CA2251249A1 - Cdc2-related kinase associated with acute leukemia - Google Patents

Description

2-RELATED KINASE ASSOCIATED WITH ACUTE
LEUKEMIA
Field of the Invention This invention relates to cdc2-related kinases and in particular to cdc2-related kinases which are putative tumour suppressors.
Background of the Invention Intracellular signaling cascades are manipulated by a range of extracellular stimuli resulting in diverse outcomes such as cell division and differentiation. These stimuli-sensitive signaling cascades focus on key cell cycle regulatory molecules which in turn directly control cell division.
One group of stimulus-sensitive cascades activates the MAP
kinases, which control cell cycle progression by regulating, directly or indirectly, the activities of the cyclin-dependent kinases (CDKs). CDK
activity is partially dependent on binding to cyclin regulatory partners which induce an activated conformation (1). Mitogenic stimuli promote entry into S phase from G1 by activating CDK4 and CDK6 which bind to members of the cyclin D family. Activation of these cyclin D-associated CDKs is the earliest identified point of S phase control and may be a common target of activated MAP kinases.
Both the MAP kinases (MAPKs) and the cyclin dependent kinases have characteristic sites of phosphorylation which distinguish them (10-14). The MAPKs have a specific dual phosphorylation motif, consisting of a threonine/tyrosine pair, located in a regulatory loop of kinase domain VIII (15-17). These regulatory phosphoamino acids, which are separated by only one variable residue, are positioned on a lip at the entry into the kinase active site. When phosphorylated, a conformational change occurs exposing the kinase to ATP and substrate (18).

Characteristic of the cyclin-dependent kinases are a threonine and tyrosine pair embedded within the glycine-rich ATP-binding sequence found in kinase domain I. Negatively charged phosphates on these residues repel ATP and prevent kinase activity (18). In addition, cyclin-dependent kinases have a single site of activating phosphorylation within domain VIII, analogous to the MAPKs (19).
These regulatory sites are conserved within these two kinase families and are critical for their respective roles in cell cycle control.
Two protein kinases have been identified which have sequence similarity both to both the MAP kinases and to the cyclin dependent kinases (20,21). These cdc2-related kinases, named KKIAMRE and KKIALRE, after the amino acid sequences of the putative cyclin-binding domains, are expressed primarily in kidney, brain, lung and testis (p56 KKIAMRE) or ovary (p42 KKIALRE). Both of these molecules also have the conserved MAP kinase dual phosphorylation motif, TDY, located in kinase subdomain VIII. Activated MAPK/Erk Kinase (MEK) can phosphorylate both of these residues in KKIAMRE
in vatro, though less efficiently than similar sites on ERK (20). The kinase activity of KKIAMRE is increased by Epidermal Growth Factor (EGF) stimulation, independent of phosphorylation at these putative regulatory sites, suggesting an alternative mechanism of kinase regulation. Although expression of these two kinases in germinal tissues suggests their role in the normal function of these tissues, physiologic substrates have not yet been identified.
Mammalian cells respond to many complex stimuli and additional kinase regulators of cell cycle control remain to be described.
Patients with acute myeloid leukemia or myelodysplastic syndromes usually have many genetic abnormalities (47,48) and have a poor clinical prognosis. Small deletions in chromosome 5q31, in a region adjacent to the IL5 gene, have been found in many patients with

3 these disorders (48,48). The presence of these conserved losses has suggested that a tumour suppressor gene, important in the development of leukemia, may be located in this region. Several groups have attempted to define the minimal region of chromosome 5q31 loss by studying small groups of patients with unusually small deletions of this chromosomal band (49). While mutually exclusive regions have been identified by different groups, all sites map within a 10 Mb region flanked by the genes for IL5 and NKSF1 (47-52). Contained within this segment are genes for many cytokines or their receptors, including GSF
1R, IL9, CSF-2 and TCF7 (49). To date, candidate tumour suppressor genes have not been identified in this region.
Summary of the Invention A previously undescribed cdc2-related kinase involved in cell growth and in the control of the cell cycle has been identified. The gene for the new kinase, designated herein as LLK (lost in leukemia kinase), has been cloned and its cDNA sequence has been obtained and the encoded amino acid sequence determined.
The LLK gene has been found to be associated with acute leukemia in humans.
In accordance with one embodiment, the invention provides isolated polynucleotides comprising nucleotide sequences encoding LLK
proteins.
In accordance with a further series of embodiments, the invention provides an isolated polynucleotide selected from the group consisting of (a) a nucleotide sequence encoding a mammalian LLK protein;
(b) a nucleotide sequence encoding a human LLK protein;
(c) a nucleotide sequence encoding a rat LLK protein;

4 (d) a nucleotide sequence encoding a non-mammalian LLK
protein;
(e) a nucleotide sequence encoding the LLK amino acid sequence of Table 2; and (fj a nucleotide sequence encoding the LLK amino acid sequence of Table 5.
In accordance with a further embodiment, the invention provides the nucleotide sequences of Table 1 (rat LLK) and Table 4 (human LLK).
In accordance with a further embodiment, the invention provides recombinant vectors including the polynucleotides disclosed herein and host cells transformed with these vectors.
The invention further provides a method for producing LLK
proteins, comprising culturing such host cells to permit expression of a LLK protein-encoding polynucleotide and production of the protein.
The invention also includes polynucleotides which are complementary to the disclosed nucleotide sequences, polynucleotides which hybridise to these sequences under high stringency and degeneracy equivalents of these sequences.
In accordance with a further embodiment, the invention provides antisense molecules which may be used to prevent expression of a LLK
protein. Such antisense molecules can be synthesised by methods known to those skilled in the art and include phosphorothioates and similar compounds.
The invention further includes polymorphisms and alternatively spliced versions of the LLK genes and proteins wherein nucleotide or amino acid substitutions or deletions do not substantially affect the functioning of the gene or its encoding protein.
The invention also enables the identification and isolation of allelic variants or homologues of the described LLK genes, and their corresponding proteins, using standard hybridisation screening or PCR
techniques.
The invention provides a method for identifying allelic variants or homologues of the described LLK genes, comprising

5 choosing a nucleic acid probe or primer capable of hybridising to a LLK gene sequence under stringent hybridisation conditions;
mixing the probe or primer with a sample of nucleic acids which may contain a nucleic acid corresponding to the variant or homologue;
and detecting hybridisation of the probe or primer to the nucleic acid corresponding to the variant or homologue.
In accordance with a further embodiment, the invention provides fragments of the disclosed polynuc:leotides, such as polynucleotides of at least 10, preferably 15, more preferably 20 consecutive nucleotides of the disclosed polynucleotide sequences. These fragments are useful as probes and PCR primers or for encoding fragments, functional domains or antigenic determinants of LLK proteins.
In accordance with a further embodiment, the invention provides substantially purified LLK proteins, including the protein of Table 2 and the protein of Table 5.
The invention further provides a method for producing antibodies which selectively bind to a LLK protein comprising the steps of administering an immunologically effective amount of a LLK
immunogen to an animal;
allowing the animal to produce antibodies to the immunogen;
and obtaining the antibodies from the animal or from a cell culture derived therefrom.
The invention further provides substantially pure antibodies which bind selectively to an antigenic determinant of a LLK protein.

6 The antibodies of the invention include polyclonal antibodies, monoclonal antibodies and single chain antibodies.
The invention includes analogues of the disclosed protein sequences, having conservative amino acid substitutions therein. The invention also includes fragments of the disclosed protein sequences, such as peptides of at least 5, preferably 10, more preferably 20 consecutive amino acids of the disclosed protein sequences.
The invention further provides polypeptides comprising at least one functional domain or at least one antigenic determinant of a LLK
protein.
In accordance with a further embodiment, the invention provides a method for preventing or treating diseases associated with an abnormal LLK gene.
The invention further provides a method for identifying acute leukemias associated with loss or mutation of the LLK gene.
In accordance with a further embodiment, the invention provides non-human transgenic animals and methods for the production of non-human transgenic animals which afford models for further study of LLK protein-associated signalling and tools for screening of candidate compounds as therapeutics. For example, knock-out animals, such as mice, may be produced with deletion of a LLK gene. These animals may be used to screen candidate compounds for effectiveness to reverse the phenotype produced.
In accordance with a further embodiment, the invention provides methods for screening candidate compounds to identify those able to modulate signalling through a pathway involving a LLK protein.
The invention further provides screening methods for candidate compounds able to bind to a LLK protein, these being therefore candidates for modifying the activity of a LLK protein. Various suitable screening methods are known to those in the art, including

7 immobilisation of a LLK protein on a substrate and exposure of the bound LLK protein to candidate compounds, followed by elution of compounds which have bound to the LLK protein.
In accordance with a further embodiment of the invention, the invention provides a method of gene therapy comprising administering an effective amount of a LLK nucleic acid sequence to a cell or organism which has a defective or absent LLK gene.
Summary of the Drawings Figure lA shows a southern analysis of rat genomic DNA probed with full length LLK. 15 ~g samples of genomic DNA were digested with EcoRI, BamH1 or HindIII. Molecular weight is shown in kilobases.
Figure 1B shows a phylogenetic southern analysis. Genomic DNA from rat, pig, mouse, human and Drosophila were digested with EcoR1 and analyzed and probed with full length LLK. Cross hybridization is observed most strongly in mouse, human and Drosophila samples.
Figure 2 shows a northern analysis of rat LLK. Full length cDNA was used to probe poly A RNA samples from various tissues.
Two prominent bands were observed at 4.4 kB and at 2.6 kB suggesting alternatively spliced forms. LLK expression was observed most strongly in brain, muscle and heart.
Figure 3A shows LLK activity in Hela cells exposed to the indicated stimuli (FCS O/N = incubation with FCS overnight).
Figure 3B shows LLK activity in Hela cells exposed to pcDNA3 vector only (vec), Serum, PMA or EGF at 20, 40 or 60 ng/ml.
Figure 4 shows progression into S phase, expressed as incorporation of BrdU (%BrdU+), of RAT-1 cells transiently transfected with LLK (MKH), mutant LLK deficient in kinase activity (MKH-KD),

