to a molecule which has at least one carbon atom in its structure.
  • complex refers to an assembly of at least two molecules bound to one another.
  • Signal transduction complexes often contain at least two protein molecules bound to one another.
  • a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
  • contacting refers to any touching between a compound and a protein, preferably the mixing of a solution comprising a compound with a liquid medium bathing the protein of the methods.
  • the touching may involve interaction between the compound and the protein.
  • the solution comprising the compound may be added to the medium bathing the protein by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
  • protein refers to a naturally occurring or chemically modified polypeptide chain that has distinct secondary and tertiary structures. The chemical modification may be point mutations.
  • protein as used herein does not include a polypeptide chain which is covalently fused or otherwise joined through human intervention with another distinct polypeptide chain.
  • a GST-fusion protein is not included under the term “protein” as used herein.
  • captured protein refers to a protein that has come to contact with one of its natural binding partners and has formed a complex with the natural binding partner. The natural binding partner may be free in the solution, bound to a solid support, or free in the solution with the ability to bind to a solid support.
  • test compound refers to a compound under study for its potential effect on the catalytic activity of a protein.
  • control protein refers to a protein which has the same amino acid sequence of the captured protein but is not being modulated by a test compound, nor has it come in contact with a test compound, nor is it bound to a natural binding partner.
  • the activity of a control protein can be measured using the techniques of the invention, and such activity may be compared with the activity of a modulated protein. A difference between the levels of the two measured activities determines the extent of modulation by the modulators.
  • the invention provides a method for identifying modulators of protein activity, where the method is preferably a non- radioactive method.
  • the protein is preferably not a fusion protein. Most preferably, the protein is not a GST-fusion protein.
  • the protein is preferably an enzyme, a receptor enzyme, or a non-receptor enzyme, more preferably a protein kinase, and most preferably a protein tyrosine kinase.
  • the protein tyrosine kinase is preferably Zap70 or Syk.
  • the protein is a protein tyrosine phosphatase, and more preferably the protein is PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7.
  • fusion protein refers to a heterologous protein formed by the covalent linkage of two distinct polypeptides.
  • GST-fusion protein refers to a heterologous protein formed by the covalent linkage of a polypeptide and glutathione
  • GST S-transferase
  • enzyme refers to a protein that can act as a catalyst for biological reactions.
  • Examples of catalyzed biological reactions include, but are not limited to, formation of new bonds, addition of water, addition of a phosphoryl group, and isomerization of an organic molecule.
  • catalyst refers to a compound or a dissolved metal ion that increases the rate of a chemical reaction without being consumed in the reaction.
  • receptor enzyme refers to an enzyme that has a portion of its amino acid sequence within the cell membrane.
  • non-receptor enzyme refers to an enzyme that has none of its amino acid sequence within the cell membrane.
  • the non-receptor enzyme may be associated with the membrane via interactions, such as covalent linkage with fatty acids of the membrane .
  • protein kinase refers to an enzyme that transfers the high energy phosphate of adenosine triphosphate to an amino acid residue, either tyrosine, serine, or threonine, located on a protein target.
  • protein tyrosine kinase refers to an enzyme that transfers the high energy phosphate of adenosine triphosphate to a tyrosine residue located on a protein target.
  • Zap70 and “Syk” are protein tyrosine kinases of the Syk family which is characterized by the presence of two tandemly arranged Src-homology 2 (SH2) domains and no membrane localization motifs. These proteins are probably phosphorylated by the Src family of protein tyrosine kinases at the two tyrosine residues within the ITAM motif.
  • SH2 Src-homology 2
  • ITAM motif stands for "immunoreceptor tyrosine- based activation motif” and refers to a 16 amino acid motif
  • protein tyrosine phosphatase refers to an enzyme that removes a phosphate group from a phosphotyrosine in a protein target.
  • the natural binding partner of one of the above proteins is capable of binding to a solid support.
  • the natural binding partner is preferably a peptide, more preferably a phosphopeptide, and most preferably the phosphopeptide comprises an ITAM motif.
  • the natural binding partner comprises a lipid.
  • solid support refers to an insoluble surface to which a molecule can be bound.
  • solid supports include, but are not limited to, well plates (i.e. 96-well plates), glass beads, or resins (i.e. cellulose, agarose, polypropylene, polystyrene, etc.).
  • Natural binding partners can be attached, through either covalent or non- covalent interactions, to the solid support prior to or after binding a protein.
  • non-covalent interactions include, but are not limited to, hydrogen bonds, electrostatic interactions, and hydrophobic interactions.
  • peptide refers to an arrangement of two or more amino acids, linked together through an amide bond between the carboxyl end of one amino acid and the amino end of another.
  • phosphopeptide refers to a peptide that has a phosphate group chemically attached to one of its amino acid residues.
  • lipid refers to a water-insoluble substance that can be extracted from cells by organic solvents of low polarity.
  • examples of lipids include, but are not limited to, glycerides, steroids, and terpenes.
  • the modulators of protein activity being identified by the methods of the invention preferably modulate the autocatalytic activity, catalytic activity, or binding of a second natural binding partner.
  • the activity of an enzyme is "autocatalytic activity" when the enzyme and its substrate are identical.
  • Some receptor protein tyrosine kinases are capable of exhibiting autocatalytic activity.
  • the invention provides a method for identifying modulators of protein activity, comprising the step of contacting the captured protein with one or more components of the group consisting of a substrate, a second natural binding partner, and an antibody.
  • the method preferably further involves the step of lysing cells before forming the captured protein.
  • the method involves the step of washing the solid support after capturing the protein and binding the protein:natural binding partner complex to the solid support and prior to measuring the protein activity.
  • the invention provides a kit for the identification of modulators of non-receptor enzyme activity comprising: a) a natural binding partner; b) a solid support; and c) one or more components selected from the group consisting of a substrate, a second natural binding partner, and an antibody.
  • the natural binding partner in the above kit is preferably a peptide, more preferably a phosphopeptide. Even more preferably the phosphopeptide comprises an ITAM motif. In other preferred embodiment, the natural binding partner comprises a lipid.
  • Figure 1 shows a comparison between the amino acid sequence of human PTP04 and the amino acid sequence of the protein to which it is most closely related, murine ZPEP.
  • the relative homology between the two suggests that the two proteins are members of the same PTP family but are not species orthologs.
  • the present invention relates to the isolation and characterization of new proteins which we have called PTP04, SAD, PTP05, PTPIO, ALP, and ALK-7, nucleotide sequences encoding PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7, various products and assay methods that can be used to identify compounds useful for the diagnosis and treatment of various PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 related diseases and conditions, for example cancer.
  • Polypeptides derived from PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 and nucleic acids encoding such polypeptides may be produced using well known and standard synthesis techniques when given the sequences presented herein.
  • PTP04 is a tyrosine phosphatase with an apparent molecular weight of approximately 100 kDa. Primary sequence analysis shows that PTP04 is comprised of three domains: an N-terminal domain, a catalytic domain, and a C-terminal domain. The lack of a hydrophobic stretch of amino acids generally characterized as a transmembrane region indicates that PTP04 is a non- receptor tyrosine phosphatase.
  • PTP04 plays an important role in the growth and persistence of these cancers.
  • SAD is a tyrosine kinase with an apparent molecular weight of approximately 55 kDa.
  • Primary sequence analysis shows that SAD is comprised of four domains: a domain at the N-terminus that shows no homology to any known sequence (the unique domain) , an SH3 domain, an SH2 domain and a catalytic domain.
  • the lack of a hydrophobic stretch of amino acids generally characterized as a transmembrane region indicates that SAD is a non-receptor tyrosine kinase.
  • a comparison of the amino acid sequences suggests that SAD is a member of the Frk family.
  • SAD lacks an N-terminal myristylation site and a C-terminal regulatory tyrosine characteristic of Src family members. It is most closely related to the murine NR-TK Srm (Kohmura, et al, Mol. Cell. Bio. 14(10): 6915, 1994) with approximately 85% sequence homology in the catalytic domain. (Discussed in detail in the examples below.)
  • SAD was originally isolated from a human breast cancer cell line. Subsequent expression analysis of both normal tissues and cancer cell lines, shown in detail below, revealed that SAD has very limited expression in normal cells but is significantly overexpressed in a number of tumors. This suggests that SAD plays an important role in the growth and persistence of these cancers.
  • PTP05 is a tyrosine phosphatase with an apparent molecular weight of approximately 49 kDa. Two additional isoforms have been identified, one larger (approximately 54 kDa) and one smaller (approximately 47 kDa) . Primary sequence analysis shows that PTP05 is comprised of three domains: an N-terminal domain, a catalytic domain, and a C-terminal domain. The lack of a hydrophobic stretch of amino acids generally characterized as a transmembrane region indicates that PTP05 is a non- receptor tyrosine phosphatase. PTPIO is also a tyrosine phosphatase with significant homology to PTP05. Together they define a new family of PTPs.
  • ALP is a tyrosine phosphatase with an apparent molecular weight of approximately 160 - 200 kDa. Primary sequence analysis shows that ALP is comprised of three domains: a domain at the N-terminus that is rich in proline residues
  • proline rich regions may be protein:protein interaction sites as SH3 domains have been shown to bind to proline rich regions (Morton and Campbell, Curr. Biol. 4:614, 1994; Ren, et al. Science 259:1157, 1993).
  • the lack of a hydrophobic stretch of amino acids generally characterized as a transmembrane region indicates that ALP is a non-receptor tyrosine phosphatase.
  • ALP The full-length ALP was originally isolated from a human brain cancer cell line. Subsequent expression analysis of both normal tissues and cancer cell lines, shown in detail below, revealed that ALP has low expression in normal cells but is significantly overexpressed in a number of tumors. This suggests that ALP plays an important role in the growth and persistence of these cancers.
  • ALK-7 is a type I receptor serine/threonine kinase (STK receptor) . Proteins with some homology have been described in the rat (Ryden, et al. J. Biol. Chem. 271:30603, 1996; Tsuchida, et al . Molec. Cell. Neurosci. 7:467, 1996), however, unlike the rat proteins, the human ALK-7 is expressed in more restricted regions of the brain, notably hippocampous, hypothalamic nuclei, sustantia nigra, an pituitary. This extremely restricted expression pattern strongly suggests a role for human ALK-7 in the growth and/or survival of neurons and its relevance in treatment of such diseases as Parkinson's, Huntington's disease and Alzheimer's.
  • STK receptor type I receptor serine/threonine kinase
  • polypeptide and nucleotide sequences of the invention can be used, therefore, to identify modulators of cell growth and survival which are useful in developing therapeutics for various cell proliferative disorders and conditions, and in particular cancers related to inappropriate PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity.
  • Assays to identify compounds that act intracellularly to enhance or inhibit PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity can be developed by creating genetically engineered cell lines that express PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 nucleotide sequences, as is more fully discussed below.
  • a first aspect of the invention features nucleic acid sequences encoding a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide. Included within the scope of this invention are the functional equivalents of the herein-described isolated nucleic acid molecules. Functional equivalents or derivatives can be obtained in several ways. The degeneracy of the genetic code permits substitution of certain codons by other codons which specify the same amino acid and hence would give rise to the same protein.
  • the nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon.
  • portions or all of the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 gene could be synthesized to give a nucleic acid sequence significantly different from that shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • SEQ ID NO:l SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5 '-end and/or the 3 '-end of the nucleic acid formula shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a derivative thereof.
  • nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16 which is encoded by the nucleotide sequence.
  • the present invention is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5 ' -end of the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 nucleic acid sequence or its functional derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3 ' -end of the inventive nucleotide sequence or its derivative.
  • the nucleic acid molecule of the present invention may, as necessary, have restriction endonuclease recognition sites added to its 5 ' -end and/or 3 '-end.
  • nucleic acid sequence Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto. All variations of the nucleotide sequence of the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 genes and fragments thereof permitted by the genetic code are, therefore, included in this invention.
  • the two polypeptides are functionally equivalent, as are the two nucleic acid molecules which give rise to their production, even though the differences between the nucleic acid molecules are not related to degeneracy of the genetic code.
  • Functional equivalents or derivatives of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 can also be obtained using nucleic acid molecules encoding one or more functional domains of the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide.
  • the catalytic domain of PTP04 functions as an enzymatic remover of phosphate molecules bound onto tyrosine amino acids and a nucleic acid sequence encoding the catalytic domain alone or linked to other heterologous nucleic acid sequences can be considered a functional derivative of PTP04.
  • Other functional domains of PTP04 include, but are not limited to, the proline- rich region within the N-terminal domain, and the C-terminal domain.
  • Nucleic acid sequences encoding these domains are shown in SEQ ID NO:l as follows: N-terminal domain 53-196; catalytic domain 197-934, C-terminal domain 935-2473.
  • the SH2 domain of SAD functions as a phosphorylated tyrosine binding domain and a nucleic acid sequence encoding the SH2 domain alone or linked to other heterologous nucleic acid sequences can be considered a functional derivative of SAD.
  • Other functional domains of SAD include, but are not limited to, the unique domain, the SH3 domain, and the catalytic domain.
  • Nucleic acid sequences encoding these domains are shown in SEQ ID NO: 2 as follows: N-terminal unique domain approximately 49-213; SH3 domain approximately 214-375; SH2 domain approximately 406-684; catalytic domain approximately 736-1488.
  • the catalytic domain of PTP05 functions to remove phosphate molecules bound onto tyrosine residues and a nucleic acid sequence encoding the catalytic domain alone or linked to other heterologous nucleic acid sequences can be considered a functional derivative of PTP05.
  • Other functional domains of these proteins include, but are not limited to, the proline- rich region within the N-terminal domain, and the C-terminal domain. Nucleic acid sequences encoding these domains are shown in SEQ ID NO: 3 as follows: N-terminal domain approximately 199-759 ; catalytic domain approximately 760- 1458, C-terminal domain approximately 1459-1476.
  • the N-terminal proline-rich domain of ALP functions as a SH3 binding domain and a nucleic acid sequence encoding the N- terminal proline-rich domain alone or linked to other heterologous nucleic acid sequences can be considered a functional derivative of ALP.
  • Other functional domains of ALP include, but are not limited to, the proline-rich region within the N-terminal proline-rich domain, the C-terminal proline/serine-rich domain, the proline/serine-rich region within the C-terminal proline/serin-rich domain, and the catalytic domain.
  • Nucleic acid sequences encoding these domains are shown in SEQ ID NO: 7 as follows: N-terminal domain 313-2883; proline-rich region 1369-2643 ; catalytic domain approximately 2884-3600, C-terminal proline/serine-rich domain 3601-4134, proline/serine-rich region 3613-4456.
  • the extracellular domain of ALK-7 functions as a ligand or co-receptor binding domain and a nucleic acid sequence encoding the extracellular domain alone or linked to other heterologous nuclic acid sequences can be considered a functional derivative of ALK-7.
  • Other functional domains of ALK-7 include, but are not limited to, the signal sequence, the transmembrane domain, the intracellular domain, and the catalytic domain. Nucleic acid sequences encoding these domains are shown in SEQ ID NO: 8 as follows: signal sequence 155-229; extracellular domain 155- 493; transmembrane domain 494-568; intracellular domain 569- 1633; catalytic domain approximately 731-1609. It should be noted that the signal sequence is cleaved from the extracellular domain in the mature protein.
  • a nucleic acid probe of the present invention may be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain another nucleic acid molecule of the present invention.
  • a chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (e.g. "Molecular Cloning: A Laboratory Manual", second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989) .
  • nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the polypeptide of interest.
  • the synthesized nucleic acid probes may be used as primers in a poiymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, "A Guide to Methods and Applications", edited by Michael et al., Academic Press, 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention.
  • PCR poiymerase chain reaction
  • hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes may be visualized using known methods .
  • the nucleic acid probes of the present invention include RNA as well as DNA probes and nucleic acids modified in the sugar, phosphate or even the base portion as long as the probe still retains the ability to specifically hybridize under conditions as disclosed herein. Such probes are generated using techniques known in the art.
  • the nucleic acid probe may be immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins, such as polyacrylamide and latex beads, and nitrocellulose. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
  • test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • a Probe Based Method And Kit For Detecting the Proteins of the Invention comprises (a) contacting the sample with the above-described nucleic acid probe, under conditions such that hybridization occurs, and (b) detecting the presence of the probe bound to the nucleic acid molecule.
  • One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue.
  • PTPIO, ALP, or ALK-7 in a sample comprises at least one container having disposed therein the above-described nucleic acid probe.