8 dominant negative CDK2 (CDK2-DN), cyclin-dependent kinase inhibitor p21 (p21) or plasmid clone (pcDNA3), arrested by serum starvation and measured at 14 hours after serum replenishment.
Detailed Description of the Invention The inventors have identified a new cdc2-related kinase involved in cell growth and in the control of the cell cycle. The new kinase, designated LLK, has been demonstrated in rats and humans. The full cDNA sequences and deduced amino acid sequences of rat and human LLK are provided, as shown in Tables 1 and 2 (rat), Tables 4 and 5 (human).
The inventors have shown that a high percentage of human acute leukemia patients have both LLK alleles deleted, indicating that the LLK gene is a putative tumour suppressor gene.
In Table 3, the protein kinase consensus residues of rat LLK are shown in bold and the cyclin-binding motif, NKIATRE, and the two sites of potential regulation, the SY duplex and the TDY MAP kinase-like regulatory region, are boxed.
In Table 5, the human LLK amino acid sequence is shown, with the ATP binding site, GEGSYG, the cyclin-binding motif, NKIAMRE, and the MAP kinase activation motif TDY, boxed.
The invention provides a novel family of EGF-stimulated protein kinases with sequence similarity to both the MAP kinases and to the cyclin-dependent kinases. Overexpression of LLK leads to growth suppression, suggesting that it may function in EGF-mediated growth suppressive pathways leading to enhanced p2lw'~~~ 1 expression.
The LLK proteins have sequence similarity to both the MAPKs and the cyclin dependent kinase family of cell cycle regulators. Similar to the MAPKs, the LLK proteins have a potential MAPK activation motif, TDY, followed by the MAPK specific sequence, VATRW. In this

9 regard, they are most similar to the ERK-family of MAPKs which also have an acidic residue separating these two regulatory hydroxy aminoacids.
Unexpectedly, the new kinase also has considerable similarity to the cyclin-dependent kinases, as suggested by the presence of a putative cyclin-binding motif, NK.IATRE in rat and NKIAMRE in human, and of potential negative regulatory phosphorylation sites found within the amino-terminal glycine rich ATP-binding domain.
Cyclin-dependent kinases have the sequence GEGTYG, with dual phosphorylation on threonine and tyrosine preventing ATP binding and enzymatic inactivation. LLK has a conservative substitution of serine for threonine, suggesting a similar mechanism of regulation. In contrast, the MAPKs do not have phosphorylation sites within this motif, underscoring a divergence from the LLKs.
Analysis of the LLK sequence reveals that it contains all twelve of the invariant or nearly invariant residues that are characteristic of the kinase catalytic domain. The 12 residues that are conserved and their positions within LLK are as follows: Glycine 11 and glycine 13 within subdomain I of Hanks (31) (GXGXXGXV motif), lysine 33 in subdomain II, glutamic acid 50 in subdomain III, aspartic acid 125 and asparagine 130 in subdomain VIB, aspartic acid 143 and glycine 145 in subdomain VII (DFG motif), glutamic acid 170 in subdomain VIII (APE
motifj, aspartic acid 183 and glycine 188 in subdomain IX, and arginine 274 in subdomain XI.
In general, strong indicators of the specificity of the kinase can be found within two short motifs in subdomain VI and subdomain VIII.
In subdomain VI, the consensus DLKPEN predicts serine/threonine specificity, whereas DLRAAN or L)LAARN predicts tyrosine specificity.
LLK contains the sequence DIKPI~N (residues 125-130), which conforms most closely to the serine/threonine specific kinase consensus sequence. In subdomain VIII, the sequence GT/SXXY/FX is found to be conserved in serine/threonine specific kinases, whereas PI/VK/RWT/M
is characteristic of the tyrosine specific kinases. LLK contains the sequence ATRWYR (residues 162-167) which again is most closely 5 related to the serine/threonine specific kinases.
Comparison of the LLK proteins with all known protein kinases reveals that they are most homologous to the cdc2-related protein kinases KKIAMRE and KKIALRF: (Table 6). Each of these other kinases possesses the MAPK activation motif, TDY, putative cyclin-

10 binding sequence, and sites of potential negative regulatory phosphorylation within their ATP binding motifs. Rat LLK, for example, has 44% and 47% overall identity to KKIALRE and KKIAMRE respectively.
Within the kinase domain, ~°at LLK has 60% identity to KKIAMRE, 50% identity to KKIALRE, 33% identity to rat ERK1, 32%
identity to rat ERK2, 34% identity to mouse p38HOC and 33% identity to rat SAPKa.. Threonine 158 and. Tyrosine 161, in subdomain VIII, correspond to the sites of activating phosphorylation for the MAPKs.
Threonine 158 of LLK corresponds to a critical site of activating phosphorylation in the CDKs while serine 14 and Tyrosine 15 correspond to CDK sites of negative regulation.
KKIALRE and KKIAMRE are not dependent on phosphorylation within the TDY motif for activity, suggesting that regulation of these enzymes is unlike that of the MAF'Ks. The inventors have shown that a mutant form of LLK, in which the TDY putative activation motif has been mutated to ADF, also retains enzymatic activity, suggesting that its mechanism of regulation is also distinct from the MAPKs.
LLK is expressed strongly in muscle, heart, liver and brain, all tissues which have a very low mitotic index.

11 To investigate the effects of LLK expression on cell growth, this enzyme was transiently expressed in rat-1 cells. A technique of CD20 expression was used to tag transiently-transfected cells and DNA
synthesis was measured by BrdU incorporation flow cytometrically.
This technique can rapidly identify genes which inhibit DNA synthesis, such as p2lw'~F1 and dominant interfering mutants of CDK2. LLK
overexpression is associated with a marked decreased in DNA
synthesis, suggesting that it is a negative regulator of cell growth.
Mutants of LLK lacking the TDY MAP kinase motif are equally capable of delaying DNA synthesis, suggesting that phosphorylation at these sites is not needed for this action. These data indicate that LLK
restricts cell growth and may contribute to the maintenance of the low mitotic rate in differentiated tissues in which it is expressed.
Signaling through the epidermal growth factor receptor usually causes ERK activation, often resulting in increased cell growth. It has been shown by the inventors that LLK is both stimulated by EGF and causes suppression of cell growth, suggesting its participation in signaling pathways which are disi;inct from ERK-induced mitogenic signaling cascades. LLK overexpression may mimic hyperactivation of the EGF receptor, inducing its growth suppressive effects through p2lw'~~F1. This kinase may be a novel link between the EGF receptor and STAT1 activation.
The human LLK gene has been mapped to chromosome 5q31.1, a chromosome region implicated in human acute leukemia. More than 60% of human acute leukemia patients examined showed loss of both alleles of the LLK gene, indicating that the LLK gene is a putative tumour suppressor gene.
Isolated Nucleic Acids

12 In accordance with one series of embodiments, this invention provides isolated nucleic acids corresponding to the nucleic acid sequences encoding rat and human LLK proteins. Also provided are portions of the LLK sequence useful as probes and PCR primers or for encoding fragments, functional damains or antigenic determinants of LLK proteins.
One of ordinary skill in the art is now enabled to identify and isolate LLK genes or cDNAs which are allelic variants or homologues of the disclosed LLK sequence, using standard hybridisation screening or PCR techniques.
Depending on the intended use, the invention provides portions of the disclosed nucleic acid sequence comprising about 10 consecutive nucleotides (e.g. for use as PCR primers) to nearly the complete disclosed nucleic acid sequences. The invention provides isolated nucleic acid sequences comprising sequences corresponding to at least 10, preferably 15 and more preferably at least 20 consecutive nucleotides of the LLK genes as disclosed herein.
In addition, the isolated nucleic acids of the invention include any of the above described nucleotide sequences included in a vector.
Substantially Pure Proteins In accordance with a further series of embodiments, this invention provides substantially pure LLK protein, fragments of this protein and fusion proteins including this protein and fragments.
The proteins, fragments and fusion proteins have utility, as described herein, for the preparation of polyclonal and monoclonal antibodies to LLK proteins, for the identification of binding partners of the LLK proteins and for diagnostic and therapeutic methods, as described herein. For these uses, the present invention provides substantially pure proteins, polypeptides or derivatives of polypeptides

13 which comprise portions of the LLK amino acid sequences disclosed or enabled herein and which may vary from about 4 to 5 amino acids (e.g.
for use as immunogens) to the complete amino acid sequence of the LLK protein. The invention provides substantially pure proteins or polypeptides comprising sequences corresponding to at least 5, preferably at least 10 and more preferably 50 or 100 consecutive amino acids of the LLK proteins disclosed or enabled herein.
Preparation of Proteins LLK proteins, fragments of the proteins and fusion proteins may be isolated and purified by techniques well known to those skilled in the art. The LLK proteins may be purified from tissues (e.g. heart and muscle) in which there is a high level of expression of the protein or it may be made by recombinant techniques. Isolated proteins, or fragments thereof, can be used for the generation of antibodies, in the identification of proteins which may bind to LLK or for diagnostic or therapeutic methods and assays. Full length proteins and fragments of at least 5 amino acids may be isol;~ted and purified for various applications.
The protein may be isolated from tissue by extraction and solubilised using a detergent.
Purification can be achieved using protein purification procedures such as chromatography methods (gel-filtration, ion-exchange and immunoaffinity), by high-performance liquid chromatography (RP-HPLC, ion-exchange HPLC, size-exclusion HPLC, high-performance chromatofocusing and hydrophobic interaction chromatography) or by precipitation (immunoprecipitation or immunoaffinity SDS-Page and Page). Polyacrylamide gel electrophoresis can also be used to isolate the LLK proteins based on their molecular weight, charge properties and hydrophobicity.

14 Similar procedures may be used to purify the protein obtained from recombinant expression systems.
For protein expression, eukaryotic or prokaryotic expression systems may be generated in which a LLK gene sequence, cDNA or genomic, is introduced into a plasmid or other expression vector which is then introduced into living cells. Constructs in which a LLK cDNA
sequence containing the entire open reading frame or portions thereof is inserted in the correct orientation into an expression plasmid may be used for protein expression.
Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the gene. They may also include sequences allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow cells containing the vectors to be selected, and sequences that increase the efficiency with which the mRNA is translated. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of viruses. Cell lines may also be produced which have integrated the vector into the genomic DNA and in this manner the gene product is produced on a continuous basis.
Expression of foreign sequences in bacteria such as E. colt require the insertion of the sequence into an expression vector, usually a plasmid which contains several elements such as sequences encoding a selectable marker that assures maintenance of the vector in the cell, a controllable transcriptional promoter which upon induction can produce large amounts of mRNA from the cloned gene, translational control sequences and a polylinker to simplify insertion of the gene in the correct orientation within the vector. A relatively simple E. coli expression system utilizes the lac promoter and a neighbouring lacZ
gene which is cut out of the expression vector with restriction enzymes and replaced by a LLK gene sequence.

In vitro expression of proteins encoded by cloned DNA is also possible using the T7 late-promoter expression system. Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages such as T3, T5 and SP6 may also be used 5 for in vitro production of proteins from cloned DNA. E. coli can also be used for expression by infection with M13 Phage mGPI-2. E. coli vectors can also be used with phage lambda regulatory sequences, with fusion protein vectors, with maltose-binding protein fusions, and with glutathione-S-transferase fusion proteins.
10 Eukaryotic expression systems permit appropriate post-translational modifications of expressed proteins. This allows for studies of the LLK genes and gene products including determination of proper expression and post-translational modifications for biological activity, identifying regulatory elements in the 5' region of the gene and

15 their role in tissue regulation of protein expression. It also permits the production of large amounts of protein for isolation and purification, the use of cells expressing LLK as a functional assay system for antibodies generated against the protein, the testing the effectiveness of pharmacological agents to increase or decrease the activity of LLK, and the study of the function of the normal complete protein, specific portions of the protein, or of naturally occurring polymorphisms and artificially produced mutated proteins.
In order to produce mutated or polymorphic proteins, LLK DNA
sequences can be altered using procedures such as restriction enzyme digestion, DNA polymerase fill-in, exonuclease deletion, terminal deoxynucleotide transferase extension, ligation of synthetic or cloned DNA sequences and site-directed sequence alteration using specific oligonucleotides together with PCR. Alteration of a LLK cDNA will allow for the production of specific mutations within the cDNA

16 sequence in order to express the created mutated proteins and study their biological effects.
Once an appropriate expression vector containing a LLK gene is constructed, it is introduced into eukaryotic cells by techniques that include calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion and liposome-mediated transfection.
The host cell to be transfected with the vector of this invention may be selected from the group consisting of E.coli, Pseudornonas, Bacillus Szcbtvlis, or other bacilli, other bacteria, yeast, fungi, insect (using baculoviral vectors for expression), mouse or other animal or human tissue cells. Mammalian cells can also be used to express a LLK protein using a vaccinia virus expression system.
In order to express and purify the protein as a fusion protein, the LLK cDNA sequence is inserted into a vector which contains a nucleotide sequence encoding another peptide (eg. GST - glutathionine succinyl transferase). The fusion protein is expressed and recovered from prokaryotic (eg. bacterial or baculovirus) or eukaryotic cells. The fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the LLK protein obtained by enzymatic cleavage of the fusion protein.
Purified protein can also be used in further biochemical analyses to establish secondary and tertiary structure. The preparation of substantially purified LLK protein or fragments thereof allows for the determination of the protein tertiary structure by x-ray crystallography of crystals of LLK protein or by NMR. Determination of structure may aid in the design of pharmaceuticals which interact with the protein, alter protein charge configuration or charge interaction with other proteins, or to alter its function in the cell.

17 Antibodies The knowledge of the amino acid and nucleotide sequence of LLK
proteins allows for the production of antibodies which selectively bind the LLK protein or fragments thereof. The term antibodies includes polyclonal antibodies, monoclonal antibodies, single chain antibodies and fragments thereof such as Fab fragments.
In order to prepare polyclonal antibodies, fusion proteins containing defined portions or all of a LLK protein can be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle. Fusion proteins are commonly used as a source of antigen for producing antibodies. Alternatively protein may be isolated and purified from LLK: expressing cultures and used as a source of antigen. It is understood that the entire protein or fragments thereof can be used as a source of antigen to produce antibodies.
A purified LLK protein is purified, coupled to a carrier protein and mixed with Freund's adjuvant (to help stimulate the antigenic response by the animal) and injected into rabbits or other appropriate laboratory animals. Following booster injections at weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or purified prior to use by various methods including affinity chromatography employing Protein A-Sepharose, Antigen Sepharose or Anti-mouse-Ig-Sepharose, to give polyclonal antibodies.
Alternatively, synthetic peptides can be made corresponding to the antigenic portions of the protein, and used to inoculate the animals.
The most common practice is to choose a 10 to 15 amino acid residue peptide corresponding to the carboxyl or amino terminal sequence of a protein antigen, and to chemically cross-link it to a

18 carrier molecule such as keyhole limpet hemocyanin or BSA. However, if an internal sequence peptide is desired, selection of the peptide is based on the use of algorithms that predict potential antigenic sites.
These predictive methods are, in turn, based on predictions of hydrophilicity (Kyte and Doolittle 1982, Hopp and Woods 1983) or secondary structure (Chou and Fa.sman 1978). The objective is to choose a region of the protein that; is either surface exposed, such as a hydrophilic region, or is conformationally flexible relative to the rest of the structure, such as a loop region or a region predicted to form a ~-turn. The selection process is also limited by constraints imposed by the chemistry of the coupling procedures used to attach peptide to carrier protein. Carboxyl-terminal peptides are frequently chosen because these are often more mobile than the rest of the molecule and the peptide can be coupled to a carrier in a straightforward manner using glutaraldehyde. The amino-terminal peptide has the disadvantage that it may be modified post-translationally by acetylation or by the removal of a leader sequence. A comparison of the protein amino acid sequence between species can yield important information. Those regions with sequence differences between species are likely to be immunogenic. Synthetic peptides can also be synthesized as immunogens as long as they mimic the native antigen as closely as possible.
As will be understood by those skilled in the art, monoclonal LLK antibodies may also be produced. LLK protein isolated from tissues, or from cells recombinantly expressing the protein, is injected in Freund's adjuvant into mice. Mice are injected 9 times over a three week period, after which their spleens are removed and resuspended in phosphate buffered saline (PBS). The spleen cells serve as a source of lymphocytes, some of which are producing antibody of a selected specificity. These are then fused with a permanently growing myeloma

19 partner cell, and the products of the fusion are plated into a number of tissue culture wells in the presence of a selective agent such as HAT.
The wells are then screened by ELISA to identify those containing cells making binding antibody. These are then plated and after a period of growth, these wells are again screened to identify antibody-producing cells. Several cloning procedures are carried out until over 90% of the wells contain single clones which are positive for production of the desired antibody. From this procedure, a stable line of clones which produce the antibody is established. The monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose, ion-exchange chromatography, as well as variations and combinations of these techniques. Truncated versions of monoclonal antibodies may also be produced by recombinant techniques in which plasmids are generated which express the desired monoclonal antibody fragment in a suitable host.
Antibodies are useful in various immunoassays for detecting and quantitating relative amounts of wild type and mutant protein.
Enzyme-linked immunosorbant assays (ELISA) may be used to detect both wild type and mutant LLK as well as antibodies generated against these proteins. Commonly used ELISA systems are indirect ELISA to detect specific antibodies, direct competitive ELISA to detect soluble antigens, antibody-sandwich ELISA to detect soluble antigens and double antibody-sandwich ELISA to detect specific antibodies.
For a review of methods for preparation of antibodies, see Antibody Eogirzeering: A Practical Guide, Barreback, ed., W.H.
Freeman & Company, NY (1992) or Antibody Enganeerirzg, 2nd Ed., Barreback, ed., Oxford University Press, Oxford (1995).
Diagnostic Methods LLK genes and gene products, as well as the LLK-derived probes, primers and antibodies, disclosed or otherwise enabled herein, are useful for screening for carriers of deletions associated with acute leukemia, and for the diagnosis of acute leukemia associated with loss 5 or mutation of the LLK gene. Individuals at risk for developing acute leukemia, for example those with acute leukemia present in the family pedigree, or individuals not previously known to be at risk, may be routinely screened using probes to detect the presence of a relevant deletion in the LLK gene by a variety of techniques.
10 Diagnostic methods are provided based upon the nucleic acids (including genomic and mRNA/cDNA sequences), proteins, and/or antibodies disclosed and enabled herein, including functional assays designed to detect failure or augmentation of the normal LLK activity.
Preferably, the methods and products are based upon the human LLK
15 nucleic acids, protein or antibodies. The significant evolutionary conservation of large portions of the LLK nucleotide and amino acid sequence, even in species as diverse as humans and rats, allows the skilled artisan to make use of such non-human LLK-homologue nucleic acids, proteins and antibodies, even for applications directed toward

20 human or other animal subjects.
As will be appreciated by one of ordinary skill in the art, the choice of diagnostic methods of the present invention will be influenced by the nature of the available biological samples to be tested and the nature of the information required.
Protein Based Screens and Diagnostics When a diagnostic assay is to be based upon a LLK protein, a variety of approaches are possible. For example, diagnosis can be achieved by monitoring differences in the electrophoretic mobility of normal and mutant proteins. Such an approach will be particularly

21 useful in identifying mutants in which charge substitutions are present, or in which insertions, deletions or substitutions have resulted in a significant change in the electrophoretic migration of the resultant protein. Alternatively, diagnosis may be based upon differences in the proteolytic cleavage patterns of normal and mutant proteins, differences in molar ratios of the various amino acid residues, or by functional assays demonstrating altered function of the gene products.
In preferred embodiments, protein-based diagnostics will employ differences in the ability of antibodies to bind to normal and mutant LLK proteins. Such diagnostic tests may employ antibodies which bind to the normal proteins but not to mutant proteins, or vice versa. In particular, an assay in which a plurality of monoclonal antibodies are used, each capable of binding to a mutant epitope, may be employed.
The levels of anti-mutant antibody binding in a sample obtained from a test subject (visualized by, for example, radiolabelling, ELISA or chemiluminescence) may be compared to the levels of binding to a control sample. Alternatively, antibodies which bind to normal but not mutant LLK may be employed, and decreases in the level of antibody binding may be used to distinguish homozygous normal individuals from mutant heterozygotes or homozygotes. Such antibody diagnostics may be used for arz situ immunohistochemistry using biopsy samples of tissues obtained antemortem or postmortem.
Nucleic Acid Based Screens and Diagnostics When the diagnostic assay i.s to be based upon nucleic acids from a sample, the assay may be based upon mRNA, cDNA or genomic DNA.
Whether mRNA, cDNA or genomic DNA is assayed, standard methods well known in the art may be used to detect the presence of a particular sequence either irz situ or ira vitro {see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor

22 Press, Cold Spring Harbor, NIA. As a general matter, however, any tissue with nucleated cells may be examined.
Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material. The DNA may be isolated and used directly for detection of a specific sequence or may be amplified by the polymerase chain reaction (PCR) prior to analysis. Similarly, RNA or cDNA may also be used, with or without PCR amplification. To detect a specific nucleic acid sequence, direct nucleotide sequencing, hybridization using specific oligonucleotides, restriction enzyme digest and mapping, PCR mapping, RNase protection, chemical mismatch cleavage, ligase-mediated detection, and various other methods may be employed. Oligonucleotides specil:ic to particular sequences can be chemically synthesized and labeled radioactively or nonradioactively (e.g., biotin tags, ethidium bromide), and hybridised to individual samples immobilized on membranes or other solid-supports (e.g., by dot-blot or transfer from gels after electrophoresis), or in solution. The presence or absence of the target sequences may then be visualized using methods such as autoradiography, fluorometry, or colorimetry.
These procedures can be automated using redundant, short oligonucleotides of known sequence fixed in high density to silicon chips.
Whether for hybridisation, l~,Nase protection, ligase-mediated detection, PCR amplification or any other standards methods described herein and well known in the art, a variety of subsequences of the LLK
sequences disclosed or otherwise enabled herein will be useful as probes and/or primers. In general, useful sequences will include at least 8-9, more preferably 10-50, and most preferably 18-24 consecutive nucleotides from the LLK introns, exons or intron/exon boundaries.
Depending upon the target sequence, the specificity required, and