  • the kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabelled probes, enzymaticly labeled probes
  • affinity labeled probes biotin, avidin, or steptavidin
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross- contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like) , and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like.
  • nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art with or without a set of instructions concerning the use of such reagents in an assay.
  • V. DNA Constructs Comprising a PTP04, a SAD, a PTP05, a
  • PTPIO an ALP
  • ALK-7 Nucleic Acid Molecule and Cells Containing These Constructs.
  • the present invention also relates to a recombinant DNA molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules.
  • the present invention relates to a recombinant DNA molecule comprising a vector and a nucleic acid molecule described herein.
  • the present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to a
  • the above-described molecules may be isolated and/or purified DNA molecules.
  • the present invention also relates to a cell or organism that contains a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 nucleic acid molecule as described herein and thereby is capable of expressing a peptide.
  • the polypeptide may be purified from cells which have been altered to express the polypeptide.
  • a cell is said to be "altered to express a desired polypeptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • the precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but will in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5' -non- coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the sequence encoding a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 gene may be obtained by the above-described cloning methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ⁇ ability of the promoter region sequence to direct the transcription of a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 gene sequence, or (3) interfere with the ability of the a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 gene (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 gene.
  • Prokaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, including other bacterial strains.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors may include pBR322, pUC118, pUC119 and the like;
  • suitable phage or bacteriophage vectors may include lgtlO, lgtll and the like;
  • suitable virus vectors may include pMAM-neo, pKRC and the like.
  • the selected vector of the present invention has the capacity to replicate in the selected host cell.
  • prokaryotic hosts include bacteria such as E. coli and those from genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated.
  • the prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 sequence to a functional prokaryotic promoter.
  • promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible) .
  • constitutive promoters include the int promoter of bacteriophage 1, the bla promoter of the b-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage 1 (P., and P R ) , the trp, recA, lacZ, lad, and gal promoters of E. coli, the a- amylase (Ulmanen et at., J. Bacteriol. 162:176-182, 1985) and the sigma-28-specific promoters of B.
  • subtilis (Gilman et al., Gene sequence 32:11-20(1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203:468- 478, 1986) .
  • Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiot. 1:277-282, 1987); Cenatiempo (Biochimie 68:505- 516, 1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
  • ribosome binding site upstream of the gene sequence-encoding sequence.
  • Such ribosome binding sites are disclosed, for example, by Gold et at. (Ann. Rev. Microbiol. 35:365-404, 1981).
  • the selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
  • cell As used herein, “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations include progeny.
  • the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell.
  • Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 peptide of interest.
  • Suitable hosts may often include eukaryotic cells.
  • Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO, 3T3 or CHO-K1, or cells of lymphoid origin (such as 32D cells) and their derivatives.
  • Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 and PC12 which may provide better capacities for correct post- translational processing.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459, 1988).
  • baculovirus vectors can be engineered to express large amounts of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 in insects cells (Jasny, Science 238:1653, 1987); Miller et al .
  • yeast gene sequence expression systems Any of a series of yeast gene sequence expression systems can be utilized which incorporate promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast.
  • Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides) .
  • leader sequences i.e., pre-peptides
  • SAD SAD
  • PTP05 PTP05
  • PTPIO PTPIO
  • ALP ALP
  • ALK-7 ALK-7
  • a particularly preferred yeast expression system is that utilizing Schizosaccharmocyces pombe. This system is useful for studying the activity of members of the Src family (Superti-Furga, et al, EMBO J. 12:2625, 1993) and other NR-TKs.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • eukaryotic regulatory regions are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
  • Expression of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.
  • codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 coding sequence) .
  • a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 nucleic acid molecule and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule (a plasmid) . Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent or stable expression may occur through the integration of the introduced DNA sequence into the host chromosome .
  • a vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama, Mol. Cell. Bio. 3:280, 1983.
  • the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coil (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, pVX.
  • Such plasmids are, for example, disclosed by Sambrook (cf. "Molecular Cloning: A Laboratory Manual", second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989) ) .
  • Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307- 329) .
  • Suitable Streptomyces plasmids include plJlOl (Kendall et al., J. Bacteriol.
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982); Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204, 1982); Bollon et at., J. Clin. Hematol. Oncol. 10:39-48, 1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980) .
  • the DNA construct (s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene molecule (s) results in the production of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 or fragments or functional derivatives thereof.
  • a variety of incubation conditions can be used to form the peptide of the present invention. The most preferred conditions are those which mimic physiological conditions.
  • PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptides are also a feature of the invention.
  • a variety of methodologies known in the art can be utilized to obtain the polypeptides of the present invention. They may be purified from tissues or cells which naturally produce them. Alternatively, the above- described isolated nucleic acid sequences can be used to express a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 protein recombinantly.
  • source organism refers to the original organism from which the amino acid sequence is derived, regardless of the organism the protein is expressed in and ultimately isolated from.
  • source organism refers to the original organism from which the amino acid sequence is derived, regardless of the organism the protein is expressed in and ultimately isolated from.
  • One skilled in the art can readily follow known methods for isolating proteins in order to obtain the peptide free of natural contaminants. These include, but are not limited to: size-exclusion chromatography, HPLC, ion-exchange chromatography, and immuno-affinity chromatography.
  • proteins sorted through the so-called vesicular pathway usually have a signal sequence (also called a leader peptide) in the N- terminus, which is cleaved off after the translocation through the ER (endoplasmic reticulum) membrane.
  • a signal sequence also called a leader peptide
  • Some N-terminal signal sequences are not cleaved off, remaining as transmembrane segments, but it does not mean these proteins are retained in the ER; they can be further sorted and included in vesicles.
  • SAD protein lacks a hydrophobic signal sequence and is classified as a non-receptor protein.
  • Other motifs involved in targeting proteins to specific cellular locations include those selective for the mitochondrial matrix (Gavel and von Heijne, Prot Eng 4:33, 1990), the nucleus (Robbins, et al, Cell 64:615, 1991) , peroxisomes, endoplasmic reticulum (Jackson, et al, EMBO J 9:3253, 1990), vesicular pathways (Bendiak, Biophys Res Comm 170:879, 1990), glycosyl-phosphatidylinositol (GPI) lipid anchors, and lysosomal organelles, and motifs that target proteins to lipid membranes such as myristylation (Towler, et al, Annu Rev Biochem 57:69, 1988) and farnesylation sites.
  • the N-terminal 15 amino acids of the SAD protein conforms to the features which define a mitochondrial membrane protein with a bipartite structure of an N-terminal stretch of high arginine content involved in membrane targeting followed by the apolar sequence which signals translocation to the mitochondrial intermembrane space.
  • Non-receptor proteins generally function to transmit signals within the cell, either by providing sites for protein:protein interactions or by having some catalytic activity (contained within a catalytic domain) , often both. Methods of predicting the existence of these various domains are well known in the art. Protein:protein interaction domains can be identified by comparison to other proteins.
  • the SH2 domain for example is a protein domain of about 100 amino acids first identified as a conserved sequence region between the proteins Src and Fps (Sadowski, et al, Mol. Cell. Bio. 6:4396, 1986). Similar sequences were later found in many other intracellular signal-transducing proteins.
  • SH2 domains function as regulatory modules of intracellular signaling cascades by interacting with high affinity to phosphotyrosine- containing proteins in a sequence specific and strictly phosphorylation-dependent manner (Mayer and Baltimore, Trends Cell. Biol. 3:8, 1993).
  • Kinase or phosphatase catalytic domains can be identified by comparison to other known catalytic domains with kinase or phosphatase activity. See, for example Hanks and Hunter, FASEB J. 9:576-595, 1995.
  • Receptor proteins also have, and are somewhat defined by, a hydrophobic transmembrane segment (s) which are thought to be Alpha-helices in membranes.
  • Membrane proteins also integrate into the cell membrane in a specific manner with respect to the two sides (cytoplasmic/intracellular or exo-cytoplasmic/ extracellular), which is referred to as membrane topology.
  • Extracellular portions of integral membrane proteins often function as ligand binding domains whereas intracellula portions generally function to transmit signals within the cell, either by providing sites for protein:protein interactions or by having some catalytic activity (contained within a catalytic domain) , often both. Methods of predicting the existence of these various domains are well known in the art. See, for example, D. J.
  • PTP04 amino acid sequence shown in SEQ ID NO: 9
  • SEQ ID NO: 9 Primary sequence analysis of the PTP04 amino acid sequence (shown in SEQ ID NO: 9) reveals that it does not contain a signal sequence or transmembrane domain and is, therefore, an intracellular protein. Comparison to known protein sequences reveals that PTP04 is comprised of several unique domains. These include a 48 amino acid N-terminal domain (shown from amino acid number 1-48 of SEQ ID NO: 9), a 245 amino acid catalytic domain (shown from amino acid number 49-294 of SEQ ID NO: 9), and a 512 amino acid C-terminal domain (shown from amino acid number 295-807 of SEQ ID NO: 9).
  • SAD amino acid sequence shown in SEQ ID NO: 10.
  • SAD amino acid sequence shows that it contains four distinct domains. These include an approximately 55 amino acid N- terminal unique domain (shown from amino acid number 1-55 of SEQ ID NO: 10), an approximately 54 amino acid SH3 domain (shown from amino acid number 56-109 of SEQ ID NO: 10), an approximately 93 amino acid SH2 domain (shown from amino acid number 120-212 of SEQ ID NO: 10), an approximately 251 amino acid catalytic domain (amino acid number 230-480 of SEQ ID No: 10), and a C-terminal tail of 8 amino acids (shown from amino acid 481-488 of SEQ ID NO: 10).
  • PTP05 amino acid sequence shown in SEQ ID NO: 11 with isoforms shown in SEQ ID NO: 12 and SEQ ID NO: 13
  • Comparison to known protein sequences reveals that PTP05 is comprised of several unique domains. These include a 187 amino acid N-terminal domain (shown from amino acid number 1-187 of SEQ ID NO: 11), a 242 amino acid catalytic domain (shown from amino acid number 188- 420 of SEQ ID NO: 11), and a 5 amino acid C-terminal domain (shown from amino acid number 421-426 of SEQ ID NO: 11).
  • the "long” form has a 37 amino acid insertion in the N-terminal domain (aminoacids 44-80 of SEQ ID NO: 12) which extends this domain to 224 amino acids.
  • the catalytic domain extends from amino acid 225-457 of SEQ ID NO: 12 and the C-terminal domain extents from amino acids 458-463 of SEQ ID NO: 12.
  • the "C-trunc” form results from a deletion of nucleotides 1415-1507 of SEQ ID NO: 3, most likely due to alternative exon splicing.
  • the N-terminal domain of the "C-trunc" form extends from amino acid 1-87 of SEQ ID NO: 13, the catalytic domain from amino acids 188-405 of SEQ ID NO: 13 and the unique C-terminal domain from 406-412 of SEQ ID NO:13.
  • ALP amino acid sequence shown in SEQ ID NO: 15
  • SEQ ID NO: 15 Primary sequence analysis of the ALP amino acid sequence (shown in SEQ ID NO: 15) reveals that it does not contain a signal sequence or transmembrane domain and is, therefore, an intracellular protein. Comparison to known protein sequences reveals that ALP is comprised of several unique domains.
  • proline-rich domains shown from amino acid number 1-857 of SEQ ID NO: 15 within which is a proline-rich region (amino acid number 353-777 of SEQ ID NO: 15), a 238 amino acid catalytic domain (shown from amino acid number 858-1096 of SEQ ID NO: 15), and a 177 amino acid C- terminal proline/serine-rich domain (shown from amino acid number 1097-1274 of SEQ ID NO: 15) within which is a proline/serine-rich region (amino acid number 1101-1214 of SEQ ID NO:15) .
  • ALK-7 amino acid sequence shown in SEQ ID NO: 16
  • SEQ ID NO: 16 Primary sequence analysis for an ALK-7 amino acid sequence (shown in SEQ ID NO: 16) reveals that it contains all the motifs characteristic of a type I STK receptor. These include a 25 amino acid signal peptide (shown from amino acid number 1-25 of SEQ ID NO: 16), an 88 amino acid cysteine-rich extracellular region (shown from amino acid number 26-113 of SEQ ID NO: 16), a single 25 amino acid transmembrane domain (shown from amino acid number 114-136 of SEQ ID NO: 16), and a 355 amino acid cytoplasmic domain (shown from amino acid number 137-493 of SEQ ID NO: 16), which includes a GS domain and a catalytic domain (amino acid number 193-485 of SEQ ID NO: 16).
  • the divergent extracellular domain sequence of ALK-7 (28-30% identity to ALK- 4 and ALK-5) suggests it may have a unique ligand/type II STK receptor specificity.
  • a rat ALK-7-like protein ahs been found to bind TGFbeta and activin in a complex with the type II TGF beta receptor and ACTRII.
  • Candidate ALK-7-specific ligands include other TGFbetas such as TGFbeta 2, GDF-1, and homologues of GDNF, such as neuturin, which have been found to be expressed in neurons in a pattern similar to that of ALK-7.
  • the intracellular domain is somewhat more homologous to other ALK proteins, particularly in the catalytic domain which shows 83% identity to other type I STK receptors.
  • the 40 amino acids immediately N-terminal of the transmembrane domain are, however, quite unique in comparison with other ALKs.
  • PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 domains have a variety of uses.
  • An example of such a use is to make a polypeptide consisting of the PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 catalytic domain and a heterologous protein such as glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • Such a polypeptide can be used in a biochemical assay for PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 catalytic activity useful for studying PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 substrate specificity or for identifying substances that can modulate PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 catalytic activity.
  • one skilled in the art could create a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide lacking at least one of the three major domains.
  • Such a polypeptide when expressed in a cell, is able to form complexes with the natural binding partner (s) of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 but unable to transmit any signal further downstream into the cell, i.e.,. it would be signaling incompetent and thus would be useful for studying the biological relevance of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity. (See, for example, Gishizky, et al, PNAS :10889, 1995) .
  • the present invention also relates to an antibody having specific binding affinity to an PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide.
  • the polypeptide may have the amino acid sequence set forth in SEQ ID NO: 2, or a be fragment thereof, or at least 6 contiguous amino acids thereof.
  • Such an antibody may be identified by comparing its binding affinity to a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide with its binding affinity to another polypeptide.
  • Those which bind selectively to PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 would be chosen for use in methods requiring a distinction between PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 and other polypeptides.
  • Such methods could include, but should not be limited to, the analysis of altered PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 expression in tissue containing other polypeptides and assay systems using whole cells.
  • a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 peptide of the present invention can be used to produce antibodies or hybridomas.
  • One skilled in the art will recognize that if an antibody is desired, such a peptide would be generated as described herein and used as an immunogen.
  • the antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
  • the present invention also relates to a hybridoma which produces the above-described monoclonal antibody, or binding fragment thereof.
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • the polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or b- galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or b- galactosidase
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0- Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0- Agl4 myeloma cells
  • Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz, et al., Exp. Cell Res. 175:109-124, 1988).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", supra, 1984) .
  • antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
  • the above-described antibodies may be detectably labeled.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like) , enzymatic labels (such as horse radish peroxidase, Alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like) , paramagnetic atoms, and the like.
  • the above-described antibodies may also be immobilized on a solid support.
  • solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacoby et al., Meth. Enzym. 34, Academic Press, N.Y., 1974) .
  • the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
  • the present invention encompasses a method of detecting a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide in a sample, comprising: (a) contacting the sample with an above- described antibody, under conditions such that immunocomplexes form, and (b) detecting the presence of said antibody bound to the polypeptide.
  • the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample. Altered levels, either an increase or decrease, of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 in a sample as compared to normal levels may indicate disease. Conditions for incubating an antibody with a test sample vary.
  • Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay.
  • immunological assay formats such as radioimmunoassays, enzyme- linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays
  • Examples of such assays can be found in Chard, "An Introduction to Radioimmunoassay and Related Techniques” Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., “Techniques in Immunocytochemistry, " Academic Press, Orlando, FL Vol. 1(1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, "Practice and Theory of Enzyme Immunoassays : Laboratory Techni- ques in Biochemistry and Molecular Biology,” Elsevier Science Publishers, Amsterdam, The Netherlands (1985) .
  • the immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine.
  • the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is capable with the system utilized.
  • kits contains all the necessary reagents to carry out the previously described methods of detection.
  • the kit may comprise: (i) a first container containing an above-described antibody, and (ii) second container containing a conjugate comprising a binding partner of the antibody and a label.