23 future technological developments, shorter sequences may also have utility. Therefore, any LLK derived sequence which is employed to isolate, clone, amplify, identify or otherwise manipulate a LLK
sequence may be regarded as an appropriate probe or primer.
Methods of Treatment Therapies may be designed to circumvent or overcome a LLK
gene defect or inadequate LLK gene expression, and thus moderate and possibly prevent malignancy.
Protein Therapy Treatment or prevention of acute leukemia can be accomplished by replacing a mutant or absent LLK protein with normal protein, by modulating the function of a mutant protein, or by delivering normal LLK protein to the appropriate ce.Lls. Once the biological pathway involving the LLK protein has been completely elucidated and understood, it may also be possible to modify the pathophysiologic pathway in which the protein participates in order to correct the physiological defect.
To replace a mutant protein. with normal protein, or to add protein to cells which no longer express normal LLK, it is necessary to obtain large amounts of pure LLK protein from cultured cell systems which can express the protein. Delivery of the protein to the affected cells and tissues can then be accornplished using appropriate packaging or administration systems. The LLK can be provided in the form of a stable composition for oral or parental administration, the latter route including intravenous and subcutaneous administration. The composition containing the LLK protein of the present invention can also be administered in an solution or emulsion contained within phospholipid vesicles called liposo:mes. The liposomes may be

24 unilamellar or multilamellar and are formed of constituents selected from phosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, phosphatidylethanolamine, phosphatidylserine, demyristoylphosphatidylcholine and combinations thereof. The multilamellar liposomes comprise multilamellar vesicles of similar composition to unilamellar vesicles, but are prepared so as to result in a plurality of compartments in which the LLK protein-containing solution or emulsion is entrapped. Additionally, other adjuvants and modifiers may be included in the l.iposomal formulation such as polyethyleneglycol, or other materials. The liposomes containing the LLK protein or peptide composition may also have modifications such as having antibodies immobilized onto the surface of the liposome in order to target their delivery.
In one embodiment of the present invention, a pharmaceutical composition for administration to subjects in a biologically compatible form suitable for administration au vivo for treating malignancies characterized by abnormal LLK function comprises a safe and effective amount of LLK protein alone, or in combination with other agents and pharmaceutical carriers. Accordingly, the composition may vary to include not only LLK protein or peptides, but as well a vector containing an appropriate LLK sequence. The composition may be administered to any living organism including humans and animals.
By safe and effective as used herein is meant providing sufficient potency in order to decrease, prevent, ameliorate or treat a malignancy affecting a subject while avoiding serious side effects. A safe and effective amount will vary depending on the age of the subject, the physical condition of the subject being treated, the severity of the proliferative disorder, the duration of treatment and the nature of any concurrent therapy.

Administration of a therapeutically active amount of the pharmaceutical composition of the present invention is defined as an amount effective, at dosages and f:or periods of time necessary to achieve the desired result. This may also vary according to factors such 5 as the disease state, age, sex, and weight of the subject, and the ability of the LLK to elicit a desired response in the subject. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the 10 therapeutic situation.
By pharmaceutically acceptable carrier as used herein is meant one or more compatible solid or liquid delivery systems. Some examples of pharmaceutically acceptable carriers are sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, collagen, talc, 15 stearic acids, magnesium stearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids, pyrogen-free water, isotonic saline, phosphate buffer, and other suitable non-toxic substances used in pharmaceutical formulations. Other excipients such as wetting agents and lubricants, tableting agents, stabilizers, anti-oxidants and 20 preservatives are also contemplated.
The compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with

25 a pharmaceutically acceptable carrier. Suitable carriers are described for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA
1985). On this basis the compositions include, albeit not exclusively, solutions of the substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in

26 buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
Alternatively, small molecule analogs may be used and administered to act as LLK agonists or antagonists and in this manner produce a desired physiological effect. In order to screen for analogues, one can design functional screens based on the sequence of LLK. One can also fuse LLK to heterologous DNA binding proteins to design screens for agonists. Yeast screens can be used for small molecules that may interact by promoting or disrupting LLK binding with other proteins.
LLK expressed as a fusion protein can be utilized to identify small peptides that bind to LLK. In one approach, termed phage display, random peptides (up to 20 amino acids long) are expressed with coat proteins (geneIII or geneVIII) of filamentous phage such that they are expressed on the surface of the phage thus generating a library of phage that express random sequences. A library of these random sequences is then selected by incubating the library with the LLK protein or fragments thereof and phage that bind to the protein are then eluted either by cleavage of LLK from the support matrix or by elution using an excess concentration of soluble LLK protein or fragments. The eluted phage are then repropagated and the selection repeated many times to enrich for higher affinity interactions. The random peptides can either by completely random or constrained at certain positions through the introduction of specific residues. After several rounds of selection, the final positive phage are sequenced to determine the sequence of the peptide.
An alternate but related approach uses the yeast two hybrid system to identify binding partners for LLK. LLK or fragments are expressed in yeast as a fusion to a DNA binding domain. This fusion protein is capable of binding to target promoter elements in genes that

27 have been engineered into the yeast. These promoters drive expression of specific reporter genes (typically the auxotrophic marker HISS and the enzyme bgalactosidase). A library of cDNAs can then be constructed from any tissue or cell line and fused to a transcriptional activation domain. Transcription of HISS and b-galactosidase depends on association of the LLK fusion protein (which contains the DNA
binding domain) and the target protein (which carries the activation domain). Yeast survival on specific growth media lacking histidine requires this interaction. This approach allows for the identification of specific proteins that interact with LLK. The approach has also been adapted to identify small peptides. LLK, or its fragments, are fused with the DNA binding domain and are screened with a library of random peptides or peptides which are constrained at specific positions linked to a transcriptional activation domain. Interaction is detected by growth of the interacting peptides on media lacking histidine and by detection of b-galactosidase activii;y using standard techniques.
The identification of proteins or small peptides that interact with LLK can provide the basis for the design of small peptide antagonists or agonists of LLK function. Further, the structure of these peptides determined by standard techniques such as protein NMR or X-ray crystallography can provide the structural basis for the design of small molecule drugs.
Gene Therapy Gene therapy is another potential therapeutic approach in which normal copies of the LLK gene are introduced into selected tissues to successfully code for normal protein in affected cell types. The gene must be delivered to affected cells in a form in which it can be taken up and can code for sufficient protein to provide effective function.

28 Transducing retroviral vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression. The targeted cells must be able to divide and the expression level of normal protein should be high. The full length LLK gene, or portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest. Other viral vectors which can be used include adeno-associated virus, vaccinia virus, bovine papilloma virus, or a herpes virus such as Epstein-Barr virus.
Gene transfer could also be achieved using non-viral methods of infection arz vitro. These methods would include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for delivery of DNA into a cell. DNA itself may be taken up by cells directly.
Antisense-based strategies can be employed to explore LLK gene function and as a basis for therapeutic drug design. The principle is based on the hypothesis that sequence-specific suppression of gene expression can be achieved by intracellular hybridization between mRNA and a complementary antisense species. The formation of a hybrid RNA duplex may then interfere with the processing/transport/translation and/or stability of the target LLK
mRNA. Antisense strategies may use a variety of approaches including the use of antisense oligonucleotides, injection of antisense RNA and transfection of antisense RNA expression vectors.
Transplantation of normal genes into the affected cells of a patient with a malignancy can also be useful therapy. In this procedure, normal LLK is transferred into a cultivatable cell type, either exogenously or endogenously to a patient. These cells are then injected into the targeted tissue.

29 Animal Models The present invention also provides for the production of transgenic non-human animal models for the study of the LLK tumor suppressor gene function, to study the mechanisms of carcinogenesis as related to the LLK gene, for the screening of candidate pharmaceutical compounds, for the creation of explanted mammalian cell cultures which express the protein or mutant protein or in which the LLK gene has been inactivated by knock-out; deletion, and for the evaluation of potential therapeutic interventions.
The creation of an animal model for acute leukemia and other related cancers is important to the understanding of the LLK gene function in the cancer and for the testing of possible therapies.
Animal species which are suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates such as monkeys and chimpanzees. For initial studies, transgenic mice and rats are highly desirable due to their relative ease of maintenance and shorter life spans. For certain studies, transgenic yeast or invertebrates may be suitable and preferred because they allow for r;~pid screening and provide for much easier handling. For longer term studies, non-human primates may be desired due to their similarity with humans.
There are several ways in which to create an animal model for LLK-related malignancy. Generation of a specific mutation in a homologous animal gene is one strategy. Secondly, a wild type human gene and/or a humanized animal gene could be inserted by homologous recombination. Thirdly, it is also possible to insert a mutant (single or multiple) human gene as genomic or minigene cDNA construct using wild type or mutant or artificial promoter elements. Fourthly, knock-out of the endogenous homologous animal genes may be accomplished by the insertion of artificially modified fragments of the endogenous gene by homologous recombination. The modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the 5 inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase.
If it is desired to inactivate or replace the endogenous LLK gene, homologous recombination using embryonic stem cells may be applied.
For oocyte injection, one or more copies of the mutant or wild 10 type LLK gene can be inserted into the pronucleus of a just-fertilized mouse oocyte. This oocyte is then reimplanted into a pseudo-pregnant foster mother. The liveborn mice can then be screened for integrants using analysis of tail DNA for the presence of the LLK gene sequences.
The transgene can be either a complete genomic sequence injected as a 15 YAC, BAC, PAC or other chromosome DNA fragment, a cDNA with either the natural promoter or a heterologous promoter, or a minigene containing all of the coding region and other elements found to be necessary for optimum expression.
Retroviral infection of early embryos can also be done to insert 20 the mutant or wild type LLK. In this method, the mutant or wild type LLK is inserted into a retroviral vector which is used to directly infect mouse embryos during the early stages of development to generate a chimera, some of which will lead to germline transmission. Similar experiments can be conducted in the case of mutant proteins, using 25 mutant murine or other animal LLK gene sequences.
Homologous recombination using stem cells allows for the screening of gene transfer cells to identify the rare homologous recombination events. Once identified, these can be used to generate chimeras by injection of mouse blastocysts, and a proportion of the

30 resulting mice will show germline transmission from the recombinant

31 line. This methodology is especially useful if inactivation of the LLK
gene is desired. For example, inacaivation of the LLK gene can be done by designing a DNA fragment which contains sequences from a LLK
exon flanking a selectable marker. Homologous recombination leads to the insertion of the marker sequences in the middle of an exon, inactivating the LLK gene. DNA analysis of individual clones can then be used to recognize the homologous recombination events.
It is also possible to create mutations in the mouse germline by injecting oligonucleotides containing the mutation of interest and screening the resulting cells by PCR.
In general, techniques of generating transgenic animals are widely accepted and practiced. A laboratory manual on the manipulation of the mouse embryo, for example, is available detailing standard laboratory techniques for the production of transgenic mice (Hogan et al., 1986).
This embodiment of the invention has the most significant potential as a model for acute leukemia and related malignancies in which the function of the LLK gene could be studied as well as the effects of new therapeutic treatments and the design of new drug therapies. Animal models are also valuable in order to study the time frame in which an introduced mutation in the LLK gene causes a malignancy.
Drub Screening Cell lines may be cultured which express LLK, to which a test compound is added to the culture medium. After a period of incubation, the expression of LLK mRNA and resultant protein product can be quantified to determine any changes in expression as a result of the test compound. Cell lines transfected with constructs expressing mutant or normal LLK can also be used to test the function of

32 compounds developed to modify with the protein function.
Transformed cell lines expressing LLK protein could be mutagenized by the use of mutagenizing agents to produce a malignant phenotype in which the role of mutated LLK can be studied.
With respect to normal LLK, the effect of protein drugs or agents which interact with the protein's normal function could be studied in order to more precisely define the intracellular role of the protein.
EXAMPLES
The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.
Example 1 - Cloning and Sequencing of LLK
Methods:
A degenerate anti-sense oligonucleotide corresponding to the conserved MAPK consensus sequence YV(A/U)TRW, found in subdomain VIII, was synthesized as follows:
CC(A/I)CC(G/T)(G/A)GT(G/A)C(C/T/A)AC(G/A)TA. This oligonucleotide and the M13/pUC forward amplification primer (Gibco BRL, Gaithersburg MD, USA), were used to amplify DNA isolated from a rat brain library, constructed in ~, ZAPII (Clontech) as previously described (22). PCR was performed in a 50 ~1 reaction consisting of 10 pmol of degenerate primer, 20 pmol M13/pUC Forward amplification primer, 2 mM MgClz, 0.2 mM dNTP, 50 mM KCl, 10 mM Tris-HC1 (pH 9.0), 0.1%
Triton X-100, and 0.1 U/~l Taq DNA polymerase (Promega, Madison WI, USA). Before the addition of Taq DNA polymerase each reaction was heated to 94"C for 3 minutes. For 30 cycles the reaction mixtures were denatured at 94"C for 1 minute, allowed to anneal at 57"C for 1 minutes and 30 second, and extended at 72°C for 2 minutes. After all cycles were complete 4 U of DNA polymerase I Klenow fragment

33 (Amersham, Amersham UK) were added to create blunt termimi by incubation at 37°C for 30 minutes.. A 0.5 kb product was purified from an agarose gel using the Geneclean II kit (Bio 101, La Jolla CA, USA) and cloned into the Smal site of the pUCl8 plasmid using standard methodology.
DNA sequencing was performed using an AmpliCycle Sequencing Kit (Perkin Elmer, Branchburg NrJ, USA). Amplification of the 5' end of cDNA was accomplished with the 5' RACE System (Gibco BRL, Gaithersburg MD, USA).
cDNA library screening was performed as previously described (23). In brief, 5x10~> pfu of a rat jejunum ~, zap II cDNA library (Stratagene, La Jolla CA, USA) or of the rat brain cDNA library used for amplification were incubated with XL-1 blue bacteria and plated on each of 10, 150 mm, YT agar plates. The phage DNA from the lysed bacteria cells was transferred to duplicate Hybond-N nylon membranes (Amersham, Amersham UK). After transfer, the filters were denatured for 2 minutes in a solution of 1.5 M NaCI and 0.5 M NaOH, and neutralized for 5 minutes in a solution of 1.5 M NaCl and 0.5 M Tris-HCl (pH 8). The filters were then briefly rinsed in 2x SSC and allowed to dry at room temperature. DNA was UV crosslinked to the filters using a UV Stratalinker (Stratagene, La Jolla CA, USA). For screening, the filters were pre-hybridized for two hours at 42°C in a solution of 50% formamide, 5x SSC, 5x Denhardt's reagent, 20 mM
Na~PO:~, and 0.2 mg/ml salmon sperm DNA. Hybridization was performed overnight at 42°C in a solution of 50% formamide, 5x SSC, 5x Denhardt's reagent, 20 mM NazPO.~, 10% dextran sulphate, 0.2 mg/ml salmon sperm DNA, and 1x106 cpm/ml of probe. To prepare the probe, 25 ng of the PCR product was radiolabelled with 50 ~Ci of [a-~j'P]dCTP using the Multiprime DNA labeling system (Amersham, Amersham UK). Following hybridization, the filters were washed at

34 55°C in O. lx SSC and 0.1% SDS. Filters were exposed to film overnight at -70°C with an intensifying screen. Purification of the hybridizing plaques was performed by secondary, tertiary, and quaternary screens, as described above. Excision of the cDNA inserts from the purified phage followed the manufacturer's ,ZAP II automatic excision protocol (Stratagene, La Jolla CA, USA).
"Anchored PCR" was performed using the degenerate anti-sense oligonucleotide described above and the pUCl8 Universal Forward primer on ~,ZAPII rat brain library target DNA. Products of approximately 500 by were consistently amplified, and were batch cloned into pUCl8. Sequence analysis of 44 of these products led to the discovery of a single novel putative MAP kinase.
A rat jejunum cDNA library and a rat brain cDNA library were screened with the PCR product to isolate the complete cDNA. Three clones were isolated from the jejunum library which upon sequence analysis were shown to be identical. From the brain library two additional positive phage were isolated, each representing independent clones. Criteria for a full length cDNA included the presence of a Kozak consensus translation initiation sequence of GCCGCC(A/G)CCAUGG and the presence of an upstream stop codon in the same reading frame as the initiation codon (29). Sequencing of all clones demonstrated that none were complete by these criteria. To determine 5' sequence, "RACE" was performed on rat brain poly(A)+RNA (30) using the sequence specific anti-sense oligonucleotide CAGGCTTTATATCTCGGTGG and a standard poly T anchoring oligonucleotide. A second round of amplification was performed using a nested anti-sense oligonucleotide, GGACTAGTCTTAATGGCCACTATCCGC to ensure specificity.
Sequence analysis of the amplified products identified a putative initiation methionine with an in-frame upstream stop codon and an adjacent Kozak consensus sequence, with seven of the thirteen nucleotides conserved at this site, including a purine at position -3 and a guanine at position +4. The complete rat LLK cDNA was sequenced using 12 internal oligonucleotides.
5 The cDNA sequence and deduced amino acid sequence of rat LLK
are shown in Tables l, 2 and 3. In Table 3, protein kinase consensus residues are shown in bold face type. The cyclin-binding (NKIATRE) motif and two sites of potential regulation, the SY duplex, and the TDY
MAP kinase-like regulatory region, are boxed.
10 The cloned rat LLK cDNA described above was used to screen a human ~,gt 11 fetal heart genomic library. A human LLK genomic DNA
was cloned and the cDNA sequenced. Tables 4 and 5 show the human cDNA and deduced amino acid sequences respectively.
15 Example 2 Methods:
Southern a~ad Northern Blot, analysis: Total cellular RNA was prepared using the TRIzol Reagent (Gibco BRL, Gaithersburg MD, USA) and from this poly(A)+RNA was prepared using Oligo (dT) 20 cellulose (Boehringer Mannheim Biochemica, Laval PQ, Canada) as follows. 1 g of Oligo (dT) cellulose was suspended in 20 ml of loading buffer (400 mM NaCl, 20 mM Tris pH 7.4, 10 mM EDTA, 0.2% SDS) and mixed for 20 minutes at room temperature. Oligo (dT) cellulose was added to each sample of total RNA and each mixture was gently 25 rotated at room temperature overnight. All samples were then spun for 5 minutes at 2000 rpm, washed 2-3 times with 20 ml of loading buffer, and then 2-3 times with 20 ml of wash buffer (100 mM NaCI, 10 mM
Tris pH 7.4, 1 mM EDTA, 0.2% SDS). Cellulose samples were then resuspended in 10 ml of wash buffer and poured into columns (Bio Rad, 30 Hercules California, USA). Columns were packed with 10 ml of wash buffer and then poly(A)+RNA was eluted with five 1 ml volumes of elution buffer pre-warmed to 37°C (1 mM Tris pH 7.4, 1 mM EDTA, 0.2% SDS). Poly(A)+RNA was precipitated overnight at -20°C by the addition of 30 ~g tRNA, 0.6 ml 3 M sodium acetate, and 12 ml 99%
ethanol. Pellets were air dried and resuspended in DEPC-treated water. Ten ~g of poly(A)+RNA from rat intestine, brain, muscle, lung, spleen, heart, liver, and thymus were used to generate a rat tissue blot.
RNA was fractionated overnight by electrophoresis on a 1.2%
formaldehyde agarose gel with buffer recirculation. The gel was then washed thoroughly in DEPC treated water and the RNA was transferred to nylon membrane by capillary transfer. The blot was probed with the 1.3 kb EcoRl fragment of LLK cDNA, radiolabelled with [a-B~P]dCTP, as described above. To control for the level of RNA in each lane, a rat ~-actin probe was also used.
Souther~t blot analysis: 10 Eag of rat genomic DNA was digested overnight at 37°C with either EcoRl, BamHl or HindIII. For the cross species Southern blot, 10 ~g of either rat, pig, mouse, human or Drosophila genomic DNA was digested overnight at 37°C with EcoRl.
Each of these digests were fractionated overnight by electrophoresis on a 1.0% agarose gel. Following elecarophoresis, the gel was incubated with shaking for 15 min at room temperature in 0.1 M HC1, followed by min in a solution of 0.5 M NaOH, 1.5 M NaCl, and then for 45 min in a solution of 1 M Tris pH 7.0, 1.5 M NaCI. The genomic DNA was transferred to nylon membrane by capillary transfer. For all blots, the 25 1.3 kb EcoRl fragment (nucleotides 1-1372) of the rat jejunum cDNA
was radiolabelled with [a-B~P]dCTP, as described above, and used as a probe. Prehybridization, hybridization, and wash conditions were performed as previously described for the library screens (section 3.2.1).
Filters were visualized by Phosphor Imager (Molecular Dynamics) 30 using the Image C.~uant software (Molecular Dynamics).