  • the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies.
  • detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • the compartmentalized kit may be as described above for nucleic acid probe kits.
  • the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
  • the present invention also relates to methods of detecting natural binding partners capable of binding to a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide.
  • a natural binding partner of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 may be, for example, a substrate protein which is dephosphorylated as part of a signaling cascade.
  • the binding partner (s) may be present within a complex mixture, for example, serum, body fluids, or cell extracts.
  • methods for identifying natural binding partners comprise incubating a substance with PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 and detecting the presence of a substance bound to PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7.
  • Preferred methods include the two-hybrid system of Fields and Song (supra) and co-immunoprecipitation.
  • the present invention also relates to a method of detecting a substance capable of modulating PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity.
  • substances can either enhance activity (agonists) or inhibit activity (antagonists) .
  • Agonists and antagonists can be peptides, antibodies, products from natural sources such as fungal or plant extracts or small molecular weight organic compounds. In general, small molecular weight organic compounds are preferred.
  • Examples of classes of compounds that can be tested for PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 modulating activity are, for example but not limited to, thiazoles (see for example co-pending US applications 60/033,522, 08/660,900), and naphthopyrones (US patent number 5,602,171).
  • the method comprises incubating cells that produce PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 in the presence of a test substance and detecting changes in the level of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity or PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 binding partner activity.
  • a change in activity may be manifested by increased or decreased phosphorylation of a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide, increased or decreased phosphorylation of a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 substrate, or increased or decreased biological response in cells.
  • a method for detecting modulation of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity using the phosphorylation of an artificial substrate is shown in the examples below.
  • Biological responses can include, for example, proliferation, differentiation, survival, or motility. The substance thus identified would produce a change in activity indicative of the agonist or antagonist nature of the substance. Once the substance is identified it can be isolated using techniques well known in the art, if not already available in a purified form.
  • the present invention also encompasses a method of agonizing (stimulating) or antagonizing PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 associated activity in a mammal comprising administering to said mammal an agonist or antagonist to PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 in an amount sufficient to effect said agonism or antagonism.
  • Also encompassed in the present application is a method of treating diseases in a mammal with an agonist or antagonist of PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-related activity comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 associated function (s).
  • the particular compound can be administered to a patient either by itself or in a pharmaceutical composition where it is mixed with suitable carriers or excipient (s) . In treating a patient a therapeutically effective dose of the compound is administered.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 5 o.
  • Compounds which exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal disruption of the protein complex, or a half- maximal inhibition of the cellular level and/or activity of a complex component) .
  • IC 50 as determined in cell culture
  • levels in plasma may be measured, for example, by HPLC.
  • the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) .
  • the magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • Such agents may be formulated and administered systemically or locally.
  • Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," 1990, 18th ed., Mack Publishing Co., Easton, PA. Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intra- thecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks ' s solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks ' s solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water- soluble form. Additionally, suspensions of the active com- pounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injec- tion suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxyprop
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • transgenic animals useful for the study of PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity in complex in vivo systems.
  • a variety of methods are available for the production of transgenic animals associated with this invention.
  • DNA sequences encoding PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g.., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster, et al., Proc. Nat. Acad. Sci. USA 82 : 4438, 1985) .
  • Embryos can be infected with viruses, especially retro- viruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA) , Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
  • transgenic mouse female mice are induced to superovulate. After being allowed to mate, the females are sacrificed by C0 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice. See Hammer, et al., Cell 63:1099-1112, 1990).
  • a clone containing the sequence (s) of the invention is co-transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence (s) of the invention.
  • Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E.J. Robertson, supra) .
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination. Capecchi, Science 244: 1288-1292
  • the invention provides transgenic, nonhuman mammals containing a transgene encoding a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide or a gene effecting the expression of a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide.
  • transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introducing a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide, regulating the expression of a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes) .
  • a "transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats.
  • the transgenic DNA may encode for a human PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 polypeptide.
  • Native expression in an animal may be reduced by providing an amount of anti-sense RNA or DNA effective to reduce expression of the receptor.
  • PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 or its genetic sequences, both mutated and non-mutated, will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, (1992). Miller states that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. The basic science of gene therapy is described in Mulligan, Science 260:926-931, (1993).
  • an expression vector containing a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 coding sequence or a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 mutant coding sequence as described above is inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.
  • a DNA segment containing a promoter of choice is transferred into cells containing an endogenous PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 in such a manner that the promoter segment enhances expression of the endogenous PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 gene) .
  • a promoter of choice for example a strong promoter
  • the gene therapy may involve the use of an adenovirus containing PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 cDNA targeted to an appropriate cell type, systemic PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 increase by implantation of engineered cells, injection with PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 virus, or injection of naked PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 DNA into appropriate cells or tissues, for example neurons .
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) encoding recombinant PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 protein into the targeted cell population (e.g., tumor cells or neurons) .
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences.
  • nucleic acid molecules encoding protein sequences can be used as naked DNA or in reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (See e.g., Feigner et al., Na ture 337:387-8, 1989).
  • transfection wherein DNA is precipitated with CaPO-, and taken into cells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52, 1987); electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu G., et al., Nucleic Acids Res., 15:1311- 26, 1987); lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles which fuse with a target cell (Feigner PL., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7, 1987)); and particle bombardment using DNA bound to small projectiles (Yang NS. et al., Proc. Natl. Acad. Sci. 87:9568-72, 1990).
  • Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins.
  • adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene.
  • gene transfer means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene.
  • the product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA.
  • Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.
  • gene therapy is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vi tro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.
  • a vector having nucleic acid sequences encoding a PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 is provided in which the nucleic acid sequence is expressed only in specific tissue. Methods of achieving tissue-specific gene expression as set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.
  • nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.
  • Gene replacement means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • Gene replacement means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • substances capable of modulating PTP04, SAD, PTP05, PTPIO, ALP, or ALK-7 activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines.
  • the quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well known compounds such as those described in the literature.
  • representative publications describing quinazoline include Barker et al., EPO Publication No. 0 520 722 Al; Jones et al., U.S. Patent No. 4,447,608; Kabbe et al., U.S. Patent No. 4,757,072; Kaul and Vougioukas, U.S. Patent No. 5, 316,553; Kreighbaum and Comer, U.S. Patent No. 4,343,940; Pegg and Wardleworth, EPO Publication No. 0 562 734 Al; Barker et al., Proc.
  • the examples below are non-limiting and are merely representative of various aspects and features of the present invention.
  • the examples below show the isolation and characterization of the novel proteins, protein expression in normal and tumor cells, generation of protein specific antibodies, and recombinant expression in mammalian and yeast systems. Also shown are assays useful for identifying compounds that modulate protein activity.
  • the example below describes the isolation and identi- fication of a new PTP sequence from primary cancer tissues and the subsequent cloning of a full-length human PTP04. Also described are probes useful for the detection of PTP04 in cells or tissues.
  • RNA was isolated from 30uM cryostat sections of frozen samples from primary human lung and colon carcinomas (Micro-FastTrack, Invitrogen, San Diego, CA) . This RNA was used to generate single-stranded cDNA using the Superscript Preamplification System (GIBCO BRL, Gaithersburg, MD. ; Gerard, GF et al. (1989), FOCUS 11, 66) under conditions recommended by the manufacturer.
  • GEBCO BRL Superscript Preamplification System
  • a typical reaction used 10 ⁇ q total RNA or 2 ⁇ g poly (A) RNA with 1 . 5 ⁇ q oligo (dT) 12 - ⁇ in a reaction volume of 60 ⁇ L .
  • the product was treated with RNaseH and diluted to 100 ⁇ L with H20. For subsequent PCR amplification, 1-4 ⁇ L of this sscDNA was used in each reaction.
  • oligonucleotide primers were synthesized on an Applied Biosystems 394 DNA synthesizer using established phosphora- midite chemistry, precipitated with ethanol and used unpurified for PCR.
  • sequence of the degenerate oligonucleotide primers follows:
  • PTPDFW 5 ' -GAYTTYTGGVRNATGRTNTGGGA- (sense) (SEQ ID NO: 17)
  • PTPHCSA 5 ' -CGGCCSAYNCCNGCNSWRCARTG -3' (antisense) (SEQ ID NO:18) .
  • primers were derived from the peptide sequences DFWXMXW(E/D) (SEQ ID NO: 19) (sense strand from PTP catalytic domain) and HCXAGXG (antisense strand from PTP catalytic domain) (SEQ ID NO:20), respectively.
  • PCR reactions were performed using degenerate primers applied to the single-stranded cDNA listed above.
  • the primers were added at a final concentration of 5 ⁇ M each to a mixture containing 10 mM Tris'HCl (pH8.3), 50 mM KC1, 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U
  • Colonies were selected for mini plasmid DNA-preparations using Qiagen columns and the plasmid DNA was sequenced using cycle sequencing dye-terminator kit with AmpliTaq DNA Poiymerase, FS (ABI, Foster City, CA) . Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al., J. Mol. Biol.215:403-10) .
  • One novel clone novel clone (G77-4a-117) , designated PTP04, was isolated from human HLT370 primary lung carcinoma sample.
  • RACE rapid amplification of cDNA ends
  • sense or anti-sense oligonucleoides derived from the original PCR fragments.
  • Marathon-Ready cDNA (Clontech, Palo Alto, CA) made from human Molt-4 leukemia cells was used in the RACE reactions with the following primers:
  • Isolated cDNA fragments encoding PTP04 were confirmed by DNA sequencing and subsequently used as probes for the screening of a human leukocyte cDNA library.
  • a human leukocyte cDNA library (lTriplEx, Clontech) and a
  • Molt-4 leukemia cell library (lgtll, Clontech) were then screened to isolate full-length transcripts encoding PTP04.
  • the 5' or 3'-RACE fragments were 32 P-labeled by random priming and used as hybridization probes at 2xl0 6 cpm/mL following standard techniques for library screening.
  • the 3,580 bp human PTP04 nucleotide sequence encodes a polypeptide of 807 amino acids.
  • the PTP04 coding sequence is flanked by a 52 nucleotide 5 ' -untranslated region and a 1086 nucleotide 3 ' -untranslated region ending with a poly (A) tail. While there are no upstream in frame stop codons, the first ATG beginning at nucleotide position 53 conforms to the Kozak consensus for an initiating methionine.
  • This predicted first 6 amino acids are identical to those of murine ZPEP (SwissProt: P29352, GeneBank: M90388), further supporting this is the true translational start site.
  • One cDNA clone had an insert after nucleotide 30 in the 5'UTR, but otherwise had no sequence differences.
  • the 807 amino acid sequence shows no signal sequence or a transmembrane domain and PTP04 is, therefore, an intracellular protein.
  • PTP04 has an N-terminal region from amino acids 1-48, a catalytic domain from amino acids 49-294, and a C-terminal tail from amino acids 295-807.
  • PTP04 is most related to murine ZPEP with an overall homology of 70%.
  • ZPEP is a member of a subfamily of PTPs that includes PTP-PEST, HSC, BDP1 and PTP20, all of which are cytoplasmic PTPs with a single catalytic domain and a region rich in Pro, Glu, Ser and Thr residues (PEST domain) .
  • PTP04 also has a C-terminal PEST domain, from amino acids 495-807, where there are 57 serine residues (18%) and 35 proline residues (11%) .
  • a comparison of the amino acid sequences of PTP04 and ZPEP is shown in Figure 1.
  • the homology between PTP04 and ZPEP is concentrated in the N terminal and C-terminal ends of the proteins with significant divergence in the middle.
  • the N-terminal region of PTP04 from amino acids 1-48, is 81% homologous to murine ZPEP.
  • the catalytic domain of PTP04, from amino acids 49-294, is 89% homologous to murine ZPEP.
  • the region of PTP04 from 294-600 is approximately 50% homologous to murine ZPEP.
  • the C-terminal region of PTP04, from 680-817, is 80% homologous to murine ZPEP.
  • the human SuPTP04 sequence defines a novel member of the PTP-PEST subfamily of PTPs.
  • the example below shows the evaluation of PTP04 expression in normal human tissues and in cancer cell lines.
  • Northern blots were prepared by running 20 ⁇ g total RNA per lane isolated from 22 human adult normal tissues (thymus, lung, duodenum, colon, testis, brain, cerebellum, salivary gland, heart, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, placenta, mammary gland, bladder, lymph node, adipose tissue) , 2 human fetal normal tissues (fetal liver, fetal brain) , and 24 human tumor cell lines ( HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, SR, DU-145, PC-3) (
  • RNA samples were run on a denaturing formaldehyde 1% agarose gel and transferred onto a nitrocellulose membrane (BioRad, CA) .
  • RNA samples were hybridized with randomly primed [a- 3 P] dCTP-labeled probes synthesized from a 579 bp StuI-BstXI fragment of pCR2. I.mini298. Hybridization was performed overnight at 42°C in 4X SSPE, 2.5X Denhardt ' s solution, 50% formamide, 0.2 mg/mL denatured salmon sperm DNA, 0.1 mg/mL yeast tRNA (Boehringer Mannheim, IN) , 0.2% SDS, with 5 x IO 6 cpm/mL of [a- 32 P]dCTP labeled DNA probes on a Techne hybridizer HB-1.
  • the blots were washed with 2X SSC, 0.1% SDS, at 65 °C for 20 min twice followed by in 0.5 X SSC, 0.1% SDS at 65 °C for 20 min.
  • the blots were exposed to a phospho-imaging screen for 24 hours and scanned on a Molecular Dynamics Phosphoimager SF.
  • a 351 bp EcoRI-Hindlll fragment of G77-4a-117 was used to generate a probe for 2 ⁇ g poly A+ mRNA samples on a Clontech nylon membrane. Hybridization was performed at 42 °C overnight in 5X SSC, 2% SDS, 10X Denhardt ' s solution, 50% formamide, 100 ⁇ g/mL denatured salmon sperm DNA with 1-2 x IO 6 cpm/mL of [a-
  • P]dCTP -labeled DNA probes The membrane was washed at room temperature in 2X SSC/0.05% SDS for 30 min and followed by at 50 °C in 0.2X SSC/0.1% SDS for 30 min, twice, and exposed for 48 hours on Kodak XAR-2 film.
  • a single SuPTP04 mRNA transcript of approximately 4.5 kb was identified by Northern analysis, and found to be exclusively in the Thymus.
  • the rest of 23 human normal tissues (fetal brain, fetal liver, lung, duodenum, colon, testis, brain, cerebellum, salivary gland, heart, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, placenta, mammary gland, bladder, lymph node, adipose tissue) were all negative.
  • Six of the human tumor cell lines (CCRF- CEM, K-562, MOLT-4, HL-60, SR, Raji) were positive. The rest of 26 human tumor cell lines were negative.
  • RT-PCR with gene specific primer-pairs showed that expression of the transcripts encoding SuPTP04 confirmed the results from Northern analysis and also detected low levels in adipose, kidney, small intestine, hematopoietic tissues and various cell types (spleen, thymus, lymph node, bone marrow, peripheral leukocytes and lymphocytes.
  • the selective expression of PTP04 in cells of hematopoetic origin including normal human thymus and several leukemia cell lines suggests a potential involvement in immune regulation including T and B cell survival, differentiation or co- stimulation, and/or inflammatory, immunosuppressive or autoimmune disorders. Additionally, expression in adipose tissue suggests a possible role in metabolic disorders such as diabetes.
  • the following example illustrates the construction of vectors for expression of recombinant PTP04 and the creation of recombinant cell lines expressing PTP04.
  • Expression constructs were generated by PCR-assisted mutagenesis in which the entire coding domains of PTP04 was tagged on its carboxy-terminal end with the hemophilus influenza hemaglutinin (HA) epitope YPYDVPDYAS (SEQ ID NO: 55) (Pati, 1992) .
  • the construct was introduced into two mammalian expression vectors: pLXSN (Miller, A.D. & Rosman, G.J., Biotechniques 7, 980-988, 1989) for the generation of virus producing lines; and pRK5 for transient expression in mammalian.
  • Dominant negative (signaling incompetent) PTP04 constructs were also made in both pLXSN and pRK5 by mutation of the invariant Cys in the conserved HCSAG (SEQ ID NO: 56) motif to an Ala by PCR mutagenesis.
  • the entire PTP04 open reading frames (no HA-tag) excluding the initiating methionines were generated by PCR and ligated into pGEX vector (Pharmacia Biotech, Uppsala, Sweden) for bacterial production of GST-fusion proteins for immunization of rabbits for antibody production.
  • the entire PTP04 open reading frame excluding the initiating methionines was generated by PCR and ligated into pGEX vector for bacterial production of GST- fusion proteins for immunization of rabbits for antibody production.
  • This vector contains the glutathione-S-transferase coding sequence followed by a polylinker for generating recombinant fusion proteins.
  • the GST moiety comprises the N- terminal portion of the fusion protein.
  • the pRK5 expression plasmids (10 ⁇ g DNA/100 mm plate) containing the HA-tagged PTP04 gene can be introduced into COS and 293 cells with lipofectamine (Gibco BRL) . After 72 hours, the cells were harvested in 0.5 mL solubilization buffer (20 mM HEPES pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL aprotinin) .
  • pLXSN recombinant constructs containing the PTP04 gene were transfected into an amphotropic helper cell line PA317 using CaCl 2 .mediated transfection. After selection on G418, the cells were plated on normal media without G418 (500 ⁇ g/mL) . Supernatants from resistant cells were used to infect the ecotropic helper cell line GP+E86, and cells again selected on G418. Resistant cells were again taken off G418, and the supernatants harvested every 8-12 hours and pooled as virus stock. Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498. Viral stock titers were typically ⁇ 10 /mL.
  • NIH-3T3, and BALB/3T3 cells were grown in 100 mm plates with DMEM (Gibco) containing 10% fetal calf serum (FCS) .
  • DMEM Gibco
  • FCS fetal calf serum
  • the cells were superinfected with the PTP04 retrovirus by adding approximately 3 mL viral supernatant to 15 mL culture media for approximately 24 hours.
  • Cells expressing the retroviral constructs were then selected by growth in DMEM/10% FCS supplemented with 500 ⁇ g/mL G418.
  • PTP04-specific immunoreagents were raised in rabbits against a mixture of three KLH-conjugated synthetic peptides corresponding to unique sequences present in human PTP04.
  • the peptides (see below) were conjugated at the C-terminal residue with KLH.
  • peptide 428A SWPPSGTSSKMSLDDLPEKQDGTVFPSSLLP (SEQ ID NO:27)
  • peptide 429A YSLPYDSKHQIRNASNVKHHDSSALGVYSY (SEQ ID NO:28)
  • peptide 430A HTLQADSYSPNLPKSTTKAAKMMNQQRTKC (SEQ ID NO:29)
  • PTP04 expressed as a GST-fusion protein.
  • GST fusion proteins were produced in DH5-alpha E. coli bacteria as descaribed in Smith, et al Gene 67:31, 1988.
  • Bacterial protein lysates were purified on glutathione-sepharose matrix as described in Smith, et al, supra.
  • Recombinant wild-type and dominant negative (signaling incompetent) PTP04 were purified from bacteria as GST-fusion proteins. Lysates were bound to a glutathione-sepharaose matrix and washed twice with IX HNTG, followed by one wash with a buffer containing 100 mM 2-(N- morpholino) ethansulfonic acid (MES) , pH 6.8, 150 mM NaCl, and 1 mM EDTA.
  • MES 2-(N- morpholino) ethansulfonic acid
  • the assay for phosphatase activity was essentially done as described by Pei et al. (1993) using p-nitrophenolphosphate (PNPP) as a generic PTP substrate. Briefly, after the last washing step, reactions were started by adding 50 mL Assay Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA, and 50 mM PNPP) to the matrix bound proteins. Samples were incubated for 20 min. at 23 °C. The reactions were terminated by mixing 40 ⁇ L of each sample with 960 ⁇ L 1 N NaOH, and the absorbance of p-nitrophenol was determined at 450 nm. To control for the presence of PTP04 in the precipitates, the precipitates were boiled in SDS sample buffer and analyzed by SDS-PAGE. The presence of PTP04 was then detected by immunoblot analysis with anti-PTP04 antibodies.
  • Assay Buffer 100 mM MES pH 6.8, 150 mM Na
  • This example describes the isolation and characterization of the non-receptor tyrosine kinase SAD.
  • SAD non-receptor tyrosine kinase
  • sequence FGE/DVW (SEQ ID NO: 30) is located near the amino terminus of the kinase domain and is unique to Src family members and the Srm/Brk/Frk group.
  • sequence WTAPE (SEQ ID NO: 31) is located just amino terminal to the highly conserved DVWS motif of tyrosine kinases and is contained in the Src family and the Srm/Brk/Frk group as well as the Eph, Csk, Abl, and Fes families.
  • HME human mammary epithelial
  • a SAD probe was used to screen a cDNA library constructed from human breast cancer cell line mRNA to isolate two overlapping, independent clones spanning the kinase domain, but containing apparent introns and presumably arising from incompletely processed transcripts.
  • the 5' end of the coding region was subsequently isolated by sequential RACE reactions from MCF7 RNA, and the entire coding region was re-isolated by PCR from HME cDNA.
  • a typical reaction used 10 ⁇ g total RNA or 2 ⁇ g poly (A) + RNA with 1.5 ⁇ g oligo (dT) ⁇ 2 - ⁇ 8 in a reaction volume of 60 ⁇ L.
  • the product was treated with RNaseH and diluted to 100 uL with H 2 0.
  • 1-4 ⁇ L of these sscDNAs were used in each reaction.
  • Oligonucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer using established phosphoramidite chemistry and were used unpurified after precipitation with ethanol.
  • the degenerate oligonucleotide primers are:
  • a PCR reaction was performed using primers FGE/DVW and WTAPE applied to HME cell cDNA.
  • the primers were added at a final concentration of 0.5 uM each to a mixture containing 10 mM Tris.HCl (pH8.3), 50 mM KC1, 1.5 mM MgC12, 200 uM each deoxynucleoside triphosphate, 0.001% gelatin, and 1.5 U AmpliTaq DNA Poiymerase (Perkin-Elmer/Cetus) , and 1-4 ⁇ L cDNA.
  • PCR fragments migrating at between 450-550 bp were isolated from 2% agarose gels, phosphorylated and repaired by treatment with T4 polynucleotide kinase and Klenow fragment, and blunt-end cloned into the EcoRV site of the vector pBluescriptSK+ (Stratagene U.S.A. ) .
  • Plasmid DNAs were isolated from single colonies by DNA minipreparations using QIAGEN columns and were sequenced using cycle sequencing dye-terminator kit with AmpliTaq DNA Poiymerase, FS (ABI, Foster City, CA) . Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al., J. Mol. Biol.215:403-10, 1990). A novel clone (HME1264) was isolated by PCR with primers FGE/DVW and WTAPE on single- stranded cDNA from HME cells as a template. This clone was subsequently designated as a fragment of human SAD.
  • a lambda ZapII (Stratagene Cloning Systems, La Jolla, CA) cDNA library was constructed using mRNA from a pool of breast carcinoma cell lines as a template for first strand cDNA synthesis with both oligo- (dT) and random priming (library created by Clonetech custom library synthesis department, Palo Alto, CA) .
  • the cell lines used for the pool were MCF7, HBL100, MDA-MB231, MDA-MB175IIV, MDA-MB435, MDA-MB453, MDA-MB468, BT20, T47D and SKBR3, all of which are available from the ATCC.
  • Phage were screened on nitrocellulose filters with the random primed 32 P-labeled insert from HME1264 at 2xl0 6 cpm/mL in hybridization buffer containing 6xSSPE, 50% formamide, 2x Denhardt 's reagent, 0.1% SDS, with 0.05 mg/mL denatured, fragmented salmon sperm DNA. After overnight hybridization at 42 °C, filters were washed in lxSSC, 0.1% SDS at 65 °C. Two overlapping partial clones were isolated and sequenced through the coding region using manual sequencing with T7 poiymerase and oligonucleotide primers (Tabor and Richardson, Proc. Natl. Acad. Sci. U.S.A.
  • Single strand cDNA was prepared as described above using the Superscript Pre-amplification System (GibcoBRL) using 6 ⁇ g total RNA from MCF7 cells as a template and gene specific primers 5556 (5 ' -AGTGAGCTTCATGTTGGCT-3 ' (SEQ ID NO: 34) for RACE 1 or 5848 (5 ' -GGTAGAGGCTGCCATCAG-3 ' (SEQ ID NO:35)) for RACE 2 to prime reverse transcription.
  • Gene specific primers 5556 5 ' -AGTGAGCTTCATGTTGGCT-3 ' (SEQ ID NO: 34) for RACE 1 or 5848 (5 ' -GGTAGAGGCTGCCATCAG-3 ' (SEQ ID NO:35) for RACE 2 to prime reverse transcription.
  • sscDNA was recovered using two sequential ethanol precipitations with ammonium acetate and carrier glycogen homopolymer tail of dA was added by treatment with deoxy- terminal transferase (GibcoBRL) and two reaction mixtures diluted to 50 ⁇ L with TE.
  • Second strand cDNA synthesis by AmpliTaq DNA Poiymerase (Perkin-Elmer/Cetus) was primed with 0.2 uM PENN(dT) 17 (5'- GACGATCGGAATTCGCGA(dT) ⁇ 7 -3 ' (SEQ ID NO: 36) using 1-5 ⁇ L of tailed cDNA as a template and buffer conditions given above.
  • primers PENN (5'-GACGATCGGAATTCGCGA-3' (SEQ ID NO: 37) and 5555 (5'- CCCAGCCACAGGCCTTC-3' (SEQ ID NO: 38) were added at 1 ⁇ M and PCR done with cycling conditions of 94 °C for 30 s, 49 °C for 1 min, and 72 °C for 1 min, 45 sec for 40 cycles.
  • a second, nested PCR was done using 0.2 ⁇ L of the initial PCR reaction as a template and primers PENN (see SEQ ID NO: 37) and 5554 (5'- CCACACCTCCCCAAAGTA-3' (SEQ ID NO: 39) at 1 ⁇ M with an initial 3 min denaturation at 94 °C, followed by cycling conditions of 94 °C for 30 s, 49 °C for 1 min, and 72 °C for 1 min, 45 sec for 35 cycles.
  • PENN see SEQ ID NO: 37
  • 5554 5'- CCACACCTCCCCAAAGTA-3' (SEQ ID NO: 39) at 1 ⁇ M with an initial 3 min denaturation at 94 °C, followed by cycling conditions of 94 °C for 30 s, 49 °C for 1 min, and 72 °C for 1 min, 45 sec for 35 cycles.
  • PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining and Southern hybridization using oligonucleotide 5557 (5'- TGGGAGCGGCCACACTCCGAATTCGCCCTT-3' (SEQ ID NO: 40) end-labeled with 32 P.
  • Reaction products of 500-700 bp were digested with EcoRI and cloned into the EcoRI site of pBluescriptSK ⁇ (Stratagene U.S.A.), and positive clones were identified by colony hybridization with oligonucleotide 5557 as a probe.
  • Clone 16A1 (which encompasses nucleotides 195 to 783 of SEQ ID NO: 10) was isolated and sequenced by a combination of ABI and manual sequencing.
  • a second set of 5' RACE reactions was done based on the sequence of clone 16A1 using the procedure given above except as noted.
  • Gene specific primers were 5848 (SEQ ID NO: 35) for the first strand synthesis, 6118 (5 ' -GCCTGCGTGCGAAGATG-3 ' (SEQ ID NO: 41) for the first PCR, and 6119 (5 ' -CTTCGAGGGCACAGAGCC-3 ' (SEQ ID NO: 42) for the second PCR, and the probe for Southern and colony hybridization was random primed 32P-labeled insert from 16A1.
  • PCR fragments migrating at between 250-450 bp were isolated from 2% agarose gels, phosphorylated and repaired by treatment with T4 polynucleotide kinase and Klenow fragment, and blunt-end cloned into the EcoRV site of the vector pBluescriptSK+ (Stratagene U.S.A.).
  • Clone 20E2 (which encompasses nucleotides 1 to 267 of SEQ ID NO: 10) was isolated and sequenced by a combination of ABI and manual sequencing.
  • the coding region of SAD was subsequently isolated from HME cDNA as two overlapping PCR fragments.
  • Single stranded cDNA was prepared from poly (A) + RNA from HME cells using the Superscript Preamplification System (GibcoBRL) as described above.
  • PCR with AmpliTaq DNA Poiymerase (Perkin-Elmer/Cetus) used 1-2 ⁇ L of cDNA as a template, an initial 3 min denaturation at 94°C, followed by cycling conditions of 94oC for 30 s, 55 °C for 1 min, and 72 °C for 1 min, 45 sec for 30 cycles and the buffer conditions given above.
  • Primers 6642 (5'- ATGGAGCCGTTCCTCAGGAGG-3' (SEQ ID NO: 43) and 6644 (5'- TCACCCAGCTTCCTCCCAAGG-3' (SEQ ID NO: 44) were used to amplify an approximately 710 bp 5' fragment of SAD
  • primers 6643 (5'- AGGCCAACTGGAAGCTGATCC-3 ' (SEQ ID NO: 45) and 6645
  • 6645 5'- GCTGGAGCCCAGAGCGTTGG-3' (SEQ ID NO: 46) were used to amplify an approximately 860 bp 3' fragment of SAD.
  • PCR fragments were isolated from 1% agarose gels, phosphorylated and repaired by treatment with T4 polynucleotide kinase and Klenow fragment, and blunt-end cloned into the EcoRV site of the vector pBluescriptSK+ (Stratagene U.S.A.). Positive clones were identified by colony hybridization with the random primed 32P- labeled insert from 16A1 (for the 5' fragment) and the random primed 32P-labeled insert from HME1264 or 32P-labeled oligonucleotide 5557 (for the 3' fragment) as probes. The overlapping 5' and 3' PCR fragments were ligated together via the unique EcoRI site to give the full length SAD coding region.
  • the complete sequence of the coding region of huma SAD was determined from overlapping 5' and 3' PCR clones amplified from cDNA prepared from HME cells. 5' noncoding sequence was determined from the overlapping RACE fragment 16A1. Sequence was determined manually on both strands using cycle sequencey dye-terminator kit with AmpliTaq DNA Poiymerase, FS (ABI, Foster City, CA) .
  • SAD_h The 1,548 bp human SAD (SAD_h) nucleotide sequence shown in SEQ ID NO: 10 contains a single open reading frame encoding a polypeptide of 488 amino acids.
  • the SAD_h coding region is preceded by a 48 nucleotide 5 ' -untranslated region including an in-frame termination codon four codons before the initiating methionine and a 33 nucleotide 3 ' -untranslated region that includes two in-frame stop codons.
  • sequences of SAD cDNAs were determined from overlapping PCR-amplified fragments from normal HME cell cDNA (nucleotides 49-1548), clones from a breast carcinoma cell lambda cDNA library (nucleotides 694-1548), and overlapping 5' RACE products from MCF7 cDNA (nucleotides 1-783) with the following sequence differences including some likely polymorphic sites.
  • Ambiguities include a change of nucleotide 636 (see SEQ ID NO: 10) from a C in the HME PCR clone to a T in the MCF& RACE product, nucleotide 1477 from a T in the HME PCR clone to a C in the breast carcinoma libray, a deletion of nucleotides GT at positions 919 - 920 in the breast carcinoma library and apparent introns inserted at positions (relataive to SEQ ID NO:10) 694, 995, 1117, and 1334 in the breast carcinoma library.
  • SAD The domain structure of SAD consists of an N-terminal unique domain followed by an SH3 domain, an SH2 domain and a kinase domain. This overall topology is shared by members of the Src, Srm/Brk/Mkk3, and Csk families. SAD is most similar to mouse Srm (GeneBank Accession #D26186) (Kohmura et al., Mol. Cell. Biol. 14: 6915-6925, 1994), a distant SRC relative of unknown function. SAD and Srm share sequence identities in the individual domains of 55% (unique region), 72% (SH3 domain), 78% (SH2 domain), and 85% (kinase domain).
  • SAD and Srm lack both an N-terminal membrane attachment sequence and a potential C-terminal negative regulatory tyrosine.
  • the characteristic HRDLRXAN (SEQ ID NO: 47) sequence in the Src family kinase domain is HRDLAXRN (SEQ ID NO: 48) in SAD and other Srm/Brk/Mkk3 group members.
  • SAD and Srm both contain a potential autophosphorylation site (380Y of SAD) .
  • the N-terminal sequences of SAD and Srm are similar with twenty identical residues out of the first twenty-two amino acids, but the extreme C-termini are quite distinct.
  • SAD_h and Srm_m are distinct genes rather than species orthologues.