Phyl~o enetic conservation.
The EcoR1-EcoRl 1.3 kb fragment of the rat LLK cDNA clone was used to screen rat genomic DNA digested with three separate restriction endonucleases. Results of Southern blotting revealed that three bands hybridized to EcoRl digested DNA with approximate sizes of 7.5 kb, 5.6 kb, and 4.7 kb. Two bands of approximately 5.4 kb and 4.0 kb hybridized in BamHl digests, while two bands of 9.4 kb, and 4.7 kb were seen in HindIII digested DNA (figure 4A). Results of cross species Southern blot analysis demonstrates that rat LLK hybridizes to EcoRl digested genomic DNA from rat, pig, mouse, human, and drosophila (figure 4B). No hybridizing bands were seen in EcoRl digested DNA from either, Dictyostelaum (data not shown) or S.
cereUisaae (data not shown).
Tissue distribution of LLK.
To determine the tissue distribution of LLK, a Northern blot of poly(A)+mRNA from rat intestine, brain, muscle, lung, spleen, heart, liver, and thymus was probed with the 1.3 kb EcoR1 fragment of LLK
cDNA. Northern blotting revealed bands of 4.4 kb, 2.5 kb or 2.2 kb in brain, muscle, lung, intestine and heart (figure 5). No expression of LLK was seen in rat spleen, liver, or thymus (not shown). Quality of poly(A)+mRNA was determined by probing for rat (3-actin.
Example 3 - Stimulation by EGF
Cell culticre arid transfectaom NIH 3T3 and HeLa cells were maintained in Alpha medium (Gibco BRL) containing 10% Cool Calf serum (Gibco BRL). Semiconfluent cells grown on 10 cm plastic dishes (Nunc) were transfected with 10~g of individual expression constructs using Lipfectin (Gibco BRL) as described (25). To achieve high level expression, HeLa cells were infected with recombinant human vaccinia virus, directing the expression of T7 RNA polymerase (26) one hour prior to the introduction of T'7 polymerase-driven expression plasmids.
Transfected NIH 3T3 cells were selected in culture with 150 pg/ml 6418 (Sigma).
Imrnunoprecipitatio~a: Cells (1 x 10~) were lysed on ice for 1 hour in 500 ~l 0.1% NP40, 125 mM NaCl, 25 mM Tris, pH 7.2 with the protease inhibitors leupeptin 1 pg/ml, aprotinin 1 pg/ml and 1 mM
phenylmethylsulfonyl fluoride, followed by centrifugation.
Phosphatase activity was inhibited by 5 mM sodium orthovanadate and 50 mM dibasic sodium pyrophosphate (Sigma). Lysates were incubated on ice with 5 ~l PY20 antibody for 2 hours followed by incubation with 50 ~l of Sepharose protein A beads (Pharmacia) at 4°C with agitation.
Precipitates were washed 4 times with 1 ml of ice cold lysis buffer.
Kiraase Assay: ha vitro kinase reactions were performed using immunoprecipitated bead immobilized HA fusion proteins and myelin basic protein (Sigma) as a substrate. Each reaction was performed in a final volume of 50 ~l containing 25 pl of 2X kinase buffer (100~M
[~3zP]ATP, lOmM MgCl, 50 mM Tris HCl-pH 7.5, 1mM EDTA), 5 ~1 of myelin basic protein (1 ~g/ml) or cJun (1 ~g/ml) dissolved in distilled water and 20 ~l of immobilized kinase slury. Samples were incubated at 37° for 30 minutes. Reactions were terminated by the addition of 50 ~1 of 2X SDS protein loading buffer followed by boiling for 10 minutes.
Products were separated on an 8°/~ polyacrylamide protein gel followed by transfer to a PVDF membrane (Immobilon N, Millipore) and phosphoimaging as described above.
A characteristic of MAP kinases is activation by extracellular stimuli. To evaluate LLK activity after a variety of extracellular stimuli, (HA)LLKpcDNA3, a plasmid directing the expression of epitope tagged full length rat LLK, or the mutant (HA)LLK(ADF)pcDNA3, were transiently transfected into HeLa cells.
Cells had been infected 1 hour previously with a human recombinant vaccinia virus, engineered to express T7 RNA polymerase. After 48 hours, cells expressing wildtype protein were washed and resuspended in 0.1% fetal calf serum for 12 hours. Cells (2 x 10~) were then exposed for 30 minutes either to medium containing 10 or 20% FCS, 10 nM
PMA, 20-60 ng/ml EGF, 400 mM sorbitol and 500 ~g/ml anisomycin, or to germicidal UV light [ 254 nm (Cxeneral Electric)] for 10 minutes.
Cells were then washed, harvested and lysed. (HA)LLK was immunoprecipitated and its activity towards myelin basic protein (MBP) was evaluated in vitro as described above. Figure 3A
demonstrates that LLK is quiescent in serum starved cells and is not stimulated by brief exposures to either stress stimuli, or to growth stimulants such as FCS and PMA, however mild stimulation was observed after overnight incubatian with 10% FCS. In contrast, activation was observed after EGF stimulation in a dose dependent manner, with a 15 fold increase observed after a 30 minute exposure (Figure 3B). These observations demonstrate a very specific pattern of activation by extracellular stimuli.
Example 4 - Expression of LLK and S phase Entry Vector Corzstructiorz a~td rnu.tagenesis: Rat LLK was mutated at threonine 158 and tyrosine 160, putative sites of activating phosphorylation, to alanine and phenylalanine, respectively, using the 4 primer mutagenesis protocol, as previously described (10) and the sense oligonucleotide GGAGACGTTTACGCAGAATTCGTGGCCACCCGCTGG. Asparagine 143 of LLK was similarly mutated. to aspartic acid using the sense sequence X. LLK was cloned in frame, downstream of the sequence ATGGCTTACCCATACGATGTTCCAGAT, defining the influenza hemagglutinin epitope tag (HA) MAYPYDVPD, and was cloned into the pcDNA3 plasmid (Invitrogen). LLK was expressed as a eukaryotic GST

fusion protein in the plasmid pEBG and as a prokaryotic GST fusion using pGEX-KG as previously described (24). PCMVCD20 pcDNA3, p2l~~f~E''1 and N144CDK2 were gifts of Dr. E. Harlow (Massachusetts General Hospital, Boston MA).
5 Brd U cell stainang grad Flow Cytornetry: Analysis of growth parameters in transiently transfected cells was performed as previously described (27). In brief, cos cells were transiently transfected with both pCMVCD20 and (HA)LLKpcDNA3 or mutant forms in a molar ratio of 1:5. Thirty six hours later, cells (5 x 10~) were pulsed with 10 ~M BrdU
10 for 16 hours then trypsinized, washed in PBS and resuspended in 2 ml IFA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 4% fetal calf serum, 0.1% NaN: ) to neutralize trypsin. Cells were then centrifuged and incubated at 4°C for 1 hour in 901 of PBS containing 2% FCS (PBS-F) and 101 of anti-human CD20-RPE. Cells were washed in PBS-F and 15 resuspended in 0.5 ml ice-cold 0.15M NaCl and fixed by dropwise addition of 1.2 ml ice-cold 90% ethanol while gently vortexing on ice.
After 30 min incubation on ice, cells were washed, and resuspended in 1% paraformaldehyde and 0.01% Tween-20. Cells were centrifuged and resuspended in 1 ml freshly prepared DNAse I solution (50 Kunitz 20 U/ml bovine pancreas deoxyribonuclease I (Sigma), 4.2 mM
MgClz/0.15M NaCl pH5). Cells were incubated 10 min at room temperature, washed in pBS-F and resuspended in 10 ~1 anti-BrdU-FITC Becton-Dickinson). Samples were incubated at room temperature for 30 min, washed, and resuspended in 2 ml PBS-F. Analysis was 25 done using an EPICS-XL flow cytometer (Coulter) with 488 nm excitation and 525 nm and 575nm bandpass filters. Fluorescence was measured in at least 2000 CD20 positive cells for each sample. Results were analyzed using the WinMDI software (J. Trotter, Scripps Research Institute).

Rat-1 fibroblast cells were co-transfected with (HA)LLKpcDNA3, (HA)LLK(AEF)pcDNA3 or pcDNA3 control plasmid with pCMVCD20, in a 1:5 ratio, to allow identification of DNA uptake using a flow cytometric technique. As controls for mediators of S phase entry, samples were transfected with plasmids directing the expression of p21~~''~~t'' and a dominant negative form of CKD2. Cell transfection efficiencies were 20+/- 10% in all cases as indicated by CD20 expression. The S phase fraction among transfected cells was evaluated and compared to cells transfected with vector alone. Results are shown in Figure 4. Eighty percent of vector transfected cells were in S phase during the 16 hour period of BrdU labeling. As expected, the dominant negative CDK2 mutant and p21~~'~~E' decreased the number of S phase cells to 55% and 52% respectively. Similarly, overexpression of wild type LLK decreased the proportion of S phase cells to 60%. The kinase dead mutant of LLK also reduced the proportion of cells entering S phase, again suggesting that phosphorylation at the TDY motif is not required for this biological activity. These data show that LLK expression leads to decreased cell growth.
Example 5 - Chromosomal Localization of Human LLK
The human genomic DNA clone obtained as described in Example 1 was used as probe in Fluorescent In Situ Chromosomal Hybridization (FISH) performed on normal human peripheral lymphocytes, to determine the chromosomal location of the gene for human LLK.
The method used was based on published procedures (28).
Hybridization was carried out for 16 to 20 hours at 37°C. This was followed by three 5-min washes in 50% formamide and 2 x SSC at 42°C.
All procedures involving the handling of fluorescein isothiocyanate (FITC) were performed in subdued lighting. The slides were soaked in 3% BSA in 4 x SSC blocking solution for 30 min. before being incubated in FITC - avidin (Oncor Laboratories, Gaithersburg, MD, USA) at 5 ~g for 390 min at 37°C. Fluorophores were washed off with 4 x SSC 0.1%
Tween 20 at 42°C using three 3-minute washes. To amplify the FITC
signal biotinylated anti-avidin antibody (Oncor was added to each slide, and the slides were incubated at 37°C for 30 min. This was followed by further washes in 4 x SSC 0.1% Tween 20 at 42°C, another incubation with FITC-avidin, a second series of washes as above, and counterstaining with either 0.2~g/ml DAPI, or 0.2 ~g/ml propidium iodide (Boehringer Mannheim, Indianapolis, IN, USA) or with both counterstains as a mixture for 2 min. The slides were washed in phosphate buffered saline (PBS) for 5 min and mounted in 20 mM Tris-HCL, pH 8.0, 90% glycerol containing 2.3% DABCO antifade (1,4 diazabicyclo-[2.2.2] octane). The photomicrographs were obtained using a Nikon Microphot FXA epi~luorescence microscope equipped with dual band TITC/Texas red filters (Omega Optical Inc. FL, USA).
The images were captured by a thermoelectrically cooled charge coupled (CCD) camera (Photometrics, Tucson, AZ, USA). For these images, separate propidium iodide-stained nuclei and chromosomes, and hybridization signals were acquired and merged using image analysis software (courtesy of Tim Rand and David Ward, Yale University, New Haven, CT, USA).
Localization of the probe indicated that the human LLK gene is on chromosome 5q31.1 (Data not shown).
Example 6 - LLK Gene Loss and Acute Leukemia FISH was performed on primary acute myeloblasts from nineteen acute leukemia patients known from cytogenetic analysis to have 5q31 deletion. A bacterial artificial chromosome probe consisting of the telomeric region 5p 15 was used in combination and provided an internal control for hybridization. Scoring criteria: Each round of FISH comprised four patient samples and one normal lymphocyte control. Patient blasts previously identified to contain a single 5q31 deletion were analyzed. Only those cells that contained two red 5p 15 signals, which was used as an internal control, were scored. Each cell was then scored based on the number of green 5q31 (LLK) signals. Two green signals indicated a "contaminating" normal cell, whereas one or zero signals indicated malignant cells. If both 5q31 signals were missing cells were scored as having deleted both copies of LLK. Fifty cells were counted on each slide. Background absence of hybridization among cells displaying two 5q15 alleles was determined from each normal slide as a percentage of cells with one or zero signals. This background frequency was subtracted from the patient data to normalize for hybridization efficiency. In order to calculate the percentage of deleted cells, the number of one and zero signals were pooled defining the denominator. The number of zero signals defined the numerator. Table 7 demonstrates that malignant cells ranged from 0 to 61.5% deletion of both copies of LLK, with >60% of patients known from cytogenetic analysis to be missing one LLK locus having the second allele deleted.
Example 7 - LLK knock-out mice A LLK knock-out mouse is generated using the technique of embryonic stem (ES) cell homologous recombination, as described by Fung-Leung et al., (1992), Imnurzology, v. 4, pp. 189-192.
Briefly, the genomic sequence of mouse LLK is isolated by phage library screening using a rat LLK nucleic acid sequence as disclosed herein. The sequence surrounding exon 1 of the mouse DNA is obtained and cloned into a standard plasmid vector by conventional methods. A bacterial neomycin resistance gene is inserted in an anti-sense orientation into the coding region of exon 1 to create an inactive mutation.
The mutant LLK is introdu<:ed into the mouse embryonic stem cells by electroporation, followed by subsequent selection in neomycin containing tissue culture medium. ES cell clones harboring a single homologous integration of mutant LLK are identified by PCR screen and southern analysis. Mutant ES cells are introduced into murine blastocysts and then implanted into the uteri of hormonally manipulated mice. Chimeric mice born are bred to identify individuals in which germline heterozygous mutation of LLK has been achieved.
Heterozygous mutated mice are mated to homozygosity.
Homozygous deleted mice show development of hyperproliferation of hematopoietic tissues, resulting in acute leukemia.