  • levels of homology between SAD_h and Srm_m listed above are comparable to those of close Src family members (for example Src_h and Yes_h) , but lower than those of species counterparts (for example Src_h and Src_m) .
  • SAD_h and Srm_m also exhibit distinct expression patterns with SAD_h expression detected by PCR in the duodenum and perhaps testes, but not in other tissues tested, while the Srm_m expression was detected by Northern with highest levels in lung, liver, spleen, kidney, and testes (Kohmura et al., Mol. Cell. Biol.
  • mice 14: 6915-6925, 1994 See Example 2 below.
  • disruption of the Srm gene in mice has no detectable phenotype (Kohmura et al., Mol. Cell. Biol. 14: 6915), suggesting that other related proteins might compensate for its function.
  • RNA was isolated from a variety of human cell lines and fresh frozen normal tissues. (Tumor cell lines were obtained from Nick Scuidero, National Cancer Institute, Developmental Therapeutics Program, Rockville, MD) Single stranded cDNA was synthesized from 10 ⁇ g of each RNA as described above using the Superscript Preamplification System (GibcoBRL) .
  • the SAD expression profile in normal human tissue and multiple cell lines of diverse neoplastic origin was determined by the semi-quantitative PCR assay using primers from sequences in the kinase domain. The results are included in Tables 1 and 2.
  • Table 1 In normal tissue samples (Table 1), modest SAD expression was detected in the duodenum and possible low levels in testes with all other samples negative. Much higher expression was found in a subset of cancer cell lines (Table 2) with the highest levels in some human colon tumor cell lines (HCT-15, SW480, and HT-29) , an ovarian carcinoma (IGROV1), and an intestinal carcinoma (SNU-C2B) . Lesser expression of SAD was also seen in some other tumor cell lines derived from colon, breast, lung, ovary, and kidney as shown in Table 2.
  • a SAD-specific antisera was raised in rabbits against a
  • Expression constructs were generated by PCR-based mutagenesis in which a BamHI site was introduced upstream of the initiating Met giving a 5' untranslated sequence of 5'- GGATCCCCGGACC-3 ' (SEQ ID NO: 51).
  • An N-terminal hexahistidine tagged construct was also created by PCR with the coding sequence for MRGSHHHHHH (SEQ ID NO: 52)
  • the Spel-Xhol full length SAD constructs were also cloned from pBluescriptSK ⁇ (Stratagene U.S.A.) into the yeast expression vector pRSP (Superti-Furga et al., EMBO J. 12, 2625- 2634) .
  • This vector contains a thia ine-repressible promoter in a shuttle vector for inducible expression in Schizo- saccharomyces pombe.
  • This system has been useful in studies of SRC family members for testing negative regulation by CSK, screening for additional regulators, and purifying recombinant protein (Superti-Furga et al., EMBO J. 12, 2625-2634; Superti- Furga et al., Nature Biotech. 14, 600-605).
  • the pcDNA3 expression plasmids (5 ⁇ g DNA/60 mm plate) containing the unmodified and hexahistidine-tagged SAD genes were introduced into 293 cells with lipofectamine (Gibco BRL) .
  • the cells were harvested in 0.25 mL RIPA (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, ImM DTT, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 2 ⁇ g/mL aprotinin, 1 ⁇ g/mL leupeptin, and 25 ⁇ g/mL trypsin inhibitor) . Sample aliquots were resolved by SDS polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels and electrophoretically transferred to nitrocellulose.
  • PAGE polyacrylamide gel electrophoresis
  • Non-specific binding was blocked by preincubating blots in Blotto (Tris buffered saline containing 5% w/v non-fat dried milk and 0.1% v/v Tween-20) , and recombinant protein was detected using affinity-purified SAD-specific polyclonal antibody and peroxidase-linked secondary antibody with the ECL kit (Amersham Life Science) . Hexahistidine tagged protein was also detected using RGS»His Antibody (QIAGEN Inc.). Phosphotyrosine-containing proteins were detected by Western blotting with monoclonal antibody 4G10 (Upstate Biotechnology) with 3% BSA as the blocking agent.
  • Affinity purified antipeptide antibody raised against the C-terminus of SAD recognized a specific -55 kDa protein in transfected 293 cells with greater than 90% of the expressed protein being RIPA-insoluble. This molecular weight is consistent with the molecular weight predicted based on SAD's primary amino acid sequence (54,510 kD) .
  • SAD-transfected cells contain a prominent approximately 55 kDa tyrosine phosphorylated protein that is absent in vector controls. The phosphorylated protein is most likely SAD itself because the band is clearly detected in IP-Westerns using anti-SAD crosslinked to protein A beads and 4G10 as the blotting antibody although anti-SAD only inefficiently immuno- precipitates.
  • S. pombe was used to express recombinant SAD as an approach to studying its function and regulation since this expression system has proven useful for studying Src family members (Superti-Furga et al., EMBO J. 12, 2625-2634; Superti- Furga et al., Nature Biotech. 14, 600-605).
  • S. pombe strain SP200 h-s leul.32 ura4 ade210
  • transformations with pRSP expression plasmids were done by the lithium acetate method (Moreno et al., 1991; Superti-Furga et al., EMBO J. 12, 2625-2634).
  • Cells were grown in the presence of 1 uM thiamine to repress expression from the nmtl promoter or in the absence of thiamine to induce expression.
  • SAD-expressing strains show no growth defect compared to vector controls in either liquid culture or solid media. This result contrasts with the toxicity caused by expression of several other tyrosine kinases including Src and Frk.
  • SAD protein can be detected in these strains as a weak band on Western blots using the polyclonal antibody against the C-terminus.
  • SAD itself is the only detectable phosphotyrosine-containing protein, however in the presence of pervanadate, cellular proteins are also phosphorylated. This observation contrasts with the results seen for Src and MKK3 which phosphorylate many yeast proteins even in the absence of phosphatase inhibitors.
  • Example 10 Assay for SAD Kinase Activity
  • the example below describes an in vitro assay for SAD kinase activity.
  • the assay is useful for the identification of modulators of SAD activity.
  • Immunoprecipita- tions were done by mixing yeast extract (100 ⁇ g total protein in 100 ⁇ L NP-40 lysis buffer) with 0.6 ⁇ g the RGS»His Antibody (QIAGEN Inc.) and 10 ⁇ L Protein A/G agarose (Upstate Biotechnology) for 3 hrs at 4 °C. IP complexes were washed four times in 1 mL lysis buffer and once in 1 mL kinase buffer (20 mM Na-HEPES pH 7.5, 10 mM MnC12, 2 mM 2-mercaptoethanol, and 10 ⁇ M sodium vanadate) .
  • kinase assays were for 10 min at 30 °C in 40 ul kinase buffer containing 15 ⁇ M ATP, 0.5 uCi g-32P-ATP, and either 3 ⁇ g denatured enolase or 10 ⁇ g poly-Glu-Tyr (4:1) as the substrate. Extracts were assayed using 2-10 ⁇ g total protein per reaction and IP complexes were assayed using 5 ul Protein A/G beads per assay. Reactions were terminated by the addition of SDS sample buffer and the samples were resolved on an 10% SDS polyacrylamide gel and visualized by auto- radiography. Results
  • SAD was able to phosphorylate both denatured enolase and poly-Glu-Tyr in vitro. Phosphorylation of both substrates was detected in crude yeast lysates expressing SAD but not in lysates from vector control strains. In addition, anti-His IP complexes from SAD-expressing strains but not control strains phosphorylated both denatured enolase and poly-Glu-Tyr.
  • PTPIO The example below describes the isolation and identification of new PTP sequences from primary murine fat and rat basal forebrain and the subsequent cloning of a full-length PTP05 sequence Also described are probes useful for the detection of PTP05 and/or PTPIO in cells or tissues.
  • RNAs were isolated using the Guanidine Salts/Phenol extraction protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987) from ob/ob mouse fat and, separately, rat basal forebrain. These RNAs were used to generate single-stranded cDNA using the Superscript Preamplification System (GIBCO BRL, Gaithersburg, MD.; Gerard, et al, FOCUS 11:66, 1989) under conditions recommended by the manufacturer. A typical reaction used 10 ⁇ g total RNA with 1.5 ⁇ g oligo (dT) 12 - 1 8 in a reaction volume of 60 ⁇ L. The product was treated with RNaseH and diluted to 100 ⁇ L with H 2 0. For subsequent PCR amplification, 1-4 ⁇ L of this sscDNA was used in each reaction.
  • GEBCO BRL Superscript Preamplification System
  • oligonucleotide primers were synthesized on an Applied Biosystems 394 DNA synthesizer using established phosphoramidite chemistry, precipitated with ethanol and used unpurified for PCR.
  • sequence of the degenerate oligonucleotide primers follows:
  • PTPDFW 5 ' -GAYTTYTGGVRNATGRTNTGGGA- (sense) (SEQ ID NO: 17)
  • PTPHCSA 5 ' -CGGCCSAYNCCNGCNSWRCARTG -3* (antisense) (SEQ ID NO: 18) .
  • the primers were added at a final concentration of 5 ⁇ M each to a mixture containing 10 mM Tris'HCl (pH8.3), 50 mM KC1, 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Poiymerase (Perkin-Elmer/Cetus), and 1-4 ⁇ L cDNA. Following 3 min denaturation at 95 °C, the cycling conditions were 94 °C for 30 sec, 50 °C for 1 min, and 72 °C for 1 min 45 sec for 35 cycles.
  • PCR fragments migrating between 350-400 bp were isolated from 2% agarose gels using the GeneClean Kit (BiolOl) , and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol.
  • Colonies were selected for mini-plasmid DNA-preparations using Qiagen columns and the plasmid DNA was sequenced using cycle sequencing dye-terminator kit with AmpliTaq DNA Poiymerase, FS (ABI, Foster City, CA) . Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al., J. Mol. Biol.215: 403-10) . Several copies of a clone encoding a novel PTP (R90-2-22), designated SuPTP05, was isolated from murine adipose tissue.
  • PTPIO A related clone, PTPIO, was isolated from rat basal forebrain.
  • RACE rapid amplification of cDNA ends
  • Marathon-Ready cDNA (Clontech, Palo Alto, CA) made from mouse testis was used in the RACE reactions with the following primers:
  • 5'-CACCGTTCGAGTATTTCAGATTGTGAAGAAGTCC-3' (6595) (SEQ ID NO: 21) , 5'-GGACTTCTTCACAATCTGAAATACTCGAACGGTG-3' (6596) (SEQ ID NO: 22) , 5'-CCGTTATGTGAGGAAGAGCCACATTACAGGACC-3' (6599) (SEQ ID NO:23), 5'-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3' (6600) (SEQ ID NO: 24 ) , AP-1, and AP-2 (Clontech) .
  • RT-PCR primers for PTP05 sequeqncing 5'-CACCGTTCGAGTATTTCAGATTGTGAAGAAGTCC-3' (6595) (SEQ ID NO:21), 5'-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3' (6600) (SEQ ID NO: 24 ) .
  • Isolated cDNA fragments encoding SuPTP05 were confirmed by DNA sequening and subsequently used as probes for the screening of a murine testis cDNA library.
  • Two murine testis cDNA libraries (IZapII, Stratagene, La Jolla, CA and lgtlO, Clontech) , were screened to isolate full- length transcripts encoding PTP05.
  • the 5' or 3'-RACE fragments were 32 P-labeled by random priming and used as hybridization probes at 2xl0 6 cpm/mL following standard techniques for library screening.
  • Pre-hybridization (3 hrs) and hybridization (overnight) were conducted at 42 °C in 5X SSC, 5 X Denhart's solution, 2.5% dextran sulfate, 50 mM Na2P04/NaHP04 [pH 7.0], 50% formamide with 100 mg/mL denatured salmon sperm DNA.
  • Stringent washes were performed at 65 °C in 0.1X SSC and 0.1% SDS.
  • Several overlapping clones were isolated and found to span the collective sequences of the PCR fragment (R90-2-22) and the RACE products.
  • the primary murine PTP05 transcript is 1785 nucleotides and encodes a predicted polypeptide of 426 amino acids with a predicted molecular weight of 49122 daltons (SEQ ID NO: 3 and SEQ ID NO: 11).
  • the PTP05 coding sequence is flanked by a 198 nucleotide 5 ' -untranslated region and a 279 nucleotide 3'- untranslated region ending with a poly (A) tail.
  • the ATG beginning at nucleotide position 199 conforms to the Kozak consensus for an initiating methionine.
  • One clone (#6.1) containes an insertion of 111 bp at nucleotide 328 resulting in an addition 37 amino acids added inframe to the coding sequence.
  • a second clone (#10.1) has a deletion of 93 bp beginning at nucleotide 1415, resulting in a frame-shift and premature termination. Upstream of the 198bp 5'UTR, the numerous clones diverge into 2 groups, extending the 5'UTR an additional 98-153 bp.
  • one clone (#15.3) lacks the polyA tail at nucleotide 1758 extends the 3' UTR by another 300 nucleotides.
  • the amino acid sequence shows no signal sequence or a transmembrane domain, and PTP05 is therefore predicted to be an intracellular protein.
  • the N-terminal domain of murine PTP05 extends from amino acid 1 to 187 and is unique, i.e. contains no significant homology to any protein in the non-redundant protein database.
  • the non-redundant protein database consists of peptide sequences from GenBank Genpept, PIR, and SwissProt.
  • PTPs The C-terminal tail of PTP05 extends beyond the cata- lytic domain from amino acids 421-426 and is not homologous to other protein tyrosine phosphatases. Motifs found in the cytoplasmic domain of other mammalian PTPs that are absent from
  • PTP05 include: SH2, Talin/Ezrin-like, PEST, GLGF, and
  • Retinaldehyde-binding protein domains Owing to its divergent catalytic domain and absence of well-known non-catalytic motifs, we have designated PTP05 as a new and distinct family of protein tyrosine phosphatases.
  • murine PTP05 contains an insertion of 111-bp in the N-terminal coding region, extending the sequence by 37 aa (SEQ ID NO: 4 and SEQ ID NO: 12).
  • This 1,896 bp "long" form of murine PTP05 encodes a polypeptide of 463 amino acids with a predicted molecular weight of 53716 daltons .
  • the insertion is located at amino acid positions 44-80 and is not significantly homologous to other proteins in the non- redundant protein database.
  • a third form of PTP05 has a deletion of nucletotides 1415- 1507 resulting in a frame shift and C-terminal truncation leading to an alternate sequence from amino acids 406-412 (SEQ ID NO: 5 and SEQ ID NO: 13).
  • the 1,692 bp "C-trunc" murine PTP05 encodes a polypeptide of 412 amino acids with a predicted molecular weight of 47233 daltons.
  • the rat PTP10 clone shares 92% identity at the DNA level (320 nucleotides) and 85% amino acid identity at the protein level (107 amino acids) with murine PTP05 (See Figure 1) .
  • the level of homology of the two catalytic domains suggests that PTP05 and PTPIO are distinct but related genes, and thus PTPIO is considered to be a second member of this new PTP family.
  • Partial sequences of rat PTPIO are shown in SEQ ID NO: 6 (nucleic acid) and SEQ ID NO: 14 (amino acid).
  • a mouse normal tissue Northern blot containing 2 ⁇ g polyA+ mRNA per lane from 8 different mouse adult tissues (lung, testis, brain, heart, liver, kidney, spleen, skeletal muscle) on a charge-modified nylon membrane was obtained from Clontech (#7762-1, Palo Alto, CA) .
  • the membrane was hybridized with randomly primed [a 32 P]dCTP-labeled probe synthesized from a 241 bp EcoRI fragment of R90-2-22 (see above) . Hybridization was performed at 42 °C overnight in 5X SSC, 2% SDS, 10X Denhardt ' s solution, 50% formamide, 100 ⁇ g/mL denatured salmon sperm DNA with 1-2 x IO 6 cpm/mL of 32 P-labeled DNA probe. The membrane was washed at room temperature in 2X SSC/0.05% SDS for 30 min and followed by 50 °C in 0.2X SSC/0.1% SDS for 30 min, and exposed overnight on Kodak XAR-2 film.
  • RT-PCR with gene specific primer-pairs showed that expression of the transcripts encoding PTP05 confirmed the results from Northern analysis and also detected low levels in adipose, kidney, small intestine, and cells/tissues of hematopoietic or immune origin including spleen, thymus, lymph node, bone marrow, and peripheral blood lymphocytes) .
  • RT-PCR with rat PTPIO gene specific primers confirmed the results from the Northern analysis, detecting a strong signal only in rat testis sscDNA and not in templates corresponding to rat skeletal muscle, heart, kidney, spleen, adrenal gland, lung, liver, intestine, uterus, spinal cord, brain, cortex and ovary.
  • PTP05 reletively selective expression of PTP05 in cells of hematopoetic or immune origin suggests a potential involvement in immune regulation including T and B cell survival, differentiation or co-stimulation, and/or inflammatory, immunosuppressive or autoimmune disorders. Additionally, expression in adipose tissue (also the source from which PTP05 was originally isolated) suggests a possible role in metabolic disorders such as diabetes.
  • the following example illustrates the contruction of vectors for expression of recombinant PTP05 and the creation of recombinant cell lines expressing PTP05. Similar vectors and recombinant cell lines can be generated using PTPIO and the techniques described herein.
  • Dominant negative PTP05 constructs were also made in both pLXSN and pRK5 by mutation of the invariant Cys in the conserved His-Cys-Ser-Ala-Gly motif (SEQ ID NO: 56) to an Ala by PCR mutagenesis.
  • the entire PTP05 open reading frame excluding the initiating methionines was generated by PCR and ligated into pGEX vector for bacterial production of GST-fusion proteins for immunization of rabbits for antibody production.
  • This vector contains the glutathione-S-transferase coding sequence followed by a polylinker for generating recombinant fusion proteins.
  • the GST moiety comprises the N-terminal portion of the fusion protein.
  • the pRK5 expression plasmids (10 ⁇ g DNA/100 mm plate) containing the HA-tagged PTP05 gene can be introduced into COS and 293 cells with lipofectamine (Gibco BRL) . After 72 hours, the cells were harvested in 0.5 mL solubilization buffer (20 mM HEPES pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL aprotinin) .
  • solubilization buffer (20 mM HEPES pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/
  • pLXSN recombinant constructs containing the PTP05 gene were transfected into an amphotropic helper cell line PA317 using CaCl 2 -mediated transfection. After selection on G418, the cells were plated on normal media without G418 (500 ⁇ g/mL) . Supernatants from resistant cells were used to infect the ecotropic helper cell line GP+E86, and cells again selected on G418. Resistant cells were again taken off G418, and the supernatants harvested every 8-12 hours and pooled as virus stock. Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498. Viral stock titers were typically ⁇ 10 6 /mL.
  • PTP05-specific immunoreagents were raised in rabbits against a pool of three KLH-conjugated synthetic peptides corresponding to unique sequences present in human PTP04.
  • the peptides (see below) were conjugated at the C-terminal residue with KLH.
  • Peptides used for immunizing rabbits PTP05: peptide 433A - MSSPRKVRGKTGRDNDEEEGNSGNLNLRN (SEQ ID NO:57) peptide 431A - SPVLSGSSRLSKDTETSVSEKELTQLAQI (SEQ ID NO: 58) and peptide 432A - WDVSDRSLRNRWNSMDSETAGPSKTVSPV (SEQ ID NO:59) .
  • Additional immunoreagents were generated by immunizing rabbits with a purified preparation of a GST-fusion protein containing the entire coding region of PTP05.
  • the GST-fusion protiens were produced in DH5-alpha E. coli bacteria as described in Smith, et al Gene 67:31, 1988.
  • Bacterial protein lysates were purified on glutathione-sepharose matrix as described in Smith, et al., supra.
  • Recombinant wild-type and dominant negative (signaling incompetant) PTP05 were purified from bacteria as GST-fusion proteins. Lysates were bound to a glutathione-sepharaose matrix and washed twice with IX HNTG, followed by one wash with a buffer containing 100 mM 2- (N- morpholino) ethansulfonic acid (MES), pH 6.8, 150 mM NaCl, and 1 mM EDTA.
  • MES 2- (N- morpholino) ethansulfonic acid
  • the assay for phosphatase activity was essentially done as described by Pei et al. (1993) using p-nitrophenolphosphate (PNPP) as a generic PTP substrate. Briefly, after the last washing step, reactions were started by adding 50 mL Assay Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA, and 50 mM PNPP) to the matrix bound proteins. Samples were incubated for 20 min. at 23 °C. The reactions were terminated by mixing 40 ⁇ L of each sample with 960 ⁇ L 1 N NaOH, and the absorbance of p-nitrophenol was determined at 450 nm.
  • Assay Buffer 100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA, and 50 mM PNPP
  • the example below describes the isolation and identification of a new PTP sequence from mouse tissues and the subsequent cloning of a full-length human ALP. Also described are probes useful for the detection of ALP in cells or tissues.
  • RNAs were isolated using a commonly known guanidine salts/phenol extraction protocol from normal mouse fat and rat pituitary. Chomczynski & Sacchi, 1987, Anal. Biochem. 162: 156. These RNA extracts were used to generate single-stranded cDNA using the Superscript Pre-amplification System (GIBCO BRL, Gaithersburg, MD.; Gerard et al., 1989, FOCUS 11: 66) under conditions recommended by the manufacturer. a typical reaction used 10 ⁇ g total RNA with 1.5 ⁇ g oligo (dT) ⁇ 2 _ ⁇ 8 in a reaction volume of 60 ⁇ L. The product was treated with RNaseH and diluted to 100 ⁇ L with H 2 0. For subsequent PCR amplification, 1-4 ⁇ L of this sscDNA was used in each reaction.
  • oligonucleotide primers were synthesized on an Applied Biosystems 394 DNA synthesizer using established phosphoramidite chemistry, precipitated with ethanol and used unpurified for PCR.
  • sequence of the degenerate oligonucleotide primers were as follows:
  • PTPDFW 5'-GAYTTYTGGVRNATGRTNTGGGA-3' (SEQ ID NO: 17)
  • PTPHCSA 5'-CGGCCSAYNCCNGCNSWRCARTG-3' (SEQ ID NO: 18)
  • PTPYINA 5'-ATCCCCGGCTCTGAYTAYATHMAYGC-3' (SEQ ID NO: 60)
  • PCR reactions were performed using degenerate primers applied to the single-stranded cDNA listed above.
  • the primers were added at a final concentration of 5 ⁇ M each to a mixture containing 10 mM TrisHCl (pH8.3), 50 mM KC1, 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Poiymerase (Perkin-Elmer/Cetus), and 1-4 ⁇ L cDNA. Following 3 min denaturation at 95°C, the cycling conditions were 94 °C for 30 s, 50 °C for 1 min, and 72°C for 1 min 45 s for 35 cycles.
  • PCR fragments migrating between 350-400 bp were isolated from 2% agarose gels using the GeneClean Kit (BiolOl) , and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol.
  • Colonies were selected for mini plasmid DNA-preparations using Qiagen columns and the plasmid DNA was sequenced using cycle sequencing dye-terminator kit with AmpliTaq DNA Poiymerase, FS (ABI, Foster City, CA) . Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm. Altschul et al., J. Mol. Biol. 215: 403-410.
  • a human cDNA library was constructed in lambda ZapII (Stratagene, La Jolla, CA) from polyA+ RNA isolated from the human neuroblastoma cell line IMR32. The library was screened to isolate full-length transcripts encoding ALP. The murine ALP fragment was 32 P- labeled by random priming and used as a hybridization probe at
  • the 4,456 bp human ALP nucleotide sequence encodes a polypeptide of 1,274 amino acids.
  • the amino acid sequence shows no signal sequence or a transmembrane domain and is therefore an intracellular protein.
  • the N-terminal end extends from amino acids 1-857 and contains several putative tyrosine phosphorylation sites and a proline-rich region (30.6% prolines) from amino acids 353-777.
  • This proline-rich region is distantly related to plant extensin proteins (30.2% amino acid identity with Zea mays extensin-like protein GB:Z34465 using Smith-Waterman alignment) and may represent a protein interaction domain as well as the site for interaction with proteins containg SH3 motifs.
  • the C-terminal tail of ALP extends from amino acid 1097-1274 and contains a proline/serine rich region (45.6% serines plus prolines from amino acids 1101- 1214) resembling a PEST motif. This region also could serve as a target for binding proteins via their SH3 motifs.
  • the catalytic domain extends from amino acids 858-1096 and shares 32-37% amino acid identity to PTPs from multiple subfamilies: TC-PTP (P17706: 37.1%) PTP-BAS (D21209: 32.9%), PTP ⁇ (M34668: 34.2%), PTP ⁇ (P23467: 34.2%), PTP ⁇ (A49104: 33.2%), PTPIB (P20417: 34.9%), suggesting that it represents a new family of PTPs. While all other cytoplasmic PTPs have their catalytic domain at either the N- or C-terminal portion of the protein, ALP has a central catalytic domain flanked by large N- and C-terminal domains.
  • catalytic domain conserves most of the invariant residues present in other PTPs, but does has several atypical amino acids.
  • amino acid sequence HCSAG SEQ ID NO: 56
  • HCSSG amino acid positions 1029-1033
  • This motif is in the catalytic site of the crystal stucture of PTPIB and PTPa, and the Ala to Ser change may effect catalyitic activity or specificty.
  • ALP also has a change from WPD to WPE (amino acids positions 993 - 995) in its predicted surface loop of the catalytic domain.
  • Northern blots were prepared by running 20 ⁇ g total RNA per lane isolated from 60 different tumor cell lines (HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, A549,
  • RNA samples were run on a denaturing formaldehyde 1% agarose gel and transferred onto a nitrocellulose membrane (BioRad, CA) . Additional human normal tissue Northern blots containing 2 ⁇ g polyA+ mRNA per lane from 16 different human normal tissues
  • Nitrocellulose membranes for the total RNA samples were hybridized with randomly primed [gamma- 32 P] dCTP-labeled probes synthesized from a 1 kb fragment of EcoRI-NotI of ALP. Hybridization was performed overnight at 42 °C in 4X SSPE, 2.5X Denhardt ' s solution, 50% formamide, 200 ⁇ g/mL denatured salmon sperm DNA, 100 ⁇ g/mL yeast tRNA (Boehringer Mannheim, IN) , 0.2% SDS with 5 x IO 6 cpm/mL of [gamma- 32 P] dCTP-labeled DNA probe on a Techne Hybridizer H-l.
  • the blots were washed with 2X SSC, 0.1% SDS, at 65 °C for 20 min twice followed by 0.5 X SSC in 0.1% SDS at 65 °C for 20 min.
  • the blots were exposed to a phospho-imaging screen for 24 hours and scanned on a Molecular Dynamics Phosphoimager SF.
  • RT-PCR Reverse transcriptase PCR
  • the intensity of the fragment specific to ALP were compared among different RNA samples.
  • a rating of 4 represents large quantities of ALP transcript while a rating of 0 represents little or none of the transcript was detected. It should be noted that detection of proteins by RT-PCR indicates a relatively higher abundance than detection by Northern blot as the RT-PCR technique utilizes total RNA whereas Northern blot analysis is performed using an enriched RNA source (mRNA) .
  • a single ALP mRNA transcript of approximately 5.0 kb was visualized by Northern analysis. This transcript was identified in most of the normal tissue samples tested. However, the Northern analysis results shown in the Table 1 illustrate that the relative abundance of ALP mRNA is quite divergent. In normal tissues, ALP was identified in highest quantities in pancreas, followed by heart, testis, and skeletal muscle. Lower levels of the ALP transcript were identified in placenta, thymus, lung, brain, liver, spleen, uterus, prostate and small intestine. None of the ALP transcript was detected in colon, kidney and peripheral blood leucocytes (PBLs) . ALP expression was also detected in normal human adipocytes by RT-PCR methods.
  • the ALP RNA transcript was most abundant in NCI-H226 (lung tumor) , SK-OV-3 (ovarian tumor) , and RPMI 8226 (leukemia) cell lines.
  • the transcript was identified at lower amounts in SNB-19 (CNS tumor), SF-268 (CNS tumor), SN12C (kidney tumor), SK-MEL-2 (melanoma), UACC-62 (melanoma), and UACC-257 (melanoma) cell lines.
  • the ALP transcript was not detected in the remaining of 44 human tumor cell lines.
  • Table 1 A summary of expression of ALP is shown in Table 1 below.
  • the following example illustrates the contruction of vectors for expression of recombinant ALP and the creation of recombinant cell lines expressing ALP.
  • Expression constructs were generated by PCR-assisted mutagenesis in which the entire coding regions of ALP was introduced into the mammalian expression vectors pcDNAIII (Invitrogen) for transient expression analysis. Additional ALP constructs were made by oligonucleotide based PCR mutagenesis to convert atypical residues in the PTP-related domain back to the amino acids more commonly present in other catalytically active PTPs. These changes include: His to Tyr at amino acid 861 (See SEQ. ID. NO.:2); Ala to Gly at amino acid 902; Phe to trp at amino acid 941; Glu to Asp at amino acid 995; and Ser to Ala at amino acid 1032. Additional constructs containing paired mutations as above were generated for amino acid positions 941/1032 and 902/1032. These constructs were ligated into the pcDNAIII mammalian expression vector behind the CMV promoter.
  • the entire ALP open reading frame excluding the initiating methionines was generated by PCR and ligated into pGEX vector
  • This vector contains the glutathione-S-transferase coding sequence followed by a polylinker for generating recombinant fusion proteins.
  • the GST moiety comprises the N- terminal portion of the fusion protein.
  • the various ALP mutants were also inseted into the pGEX vecotr for production of recombinant protein reagents.
  • the pcDNAIII expression plasmids (10 ⁇ g DNA/100 mm plate) containing the wild-type and mutant forms of the ALP gene were introduced into 293 cells with lipofectamine (Gibco BRL) . After 72 hours, the cells were harvested in 0.5 mL solubilization buffer (20 mM HEPES pH7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 &g/mL aprotinin) .
  • solubilization buffer (20 mM HEPES pH7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 &g/mL
  • Endogenous ALP was detected as a 200 kD protein in Western blots of lysates from a variety of tumor cell lines including human glioblastomas (U87MG, ATCC HTB 14; U118MG, ATCC HTB 15; U138MG, ATCC HTB 16; A172, ATCC CRL 1620; Hs683, ATCC HTB 138), rodent gliomas (C6, ATCC 107), rodent pituitary tumors (ATT20, ATCC CCL 89; GH3, ATCC CCL 82.1), human neuroblastomas (SKNMC, ATCC HTB 10; IMR 32, ATCC CCL 127), and rodent adrenal pheochro ocytomas (PC12, ATCC CRL 1721) .
  • human glioblastomas U87MG, ATCC HTB 14; U118MG, ATCC HTB 15; U138MG, ATCC HTB 16; A172, ATCC CRL 1620; Hs683, ATCC HTB 138
  • ALP protein could not be immunoprecipitated from the non-transformed cell line NIH 3T3 (ATCC CRL 1658) . It is unclear why native ALP protein appears to be larger (200 kDa) than recombinant ALP detected in transfected 293 cells (180 kDa) . The difference could be the result of alternative RNA splicing, or a post-translational modification in the cell lines where it is endogenously expressed. Preliminary experiments indicate that ALP is phosphorylated on serine and threonine residues in transfected 293 cells.
  • tyrosine-phosphorylated proteins are associated with ALP since they are detected in Western blots using an anti- phosphotyrosine antibody following immunoprecipitation of endogenous ALP from human tumor cell lines such as IMR32 after treatments with the phosphatase inhibitor pervanadate.
  • pLXSN recombinant constructs containing the ALP gene are transfected into an amphotropic helper cell line PA317 using CaCl 2 -mediated transfection. After selection on G418, the cells are plated on normal media without G418 (500 ⁇ g/mL) . Supernatants from resistant cells are used to infect the ecotropic helper cell line GP+E86, and cells again selected on G418. Resistant cells are again taken off G418, and the supernatants harvested every 8-12 hours and pooled as virus stock. Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498. Viral stock titers are typically ⁇ 10 /mL.
  • NIH-3T3, BALB/3T3 or other suitable cells are grown in 100 mm plates with DMEM (Gibco) containing 10% fetal calf serum
  • FCS FCS
  • the cells are superinfected with the ALP retrovirus by adding approximately 3 mL viral supernatant to 15 mL culture media for approximately 24 hours.
  • Cells expressing the retroviral constructs are then selected by growth in DMEM/10% FCS supplemented with 500 ⁇ g/mL G418.
  • Example 19 Generation Of Anti-Alp Antibodies ALP-specific immunoreagents were generated by immunizing rabbits with the bacterially expressed N-terminal 352 amino acid portion of ALP expressed as a GST-fusion protein. Fusion protein was affinity purified using glutathione-sepharose colums (Pharmacia) . Polyclonal anti-serum against the N-termi- nal portion of ALP was generated by repeatedly immunizing rabbits with the purified GST-futions protein. Affinity- purified ALP antibody was obtained by binding serum IgG to ALP- GST-fusion protein immobilized on glutathione-sepharose and eluting with low pH and high salt.