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CGGCGGGACT ACAGGATGGG CTGAGGCGGC GGCGATTCTC AGGACGAGAA GCCAGGCTTG
AAA_ATGGAGA TGTATGAAAC CCTTGGAAAA GTAGGAGAGG GAAGTTATGG AACGGTCATG
AAATGCAAGC ATAAGGATAC TGGGCGGATA GTGGCCATTA AGATATTCTA TGAGAAACCA
GAAAAATCTG TCAACAAAAT TGCAACGAGA GAAATAAAGT TTCTAAAGCA ATTTCGTCAT
GAAAACCTGG TCAATCTGAT TGAAGTTTTT AGACAAAAAA AGAAAATCCA TTTGGTATTT
GAGTTTATTG ACCACACGGT CTTAGATGAG TTACAGCATT ATTGTCACGG ATTAGAGAGT
AAGCGACTAA GAAAGTACCT GTTCCAGATC CTCCGAGCGA TCGAGTATCT GCACAACAAC
AACATTATCC ACCGAGATAT AAAGCCTGAG AATATTTTAG TCTCCCAGTC AGGAATTACA
AAGCTCTGCG ATTTTGGGTT TGCGCGGACA CTAGCAGCTC CTGGAGACGT TTACACAGAC
TACGTGGCCA CCCGCTGGTA CAGAGCTCCA GAGCTGGTGT TGAAAGACAC CACCTATGGA
AAACCAGTGG ATATCTGGGC TTTGGGCTGT ATGATC.ATTG AAATGGCCAC TGGCAATCCC
TACCTTCCTA GCAGTTCCGA TTTGGATTTG CTCCAC.AAGA TTGTTTTAAA AGTAGGCAAC
CTGACCCCGC ACCTGCACAA TATTTTTTCC AAGAGTCCCA TCTTTGCTGG GGTGGTTCTT
CCTCAAGTTC AACATCCCAA AAACGCAAGA AAGAAATACC CAAAGCTCAA CGGATTGCTG
GCAGATATAG TTCATGCTTG TTTACAAATT GATCCTGCTG AGAGGATATC ATCCACCGAT
CTTTTGCATC ACGATTACTT TACTAGAGAT GGATTTATTG AGAAATTTAT ACCAGAGCTG
AGAGCTAAGT TATTACAGGA AGCAAAGGTT AATTCATTTA TAAAGCCAAA AGAGAATTTT
AAGGAAAATG AACCTGTGAG AGATGAGAAA AAACCAGTTT TTACCAACCC TCTGCTCTAT
GGAAACCCAA CACTTTATGG AAAGGAAGTG GACAGAGACA AAAGGGCCAA GGAGCTCAAA
GTCAGAGTCA TTAAGGCCAA AGGGGGAAAA GGAGACGTCC CAGACCTGAA GAAGACAGAG
AGTGAAGGTG AACACCGCCA GCAGGGCACA GCTGAGGACA CACACCCCAC ATCACTGGAC
AGGAAGCCTT CTGTCTCGGA ACTAACAAAC CCTGTCCATC CCAGTGCGAA TTCTGACACT
GTCAAAGAAG ACCCACACTC TGGGGGCTGT ATGATAATGC CACCTATCAA CCTGACAAGC
AGTAATTTGT TGGCCGCAAA TCCCAGTTCA AACCTCTCCC ACCCCAATTC ACGGTTAACT
GAAAGAACAA AAAAGAGACG CACCTCTTCA CAAACTATTG GACAAACTTT GTCTAATAGC
AGACAAGAGG ACACAGGTCC CACACAAGTC CAAACAGAGA AAGGTGCATT TAATGAGCGA
ACAGGTCAGA ATGCAAAATA GCAAGTGGAA ACAAAAGAAA ACTGAATTTT TCCAAATGCG
ACAGGAAAGA ATTCCATTTC CCTGAGCTGC CGTTCACAAT ACAGGCGAAG GAGATGAAAG
GGATGGAGGT TAAACAGATA AAAGTGCTGA AGAGAGAATC AAAGAAAACG GATTCACC

MEMYETLGKVGEGSYGTVMKCKHKDTGRIVAIKIFYEKPEKSVNKIATREIKFLKQFRHENL
VNLIEVFRQKKKIHLVFEFIDHTVLDELQHYCHGLESKRLRKYLFQILRAIEYLHNNNIIHR
DIKPENILVSQSGITKLCDFGFARTLAAPGDVYTDYVATRWYRAPELVLKDTTYGKPVDIWA
LGCMIIEMATGNPYLPSSSDLDLLHKIVLKVGNLTPHLHNIFSKSPIFAGWLPQVQHPKNA
RKKYPKLNGLLADIVHACLQIDPAERISSTDLLHHDYFTRDGFIEKFIPELRAKLLQEAKVN
SFIKPKENFKENEPVRDEKKPVFTNPLLYGNP~.'LYGKEVDRDKRAKELKVRVIKAKGGKGDV
PDLKKTESEGEHRQQGTAEDTHPTSLDRKPSVSELTNPVHPSANSDTVKEDPHSGGCMIMPP
INLTSSNLLAANPSSNLSHPNSRLTERTKKRR7.'SSQTIGQTLSNSRQEDTGPTQVQTEKGAF
NERTGQNAK

1 cgg gac tac agg 13 atg ggc tga ggc ggc ggc gat tct cag gac gag aag cca ggc ttg aaa ATG GAG ATG
TAT
1 * M E M Y

AAG
E T L G K V G E G S Y G T V M K C K H K

GTC AAC

GTC AAT
45 K I A T R E ~ I K F L K Q F R H E N L V N

GAC CAC

AGA AAG

CAC CGA

GAT TTT

ACC CGC

GAT ATC

AGC AGT

CAC CTG

CAA CAT

GTT CAT

CAC GAT

TTA TTA

GAA CCT

ACA CTT

ATT AAG

GAA CAC

TCT GTC

GAC CCA

TTG GCC

AAA AAG

GAC ACA

AAT GCA

caagtggaaacaaaagaaaactgaatttttcc:aaatgcgacaggaaagaattccatttccctgagctgccgt tcacaatacaggcgaaggagatgaaagggatggaggttaaacagataaaagtgctgaagagagaatcaaagaaaacgga t 1734 tcacc ATGGAGATGTATGAAACCCTTGGAAAAGTGGGAGAGGGAAGTTACGGAACAGTCATGAAATG
TAAACATAAGAATACTGGGCAGATAGTGGCCA7.'TAAGATATTTTATGAGAGACCAGAACAAT
CTGTCAACAAAATTGCGATGAGAGAAATAAAG7.'TTCTAAAGCAATTTCATCACGAAAACCTG
GTCAATCTGATTGAAGTTTTTAGACAGAAAAACJAAA.ATTCATTTGGTATTTGAATTTATTGA
CCACACAGTATTAGATGAGTTACAACATTATTC~TCATGGACTAGAGAGTAAGCGACTTAGAA
AATACCTCTTCCAGATCCTTCGAGCAATTGAC7.'ATCTTCACAGTAATAATATCATTCATCGA
GATATAAAACCTGAGAATATTTTAGTATCCCAC~TCAGGAATTACTAAGCTCTGTGATTTTGG
TTTTGCACGAACACTAGCAGCTCCTGGGGACA7.'TTATACGGACTATGTGGCCACACGCTGGT
ATAGAGCTCCCGAATTAGTATTAAAAGATACTTCTTATGGAAAACCTGTGGATATCTGGGCT
TTGGGCTGTATGATCATTGAGATGGCCACTGGAAATCCCTATCTTCCTAGTAGTTCTGATTT
GGATTTACTCCATAAAATTGTTTTGAAAGTGGC~CAATTTGTCACCTCACTTGCAGAATATCT
TTTCCAAGAGCCCCATTTTTGCTGGGGTAGTTC:TTCCTCAAGTTCAACACCCCAAAAATGCA
AG~TATCCAAAGCTTAATGGATTGTTC~GCAGATATAGTTCATGCTTGTTTACAAAT
TGATCCTGCTGACAGGATATCATCTAGTGATCTTTTGCATCATGAGTATTTTACTAGAGATG
GATTTATTGAAAAATTCATGCCAGAACTGAAAGCTAAATTACTGCAGGAAGCAAAAGTCAAT
TCATTAATAAAGCCAAAAGAGAGTTCTAAAGAAAATGAACTCAGGAAAGATGAAAGAAAA.AC
AGTTTATACCAATACACTGCTAAGTAGTTCAGTTTTGGGAGAGGAAATAGAAAAAGAGAAAA
AGCCCAAGGAGATCAAAGTCAGAGTTATTAAAC~TCAAAGGAGGAAGAGGAGATATCTCAGAA
CCAAAAAAGAA.AGAGTATGAAGGTGGACTTGGTCAACAGGATGCAAATGAAAATGTTCATCC
TATGTCTCCAGATACAA.AACTTGTAACCATTGAACCACCAAACCCTATCAATCCCAGCACTA
ACTGTAATGGCTTGAAAGAAAATCCACATTGCCsGAGGTTCTGTAACAATGCCACCCATCAAT
CTAACTAACAGTAATTTGATGGCTGCAAATCTCAGTTCAAATCTCTTTCACCCCAGTGTGAG
GTGAGCTGTAACAGAGAAGAAACCTAAATAATACAACATTCCTGTATAATGGTATTTCAAAG
AATCGTGTTCATAGTGTCTGTATGTAAACTGAACTTGAAGAAAATATATTGAAATTAAAGCT
GTATAATGGGCC