  • Recombinant wild-type and mutant ALP proteins are purified from bacteria as GST-fusion proteins. Lysates are bound to a glutathione-sepharose matrix and eluted with glutathione. The purified proteins are then washed with 2 x 1 mL HNTG, followed by one wash with 1 mL of a buffer containing 100 mM 2- (N- morpholino) ethansulfonic acid (MES), pH 6.8, 150 mM NaCl, and 1 mM EDTA.
  • MES N- morpholino
  • the assay for phosphatase activity is essentially done as described by Pei et al.(1993) using p- nitrophenolphosphate (PNPP) as a generic PTP substrate.
  • reactions are started by adding 50 mL Assay Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA, and 50 mM p-nitrophenylphosphate) to the precipitates. Samples are incubated for 20 min. at 23 °C. The reactions are terminated by mixing 40 ⁇ L of each sample (without beads) with 960 ⁇ L 1 N NaOH, and the absorbance of p- nitrophenol was determined at 450 nm. To control for the presence of ALP in the precipitates, the precipitates are boiled in SDS sample buffer and analyzed by SDS-PAGE. The presence of ALP is then detected by immunoblot analysis with anti-ALP antibodies.
  • Assay Buffer 100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA, and 50 mM p-nitrophenylphosphate
  • kinase Activity The following protocol describes the reagents and procedures used to determine Zap70 protein kinase activities measuring phosphorylation of Band III-GST as readout. This assay is used in search for inhibitors of Zap70.
  • GZH Baculovirus (Pharmingen, CA) encoding for muta- tionally activated form of Zap70, in which a tyrosine residue at position 492 is replaced with a phenylalanine residue (Y492F), containing a C-terminal HA tag and a N-terminal GST tag (GST-Zap70-HA) is used.
  • Cell lysates SF9 cells were infected with the GZH virus at MOI of 10 for 96 hours. The cells were then washed once with PBS and lysed in lysis buffer. Insoluble material was removed by centrifugation (5 min. at 10 000 x g) . Aliquots of lysates were frozen in dry ice/ethanol and stored at -80 °C until use. 3.
  • Band III-GST Band III-GST fusion protein (amino acid sequence: MEELQDYEDMMEEN (SEQ ID NO:62)) was expressed in XL1 Blue cells transformed with pGEX -2TK-Band III. Protein expression was induced by addition of 0.5 mM IPTG while shaking the bacterial culture for 18 hours at 25 °C. Band III-GST by was purified by Glutathione affinity chromatography, Pharmacia, Alameda, CA
  • Lysis buffer Kinase buffer: 10 mM Tris, pH 7.5 10 mM MgCl 2 150 mM NaCl 10 mM MnCl 2 1% NP40 10 mM DTT 1 mM PMSF 20 mM HEPES/C1, pH 7.5
  • Blocking buffer Wash buffer (TBST) :
  • Coat 1 ⁇ g/well 4G10 (Upstate Biotechnology, NY) in 100 ⁇ L of PBS overnight at 4 °C and block with 200 ⁇ L of blocking buffer for at least hour.
  • Biotinated peptide 242 was bound to the ELISA Plate by incubating 1 ⁇ g/well in 100 ⁇ L PBS overnight at 4 °C with streptavidin coated ELISA Plate (see above) . The wells were blocked with 200 ⁇ L blocking buffer for 30 minutes at room temperature, after which the blocking buffer was removed by aspiration. Insect cell lysate containing the Zap70 fusion protein (GZH) was added (30 ⁇ g/well, volume adjusted to 100 ⁇ L/well with lysis buffer) and left to incubate at 4 °C for 2 hours. The lysate was removed by aspiration and the wells washed with TBST. Substrate and test compound (if any) were added and allowed to stand for 15 minutes (GST-Band III, 5 ⁇ g/well in 90 ⁇ L final volume) . The kinase reaction was started by the addition of 10 ⁇ L of Zap70 fusion protein (GZH) was added (30 ⁇ g/well, volume
  • 0.1 mM ATP per well for a final concentration of 10 ⁇ M.
  • the 96 well plate was left for 30 minutes at room temperature (shaking) after which 90 ⁇ L of the reaction liquid was transferred to wells in a 96 well plate previously coated with an anti-phosphotyrosine antibody (UB40, Upstate Biotechnology, NY) . This plate was allowed to stand for 30 minutes at room temperature, after which the liquid was removed and the wells washed with TBST. Rabbit anti-GST antibody was added (0.1 ⁇ g/well in 100 ⁇ L blocking buffer) and incubated for 30 minutes at room temperature. The liquid was again removed and the wells washed with TBST.
  • Goat anti-Rabbit-IgG-HRP was added at 1:40,000 dilution in 100 ⁇ L of blocking buffer for 30 minutes at room temperature, after which it was removed and the wells washed with TBST and developed with ABTS.
  • the plate is then read in an ELISA plate reader at 410 nm. If the protein being tested is a captured protein, the reading from the ELISA plate reader can be related to the modulating activity of the test compound when it is compared with the activity of a control protein.
  • Example 22 Isolation And Characterization Of ALK-7
  • oligonucleotides encoding amino acid motifs within kinase subdomains II and VI common to all known mammalian STK receptors.
  • Subdomain II is at the N- terminus of the kinase domain and contains the invariant lysine residue that is essential for enzyme activity and is involved in ATP binding by interacting with the a- and b-phosphates of all kinases whose structure has been elucidated.
  • Subdomain VI is referred to as the catalytic loop and contains the consensus motif HRDLKXXN (SEQ ID NO: 64).
  • the Asp residue is involved in accepting the proton from the hydroxyl group during the phosphotransfer process key to all protein kinases. Based on comparison of all STK receptors, we designed degenerate oligonucleotide primers to these subdomains that would recognize both type I and type II STK receptors.
  • ALK-7 novel DNA fragment
  • RNAs were isolated using the Guanidine Salts/Phenol extraction protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987) from normal human tissues, from regional sections of human brain, from cultured human tumor cell lines, and from primary neonatal rat sympathetic, motor, and sensory neuronal cells, as well as mesothalamic dopaminergic neurons.
  • RNAs were used as templates to generate single- stranded cDNAs using the Superscript Preamplification System for First Strand Synthesis kit purchased from GibcoBRL (Life Technologies, U.S.A.; Gerard, G.F. et al . (1989), FOCUS 11, 66) under conditions recommended by manufacturer.
  • a typical reaction used 10 ⁇ g total RNA or 2 ⁇ g poly (A) + RNA with 1.5 ⁇ g oligo (dT) 12- ⁇ in a reaction volume of 60 ⁇ L.
  • the product was treated with RNaseH and diluted to 100 ⁇ L with H 2 0.
  • 1-4 ⁇ L of these sscDNAs were used in each reaction.
  • Oligonucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer using established phosphoramidite chemistry and were used unpurified after precipitation with ethanol.
  • the degenerate oligonucleotide primers are:
  • STK3- 5'-TTRATRTC6CKRTG6GM6AT6GM6GGYTT-3' (SEQ ID NO: 66) (antisense).
  • primers were derived from the peptide sequences E(K/E)VAVK(V/I)F (SEQ ID NO: 67) (sense strand from kinase subdomain II) and KP(A/S)I(A/S)HRDIK (SEQ ID NO: 68 ) (antisense strand from kinase subdomain VI), respectively.
  • ALK1 as a template, these primers produce a product of 321 bp.
  • a PCR reaction was performed using primers STK1 and STK3- applied to the single-stranded sources listed above.
  • the primers were added at a final concentration of 5 ⁇ M each to a mixture containing 10 mM Tris HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , 200 uM each deoxynucleoside triphosphate, 0.001% gelatin, and 1.5 U AmpliTaq DNA Poiymerase (Perkin-Elmer/Cetus), and 1-4 ul cDNA.
  • PCR fragments migrating at -320 bp were isolated from 2% agarose gels using GeneClean (BiolOl) , and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol.
  • Colonies were selected from mini plasmid DNA-preparations using Qiagen columns and the plasmid DNAs were sequenced using cycle sequencing dye-terminator kit with AmpliTaq DNA Poiymerase, FS (ABI, Foster City, CA) . Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al . , J. Mol. Biol. 215:403-10). A novel clone (STKR6.22) was isolated by PCR with primers STKl and STK3- on single-stranded cDNA from human SY5Y cells as a template. This clone was subsequently designated as a fragment of human ALK-7.
  • a lambda gtll (Clontech, Palo Alto, CA) cDNA library was constructed using mRNA from a pool of nine whole human pituitary glands. Phage were screened on nitrocellulose filters with the random primed 32 P-labeled insert from STKR6.22 encoding human ALK-7 at 2xl0 6 cpm/mL in hybridization buffer containing 6xSSC, lx Denhardt ' s reagent, 0.1% SDS, with 0.1 mg/mL denatured, fragmented salmon sperm DNA. After overnight hybridization at 65 °C, filters were washed in O.lxSSC, 0.1% SDS at 65 °C.
  • the 493 amino acid human ALK-7 sequence contains two hydrophobic regions from 1-25 and 114-138. (See SEQ ID NO: 16)
  • the second hydrophobic region generates a likelihood score of -9.34, using the ALOM method of Klein et al . (P. Klein, M. Kanehisa, and C.
  • ALK-7 is predicted to be a type la integral membrane protein with a molecular weight of 52.35 kD after cleavage of the N-terminal signal peptide.
  • RNAs using from a large number of tumor cell lines and multiple human tissues, demonstrating an apparent selectivity in expression of ALK-7 in neuronal cells from the pituitary and substantiate nigra.
  • Northern blots were obtained from Clontech (Palo Alto, CA) containing 2 ⁇ g polyA+ RNA from 16 different adult human tissues (spleen, thymus, prostate, testis, ovary, small intestine, colonic mucosa, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and peripheral blood leukocytes) , and four different human fetal tissues (brain, lung, liver, and kidney), on a charge-modified nylon membrane. Additional Northern blots were prepared by running 20 ⁇ g total RNA on formaldehyde 1.2% agarose gel and transferring to nylon membranes.
  • Filters were hybridized with random prime [ 32 P] dCTP-labeled probes synthesized from the 320 bp insert from human ALK-7 clone STKR6.22. Hybridization was performed at 60 °C overnight in 6XSSC, 0.1% SDS, IX Denhardt ' s solution, 100 mg/mL denatured herring sperm DNA with 1-2 x IO 6 cpm/mL of 32 P-labeled DNA probes. The filters were washed in O.lXSSC/0.1% SDS, 65 °C, and exposed overnight on Kodak XAR-2 film.
  • ALK-7a 5'-AACTTTGGCTGGTATCTGAATATC-3' (SEQ ID NO: 69)
  • ALK-7b 5'-CCTTGTGTACCAACAATCTCCATA-3' (SEQ ID NO: 70). Reaction products were electrophoresed on 2% agarose gels, stained with ethidium bromide and photographed on a UV light box. The relative intensity of the -150-bp ALK-7-specific bands were estimated for each sample. A similar pair of oligonucleotides was designed for detection of rat ALK-7:
  • ALK-7 mRNA transcript was not detectable by Northern analysis from multiple human tissue sources, suggesting its expression is highly restricted. Using a more sensitive PCR- based detection, ALK-7 was found to be expressed in human substantia nigra, anterior pituitary, and Calu-6 lung carcinoma cell line (see below) . Weak expression was found in several other locations including whole brain, cerebellum, and prostate. Multiple other normal human tissues and tumor cell lines showed no detectable ALK-7 expression. HUMAN ALK-7 RNA EXPRESSION ANALYSIS
  • HUVEC h. endothelial
  • ALK-7 The neuronal expression of ALK-7 was assessed by in situ analysis in sagittal and coronal sections from neonatal and adult rat brains using a fragment of the extracellular domain of rat ALK-7 as a probe. This region was selected because its dissimilarity with the related ALK-4 and ALK-5. Other groups have performed in situs with the catalytic domain of rat ALK-7 demonstrating specific expression in neuronal tissues (cerebellum, hippocampus, and brainstem nuclei) , kidney, testis, lung, dorsolateral and anterior prostate, and adipose tissue.
  • the probe used in these studies contained an ALK-7 catalytic domain which may cross-react with the related ALK-4 and ALK-5 (77% nucleotide sequence identity with stretches of 27/29 and 25/26 bp identity to rat ALK-7) and thereby broaden the expression profile.
  • ALK-7 catalytic domain which may cross-react with the related ALK-4 and ALK-5 (77% nucleotide sequence identity with stretches of 27/29 and 25/26 bp identity to rat ALK-7) and thereby broaden the expression profile.
  • ALK-7 Using a more selective ALK-7 probe our analysis revealed the more restricted expression.
  • a moderate strength granular band was visible in the CA2 and CA3 regions of the hippocampus, dentate dyrus, olfactory tubercle, dorsal outer layer of the cortex, and in a band crossing the frontal cortex area 2 from the exterior to the corpus callosum.
  • a moderate signal was detected in the caudate putamen and thalamic nuclei.
  • signals of moderate strength were detected in the region of the magnocellular nucleus of the lateral hypothalamus and the medial tuberal nucleus.
  • a similar signal was observed in the region of the cuneiform nucleus on the anterior border of the cerebellum.
  • the cerebellum was devoid of hybridizing ALK-7.
  • ALK-7-specific immunoreagents were raised in rabbits against KLH-conjugated synthetic peptide YRKKKRPNVEEPL (SEQ ID NO: 76) from the juxtamembrane portion of the cytoplasmic domain of ALK-7. This region is unique to ALK-7 compared to other type I STK receptors, thereby allowing for the generation of ALK-7 specific antisera.
  • the N-terminal extracellular domain of ALK-7 expressed as a GST-fusion was also used as an immunogen to raise polyclonal antibodies in rabbits and to generate monoclonal antibodies in mice using the techniques described above. These antibodies were used to localize expression of the endogenous and recombinant protein as describe below.
  • the following example describes the construction of vectors for transient and stable expression in mammalian cells.
  • Expression constructs were generated to make wild type ALK-7 as well as a signaling incompetent ALK-7 (ALK-7DN) and a constitutively activated ALK-7 (ALK-7TA) .
  • pAdRSVOES- a modified adenovirus vector for the generation of virus producing recombinant protein
  • pRK5 a modified adenovirus vector for the generation of virus producing recombinant protein
  • Recombinant adenoviruses were generated by in vivo ligation as follows.
  • the transfer vector used Contains the following DNA sequences in order: The left terminal region of adenovirus type 5 encoding the packaging sequences (adenovirus type 5 nucleotides 1-454); the Rous Sarcoma Virus long terminal repeat promoter and the SV40 polyA region, isolated as an expression cassette from the plasmid pREP (Invitrogen Corporation) ; nucleotides 3320-5790 of the type 5 adenoviral genome; and the ori and beta-lactamase genes derived from the E. coli plasmid pBluescript. Two additional forms of the plasmid were generated.
  • the first, pAdRSVlacZ was prepared by the insertion of a double stranded synthetic oligonucleotide into the BamHI restriction site between the RSV promotor and the SV40 polyA sequence with the following nucleotide sequence (upper strand shown) : 5'
  • the E. coli lacZ gene was excised from the expression plasmid pCMVb (Clontech, Inc.) with the enzyme Not I and inserted into the Not I site between the promoter and the polyA sequence. This generated a plasmid that expressed the lacZ gene, and had two BstBI restriction sites between the lacZ gene and the polyA region.
  • the second plasmid (pAdRSVOES-) was generated by inserting a double stranded synthetic oligonucleotide into the same region as above. Its nucleotide sequence was the following: 5'
  • This plasmid allowed the introduction of exogenous cDNAs into the plasmid for expression purposes.
  • the viral DNA used for generation of recombinant viruses was derived from a virus (AdlacZBstBI) in which the left end of the adenovirus genome has been replaced by the homologous region of pAdRSVlacZ.
  • DNA vas isolated from the Ad5 dl327 strain of adenovirus (Jones and Shenk, Cell, 1978) (deleted in the E3 region), cleaved with Clal enzyme, and cotransfected into the HEK2934 cell line via calcium phosphate coprecipitation with the pAdRSVlacZ plasmid. Recombinant adenovirus plaques resulting from this transfection were screened for the ability to express the lacZ gene by histochemical staining with X-Gal.
  • AdlacZBstBI The resulting recombinant adenovirus, AdlacZBstBI, provided the backbone for additional adenovirus constructs, allowing a screen for recombinant plaques based on the presence or absence of lacZ activity in that further recombination would replace the lacZ gene with the cotransfected cDNA.
  • the transfer vector construct is linearized by digestion with BstBI, and cotransfected with AdlacZBstBI DNA which has also been cleaved with BstBI.
  • transfer vector plasmid DNA typically, 5 mg are corecipitated with 2 mg of viral DNA for the transfection; in vivo ligation of viral DNA and linearized transfer vector produces a novel recombinant virus directing expression of the new transgene.
  • a signaling incompetent ALK-7 construct was also made in both vectors pAdRSVOES- and pRK5 by insertion of an HA-tag at aa 230 in the ALK-7 coding region just after catalytic domain II. Truncation of other Type I STKRs in an analogous location has functioned in a dominant negative manner. This construct was called ALK-7DN.
  • a constitutively active form of ALK-7 was generated by a Thr to Asp mutation at amino acid 194 just upstream of the catalytic domain I GXGXXG motif.
  • this residue undergoes ligand-dependent trans- phosphorylation by the associated Type II STKR, resulting receptor activation and initiation of a signaling cascade.
  • a similar mutation in other Type I STKR's results in a ligand- independent, constitutively activated receptor. This construct was called ALK-7TD.
  • HEK293 cells (Graham, et al . , J. Gen. Virol. 36:59, 1977) were maintained in Dulbecco's modified Eagles medium + 10% calf serum. HEK293 monolayers were transfected with the ALK-7-encoding transfer vectors and cultured from five to seven days to allow plaques to appear. The monolayers were then stained with 25 mg/mL 5-bromo-4- chloro73-indolyl-b-D-galactopyranoside for several hours to identify non-recombinant (blue-stained) plaques.
  • Putative recombinant plaques were screened for expression of the transgene by infection of HEK293 cultures followed by immunohistochemistry with the monoclonal antibody recognizing the HA epitope.
  • Viruses which were positive for transgene protein expression were picked and subjected to several rounds of claque purification prior to amplification and purification on cesium chloride gradients. Banded viruses were diluted five-fold with dilution buffer (Curiel et ai., Proc. Natl. Acad. Sci., USA 88:8850-8854, 1991) and stored at -80 °C. Approximate titers of the virus preparations were determined immunohistochemically on HEK293 cultures. The following viruses were generated: AdRSVALK-7-HA; AdRSVALK-7-DN; and AdRSVALK-7-TD.
  • the pRK5 expression plasmids (10 ⁇ g DNA/100 mm plate) containing the KA-tagged ALK-7, the ALK-7DN, and ALK-7TD constructs were introduced into COS and 293 cells with lipo- fectamine (Gibco BRL) . After 72 hours, the cells were harvested in 0.5 ml solubilization buffer (20 mM HEPES pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL aprotinin) .
  • ALK-7 protein described above were expressed in PC12 cells and primary rat neuronal cultures by adenovirus mediated infection. These cells will allow further investigation into ALK-7 function. Recombinant protein expression was confirmed by immunostaining with an anti-HA antibody.
  • PC12 cultures (Greene, et al . , Methods Enzymol. 147:207, 1987) were maintained in RPMI medium containing 10% horse serum and 5% fetal calf serum.
  • Four differentiation experiments the medium was changed to RPMI containing IX N2 supplement and 0.1% BSA, and the cells were grown on a collagen I substrate.
  • For PC12 cell survival the cells were grown in RPMI containing 0.1% BSA. All cultures also contained IX penicillin/ streptomycin.
  • PC12 cells were incubated overnight with recombinant viruses at a multiplicity of infection (MOI) between 1 and 10. The cells were then washed and replated either into differentiation or survival conditions for two days.
  • MOI multiplicity of infection
  • Nerve Growth Factor 50 ng/mL served as a positive control.
  • the cultures were fixed with 2% paraformaldehyde and the percentage of cells bearing processes longer than 1 cell diameter was determined.
  • Sympathetic and sensory neurons were isolated as described (Hawrot and Patterson, Methods Enzymol. 53:574, 1979; Fields et al . , Cell 14:43, 1978) and cultured in a defined medium (Hawrot and Patterson, supra ) .
  • Sympathetic neurons were isolated from superior cervical ganglia dissected from E20 - E21 rat fetuses, while dorsal root ganglion sensory neurons were obtained from E16 - E18 rats. The ganglia were treated with 0.25% trypsin for 10 minutes, washed, and triturated to obtain a single cell suspension. Sensory neurons were preplated for 1 hour on tissue culture plastic to deplete adherent cells.
  • Dopaminergic neurons were isolated as described (Shimoda, et al . , Brain Research 586:319-331, 1992) and cultured in Neurobasal medium, supplemented with B27 supplements (Life Technologies) . Neurons were infected with adenoviruses for two hours on collagen I- coated tissue culture plastic (supplemented with NGF for sensory and sympathetic neurons) , and the cells were then washed and allowed to recover for two to four additional hours (with NGF if appropriate) . After the recovery period, the cells were washed extensively to remove the growth factor, and plated onto polylysine-laminin coated chamber slides. The addition of NGF at 50 ng/mL served as a positive control for survival of sensory and sympathetic neurons.
  • Recombinant ALK-7 protein expressed in COS cells migrated with apparent Mr of 52kD-63kD, consistent with its predicted molecular weight of 54kD based on its primary amino acid sequence and the presence of multiple glycosylation sites.
  • the ALK-7TD constitutive active form produced proteins indistinguishable from the wild type construct on SDS-PAGE.
  • the ALK-7DN construct expressed proteins of Mr 23.5 kd, 28 kD and 32 kD consistent with the presence of varying amounts of glycosylation on this truncated receptor. This analysis confirms the recombinant protein can be stably produced in mammalian cells.
  • nucleic acids also encode the claimed peptides and proteins of the invention.
  • all four nucleic acid sequences GCT, GCC, GCA, and GCG encode the amino acide alanine. Therefore, if for an amino acid there exists an average of three codons, a polypeptide of 100 amino acids in length will, on average, be encoded by 3 100 , or 5 x IO 47 , nucleic acid sequences.
  • nucleic acid sequence can be modified to form a second nucleic acid sequence, encoding the same polypeptide as endoded by the first second nucleic acid sequences, using routine procedures and without undue experimentation.
  • nucleic acids that encode the claimed peptides and proteins are also fully described herein, as if all were written out in full taking into account the codon usage, especially that preferred in humans.
  • changes in the amino acid sequences of polypeptides, or in the corresponding nucleic acid sequence encoding such polypeptide may be designed or selected to take place in an area of the sequence where the significant activity of the polypeptide remains unchanged.
  • an amino acid change may take place within a ⁇ -turn, away from the active site of the polypeptide.
  • changes such as deletions (e.g. removal of a segment of the polypeptide, or in the corresponding nucleic acid sequence encoding such polypeptide, which does not affect the active site) and additions (e.g.
  • AAGAGAAATT CTGCAGAAGT TCCTGGATGA GGCCCAAAGC AAGAAAATTA CTAAAGAGGA 120
  • TAGCTATCCT GACACCATGG AAAATTCAAC ATCTTCAAAA CAGACACTGA AGACTCCTGG 2280 AAAAAGTTTC ACAAGGAGTA AGAGTTTGAA AATTTTGCGA AACATGAAAA AGAGTATCTG 2340 TAATTCTTGC CCACCAAACA AGCCTGCAGA ATCTGTTCAG TCAAATAACT CCAGCTCATT 2400 TCTGAATTTT GGTTTTGCAA ACCGTTTTTC AAAACCCAAA GGACCAAGGA ATCCACCACC 2460 AACTTGGAAT ATTTAATAAA ACTCCAGATT TATAATAATA TGGGCTGCAA GTACACCTGC 2520 AAATAAAACT ACTAGAATAC TGCTAGTTAA AATAAGTGCT CTATATGCAT AATATCAAAT 2580 ATGAAGATAT GCTAATGTGT TAATAGCTTT TAAAAGAAAA GCAAAATGCC AATAAGTGCC 2640 AGTTTTGCAT TTTCATATCA TTTGCATTGA GTTGAAAACT GCAA
  • CTCATCCGGC CCAGCGAGAG CAGCCTCGGG GGCTACTCAC TGTCAGTCCG GGCCCAGGCC 540
  • AAACCCTCCC ATCACCTGTC ATTCTCAGAT AAGTATGAGC TTGTTTACCC AGAGCCTTTG 420
  • CTCGAGGTTA CCGAGCAGCT TGGAGCCTGA GCCGTGCTGA AGCGTCTGCG GGCCGTGCAG 1560
  • GGTTATGTCT GACTCACTGC ACTGGAGTTT GGCAAAAGCA TCTCAGAAGT GGTTGTGCTT 60 TTTTGAATGA AATGATCAAT GGAGTGCTCC AGTTGTATGC TGGCCTCTGG ATACTAACTA 120 GACCTGCCTG ACTCCAGGAA CTAAGGCTCA GTATCTGCAG AAGCTTTTTG CCCATCTCAT 180 TCCGGCTATG GGGACAACAT GTCTTCACCC AGGAAGGTTA GAGGAAAAAC TGGAAGAGAT 240 AATGATGAAG AGGAGGGTAA TTCAGGTAAC CTGAATCTCC GCAACTCTTT GCCTTCATCG 300 AGTCAGAAAA TGACGCCTAC GAAGCCGGTA CAAAATAAAA ATCTCATGAA GTATGAAGAA 360 CACTTAGATA TATTGATGGT GTTTTTATTG ATAAAAACCA TATGGTATAA TGTCTTCAAA 420 TTATGGAAAG GCAAGCTTAT TTGGGA
  • AAACCCTCCC ATCACCTGTC ATTCTCAGAT AAGTATGAGC TTGTTTACCC AGAGCCTTTG 420
  • GAAGGTAGCA CTACTGGGCA CAGAGTGAAC TGTTTCCACT TGATCTTTCT GAACAAGAGC 1560
  • GCCCTGCCCA CACCGGCCCT CTCCCCAGAG GACAAGGCCG TGCTGCAAAA CCTAAAGCGC 900
  • CCTCAGCCCC ATCCTTCACA AGCGTTTGGG CCTCAGCCCC CACAGCAGCC CCTTCCACTC 2160
  • CAGCATCCAC ATCTCTTCCC ACCCCAGGCC CCAGGACTCC TACCCCCACA ATCCCCCTAC 2220
  • Lys Lys lie Thr Lys Glu Glu Phe Ala Asn Glu Phe Leu Lys Leu Lys 20 25 30 Arg Gin Ser Thr Lys Tyr Lys Ala Asp Lys Thr Tyr Pro Thr Thr Val 35 40 45

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Abstract

La présente invention concerne des phosphatases et des kinases, des acides nucléiques codant de tels polypeptides, des cellules, des tissus et des animaux contenant de tels acides nucléiques, des anticorps dirigés contre de tels polypeptides, des essais utilisant de tels polypeptides et des procédés se rapportant à ce qui a été énoncé précédemment. L'invention concerne également des procédés permettant de traiter, de diagnostiquer et de rechercher les affections ou états concernés par les phosphatases et les kinases et caractérisés par une interaction anormale entre une phosphatase ou une kinase et son partenaire de liaison.
PCT/US1998/008439 1997-04-28 1998-04-27 Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase WO1998049317A2 (fr)

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EP98919919A EP0979288A2 (fr) 1997-04-28 1998-04-27 Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase
JP54724498A JP2002513289A (ja) 1997-04-28 1998-04-27 チロシンホスファターゼ関連疾患の診断および治療および関連する方法
AU72600/98A AU7260098A (en) 1997-04-28 1998-04-27 Diagnosis and treatment of phosphatase 0r kinase-related disorders
CA002288221A CA2288221A1 (fr) 1997-04-28 1998-04-27 Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase

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US4442897P 1997-04-28 1997-04-28
US60/044,428 1997-04-28
US4722297P 1997-05-20 1997-05-20
US60/047,222 1997-05-20
US4975697P 1997-06-11 1997-06-11
US4947797P 1997-06-11 1997-06-11
US60/049,756 1997-06-11
US60/049,477 1997-06-11
US4991497P 1997-06-18 1997-06-18
US60/049,914 1997-06-18
US6359597P 1997-10-23 1997-10-23
US60/063,595 1997-10-23

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WO1999036548A1 (fr) * 1998-01-16 1999-07-22 Hsc Research And Development Limited Partnership Tyrosine phosphatases proteiniques lymphoides humaines
EP1046715A1 (fr) * 1999-04-23 2000-10-25 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Interaction de la protéine vasculaire endothelial tyrosine phosphatase et le récepteur Tie-2 d'angiopoietine
WO2000063392A1 (fr) * 1999-04-16 2000-10-26 Kyowa Hakko Kogyo Co., Ltd. Nouvelle tyrosine phosphatase
WO2001053530A1 (fr) * 2000-01-18 2001-07-26 Human Genome Sciences, Inc. Polynucleotides et polypeptides de la protein thyrosine phosphatase humaine, et anticorps
WO2002042435A2 (fr) * 2000-11-27 2002-05-30 Bayer Aktiengesellschaft Regulation de la tyrosine phosphatase humaine
EP1454992A1 (fr) * 2003-03-07 2004-09-08 Istituto Nazionale Per Lo Studio E La Cura Dei Tumori Essai de kinase lymphome anaplastique, ses réactifs et compositions
WO2005084699A1 (fr) * 2004-03-02 2005-09-15 Acceleron Pharma Inc. Inhibiteurs d'alk7 et de la myostatine et leurs utilisations

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WO2007037532A1 (fr) * 2005-09-30 2007-04-05 Link Genomics, Inc. Application therapeutique ou diagnostique du gene srms

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036548A1 (fr) * 1998-01-16 1999-07-22 Hsc Research And Development Limited Partnership Tyrosine phosphatases proteiniques lymphoides humaines
WO2000063392A1 (fr) * 1999-04-16 2000-10-26 Kyowa Hakko Kogyo Co., Ltd. Nouvelle tyrosine phosphatase
EP1046715A1 (fr) * 1999-04-23 2000-10-25 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Interaction de la protéine vasculaire endothelial tyrosine phosphatase et le récepteur Tie-2 d'angiopoietine
WO2000065085A1 (fr) * 1999-04-23 2000-11-02 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Interaction de proteine-tyrosine phosphatase vasculaire-endotheliale avec le recepteur angiopoietine tie-2
WO2001053530A1 (fr) * 2000-01-18 2001-07-26 Human Genome Sciences, Inc. Polynucleotides et polypeptides de la protein thyrosine phosphatase humaine, et anticorps
WO2002042435A3 (fr) * 2000-11-27 2003-01-23 Bayer Ag Regulation de la tyrosine phosphatase humaine
WO2002042435A2 (fr) * 2000-11-27 2002-05-30 Bayer Aktiengesellschaft Regulation de la tyrosine phosphatase humaine
EP1454992A1 (fr) * 2003-03-07 2004-09-08 Istituto Nazionale Per Lo Studio E La Cura Dei Tumori Essai de kinase lymphome anaplastique, ses réactifs et compositions
WO2004079326A2 (fr) * 2003-03-07 2004-09-16 Istituto Nazionale Per Lo Studio E La Cura Dei Tumori Bioanalyse de lymphome kinase anaplasique, reactifs et compositions en contenant
WO2004079326A3 (fr) * 2003-03-07 2004-12-09 Ist Naz Stud Cura Dei Tumori Bioanalyse de lymphome kinase anaplasique, reactifs et compositions en contenant
WO2005084699A1 (fr) * 2004-03-02 2005-09-15 Acceleron Pharma Inc. Inhibiteurs d'alk7 et de la myostatine et leurs utilisations
US7456149B2 (en) 2004-03-02 2008-11-25 Acceleron Pharma, Inc. ALK7 and myostatin inhibitors and uses thereof
US7807631B2 (en) 2004-03-02 2010-10-05 Acceleron Pharma Inc. Methods of promoting growth of muscle tissue using ALK7
US8637450B2 (en) 2004-03-02 2014-01-28 Acceleron Pharma Inc. Methods of promoting fat loss comprising administering an ALK7 inhibitor
US9255137B2 (en) 2004-03-02 2016-02-09 Acceleron Pharma Inc. Methods of promoting fat loss comprising administering an ALK7 inhibitor

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