TART~F 5 NKIATRE ..............................MEmYetLgkVGEGsYGtVmKckhkdTGrIV
KKIALRE .............................mMEkYekigkIGEGsYGvVFKcrnrdTGqIV
KKIAMRE ..............................MEkYenLgIVGEGsYGmVmKcrnkdTGrIV
CDK1 ..............................MEdYtkiekIGEGtYGvVYKgrhktTGqW
CDK2 ................-.............MEnFqkvekIGEGtYGvVYKArnklTGeW
ERK1 eprgtagvvpvvpgevevvkgq....pfdvgprYtqLqyIGEGaYGmVssAydhvrktrV
ERK2 .......maaaaaagpemvrgq....vfdvgprYtnLsyIGEGaYGmVCsAydnlnkvrV
SAPK ........msdsksdgqfysvqvadst:ftvLkrYqqLkpIGsGaqGiVCaAfdtvlGinV
p38 ..........msqerptfyrqelnktiwevpErYqnLspVGsGaYGsVCaAfdtkTGhrV
Consensus -------------------------w ---ME-Y--L--IGEG-YG-VCKA----TG-IV
NKIATRE AIKiFye.kpeksVnKiAtREIkFLKqFRHENIVnLiEVFrqkkki......hLVFEFiD
KKIALRE AIKKFleseDdpvIkKiAlREIrMLKqLkHpNIVnLLEVFrrkrrL......hLVFEYcD
KKIAMRE AIKKFIesdDdkmVkKiAmREIkLLKqLRHENIVnLLEVckkkkrw......YLVFEFvD
CDKl AmKKirlesEeegVpstAiREIsLLKeLRHpNIVsLqDVLmqdsrL......YLIFEFLs CDK2 AlKKirxdtEtegVpstAiREIsLLKeLnHpNIVkLLDVihtenkL......YLVFEFLh ERK1 AIKKis.pfEhqtycqrtlREIqiLIgFRHENVIgirDIL.raptLeamrdvYiVqDLME
ERK2 AIKKis.pfEhqtycqrtlREIkiLl~:FRHENIIginDIi.raptieqmkdvYiVqDLME
SAPK AVKKLsrpfqnqthaKrAyRElvLLK<:vnHkNIIsLLnVFtpqktLeefqdvYLVmELMD
p38 AVKKLsrpfqsiihaKrtyRElrLLKhMkHENVIgLLDVFtparsLeefndvYLVthLMg Consensus AIKKF----E---V-K-A-REI-LLK--LRHENIV-LLDVF-----L------YLVFE-MD
NKIATRE htvldeLqhychg..LeskrlrkYLFQILraIeYlHnnNIIHRDiKPeNiLVsqsgitKL
KKIALRE htvlheLdryqrg..vpehlVksitWQtLqaVnFcHkhNcIHRDvKPeNiLItkhsviKL
KKIAMRE htilddLelfpng..LdyqvVqkYLFQIinGIgFcHShNIIHRDiKPeNiLVsqsgvvKL
CDK1 mDLkkyLdsippgqyMdsslVksYLYQILqGIvFcHSrrVIHRDLKPqNLLIddkgtiKL
CDK2 qDLkkfMdasaltg.iplplIksYLFQILqGlaFcHShrVIHRDLKPqNLLIntegaiKL
ERK1 tDLyklLk....sqqLsndhIcyFLYQILrGIkYiHSaNVIHRDLKPsNLLInttcdlKi ERK2 tDLyklLk....tqhLsndhIcyFLYQILrGIkYiHSaNVIHRDLKPsNLLlnttcdlKi SAPK anLcqvih.....meLdhermsyLLYQmLcGIkhlHSagIIHRDLKPsNivVksdctlKi p38 aDLnnivk....cqkLtddhVqfLiYQILrGIkYiHSadIIHRDLKPsNLaVnedcelKi Consensus -DL---L--------L----V--YLYQIL-GI---HS-N-IHRDLKP-NLL-------KL
NKIATRE CDFGFARtLaa... PgdvYTDYVATRWYRAPEIvLkdttYgkpVDIWalGCmiiEMatgn KKIALRE CDFGFARILtg... PsdyYTDYVATRWYRsPEILvGdtqYgppVDVWaIGCVFAELLsgv KKIAMRE CDFGFARtLaa... PgevYTDYVATRWYRAPEILvGdvkYgkaVDVWaIGCIvtEMFmge CDK1 aDFGLARaFgi... PirvYTheVvTlWYRsPEVLLGsarYstpVDIWSIGtIFAELatkk CDK2 aDFGLARaFgv... PvrtYTheVvTIWYRAPEILLGckyYstaVDIWS1GCIFAEMvtrr ERKl CDFGLARiadpehdhtgflTEYVATRWYRAPEIMLnskgYtksIDIWSVGCILAEMLsnr ERK2 CDFGLARvadpdhdhtgflTEYVATRWYRAPEIMLnskgYtksIDIWSVGCILAEMLsnr SAPK 1DFGLARtactnf. ...mmTpYVvTR'YYRAPEViLG.mgYkenVDIWSVGCIMAEMvlhk p38 1DFGLARhtdde.. ....mTgYVATRWYRAPEIMLnwmhYnqtVDIWSVGCIMAELLtgr Consensus CDFGLAR-L----- P---YTDYVATRWYRAPEILLG---Y---VDIWSVGCIFAEML---NKIATRE PyLPssSDIDILhkIvlkvGnltphlhniFskspiFagvvlpqvq.hpknarkkYPkl..
KKIALRE PLWPGkSDVDQLylIrktLGdliprhqqvFstnqyFsgvkipdPe.dmepLelkFPni..
KKIAMRE PLFPGdSDIDQLyhIMmcLGnliprhqelFnknpvFagvrlpeik.erepLerrYPkl..
CDK1 PLFhGdSEIDQLfrIFraLG...tpnnevWpevesLqdYkntFPkwkpgsLashvknl..
CDK2 aLFPGdSEIDQLfrIFrtLG...tpdevvWpgvtsMpdYkpsFPkwarqdFskvvPpl..
ERK1 PiFPGkhylDQLnhILgiLGspsqed7_nciinmka.rnYlqsLPsktkvaWaklFPk...
ERK2 PiFPGkhylDQLnhILgiLGspsqedlnciinlka.rnYllsLPhknkvpWnrlFPn...
SAPK scsPGrdyIDQwnkVieqLGtpsaefmkkL.qptv.rnYvenrPkypgikFeelFPdwif p38 tLFPGtdhIDQLkIILrlvGtpgaellkkissesa.rnYiqsLaqmpkmnFanvFig...
Consensus PLFPG-SDIDQL--IL--LG----------F-----F--Y---LP----------FP----NKIATRE ..........ngllaDivhacLqiDPaeRIsstdlLhHdYFtr....dgfiekfipeLra KKIALRE ..........sypAlgLLkgcLhmDPt;eRltceqlLhHPYFenirEieDlakehdkptrk KKIAMRE ..........sevviDLakKcLhiDPdKRpfcaelLhHdFF....qmdgfaerfsqeLql CDK1 ..........denglDLLsKMLiYDPaKRIsgkmALnHPYFnd.......ldnqikkm..
CDK2 ..........dedgrsLLsqMLhYDPnKRIsakaALaHPFFqd.......vtkpvphLrl ERK1 .........sdskAlDLLdrMLtFnPnKRItveeALaHPYLeqyyDptD.epvaeepFtf ERK2 .........adskAlDLLdKMLtFnPhKRIeveqALaHPYLeqyyDpsD.epiaeapFkf SAPK pseserdkiktsqArDLLsKMLviDPdKRIsvdeALrHPYitvwyDpaEaeapppqiYda p38 .........anplAvDLLeKMLvLDsdKRItaaqALaHaYFaqyhDpdD.epvad.pYdq Consensus -------------A-DLL-KML--DP--KRI----AL-HPYF----D--D--------L
NKIATRE kLlqeakvnsfiKpkenfkenepvrdekkpvftnpllygnptlygkevdrdkrakelkvr KKIALRE tLrksrkhhcftetskL........qyrlpqltgssil...paldnkkyycdtkklnyrfp KKIAMRE kvqkdarnvslsKksqn........rkkekekddslveerktlvvqdtnadpkikdyklf CDK1 ............................................................
CDK2 ............................................................
ERK1 dMelddlpkerlKeliFqetarfqpgapeap.............................
ERK2 dMelddlpkeklKeliFeetarfqpgyrs...............................
SAPK qLeerehaieewKeliYkevmdweerskngvkdqpsdaavsskatpsqsssindissmst p38 sFesrdllidewKsltYdevisfvpppldqeemes.........................
Consensus -L-_________K_____________.__________________________________ NKIATRE vikakggkgdvpdlkktesegehrqqgtaedthptsldrkpsvselt.............
KKIALRE ni..........................................................
KKIAMRE kikgskidgekaekgnrasnasclhdsrtshnkivpstslkdcsnvsvdhtrnpsvaipp CDK1 ............................................................
CDK2 ............................................................
ERKl ............................................................
ERK2 ............................................................
SAPK ehtlasdtdssldastgplegcr.....................................
p38 ............................................................
Consensus -_____________-___________._____-_____________-______________ CDKl: human Cyclin Dependent Kinase 1 CDK2: human Cyclin Dependent Kinase 2 ERK1: human Extracellular stimulus Regulated Kinase 1 ERK2: human Extracellular stimulus Regulated Kinase 2 SAPK: human Stress Activated Protein :Kinase P38: human p38HOG kinase Patient Number Percent Deletion of NKIATRE

1 26.4 2 3.7 3 61.5 4 26.2 5 56. 5 6 56.6 7 25.4 8 38.1 9 0.0 10 68.0 11 20.6 12 8.8 13 0.0 14 48.3 15 30.3 16 0.0 17 0.0 18 36.6 19 0.0

Claims