The U.S. Government has certain rights in the invention based upon research support provided by National Institutes of Health Grant No. GM 47017.

Field of the Invention The invention relates to nucleotide sequences encoding a protein known to function in regulating cell division.

Background of the Invention and Prior Art

The cells of eukaryotes, including humans and other mammals, replicate themselves by carrying out an ordered sequence of events, which are cyclically repeated in each successive cell division. In somatic (non germ-line) cells, a typical cycle has four characterized phases: G l , an interval following the completion of mitosis, also termed first gap phase; S, a period during which the cell undergoes DNA synthesis; G2 or second gap phase following completion of DNA synthesis and preceding mitosis; and M, mitosis, where separation of complete sets of replicated DNA occurs. The end result of this process is the generation of two daughter cells that are equivalent both in genetic makeup and in size to the original parent cell. A complex series of biochemical interactions act to control the cell cycle through a series of checkpoints or gating reactions which function to ensure that the requisite precursor phases are completed before the ensuing phase begins. In particular, the checkpoints ensure accurate reproduction and dispersion of the cell's genetic material. In a metazoan organism with differentiated tissues, such as a human being, cells of different tissues replicate at vastly different rates at different life stages. Early embryonic cells replicate rapidly and synchronously, whereas at later stages of development and during adulthood, some cells, such as muscle and nerve cells stop replicating while others, such as epithelial cells, continue to divide throughout the organism's life. Failure of cells to precisely control their replicative state therefore leads to a variety of diseases of pathological proliferation, including cancers and atherosclerosis.

Progress over the past several years has greatly advanced the general understanding of the biochemical reactions which regulate the cell cycle. A general paradigm for cell cycle regulation has emerged in which complexes composed of cyclins and cyclin- dependent kinases (CDKs) regulate progression through stages of the cell cycle. Several mechanisms exist to keep the activity of the cyclin/CDK complexes turned off until the appropriate stage of the cell cycle. Known mechanisms include reversible phosphorylation, binding to small molecular weight inhibitors, transcription control, intracellular location and protein degradation. In yeast, there are multiple cyclins but only a single CDK. The CDK of fission yeast is encoded by the cdc2 gene, that of budding yeast by the cdc28 gene. In higher eukaryotes, including humans, there are multiple

CDKs as well as multiple cyclins. Despite the greater complexity of the higher eukaryotes, the overall scheme for cell cycle progression involving cyclins and CDKs is conserved. Deregulation of components of these regulatory pathways has been implicated in human cancer. For a recent general review, see Hunter, T. et al. (1994) Cell 79:573- 582.

In humans, there are three known Cdc25 phosphatases, denoted Cdc25A, Cdc25B and Cdc25C. Cdc25B has been shown to be overexpressed in certain breast cancers. In human cells, Cdc25C is present throughout the cell cycle. Its substrate, Cdc2/cyclin B, accumulates throughout the S and G2 phases of the cell cycle. Cdc25C is itself regulated by phosphorylation. The major site of Cdc25C phosphorylation is serine 216 (Ogg, S. et al. (1994) J. Biol. Chem. 269:30461-30469). The protein kinase that acts on Cdc25C was purified over 8000-fold from rat liver. [Ogg, S. et al. (1994) J. Biol. Chem. 269:30461.] It was shown to phosphorylate a peptide substrate having the sequence of amino acids 210- 231 of human Cdc25C, but not the equivalent peptide in which serine 216 had been changed to a threonine. The kinase is referred to herein as TcAKl , an acronym for twenty-five C associating kinase.

A family of proteins known as 14-3-3 proteins was first identified by Moore, B.F. et al. (1967) as very abundant 27-30kD acidic proteins of brain tissue (Physiological and Biochemical Aspects of Nervous Integration. F.D. Carlson, Ed. , Prentice-Hall, Englewood Cliffs, NJ, 1967). Their name reflects the original investigators'

nomenclature. Recent work has implicated the participation of 14-3-3 proteins in cell cycle control (Ford, J.C. et al. ( 1994) Science 265:533). (For a general review, see Morrison, D. (1994) Science 266:56-57 and Aitken, A. (1995) TIBS 20). A variety of functions have been ascribed to the 14-3-3 proteins. However, several lines of evidence suggest that they link signal transduction cascades with the cell cycle. Various 14-3-3 isoforms have been found in complexes with proteins that transform cells, e.g. , the middle T antigen of polyoma virus and Bcr-Abl , with signaling molecules, e.g. , c-Rafl , c-Bcr and P13k, and with two cell cycle regulators, e.g. , Cdc25A and CdcB. The ability of a protein to bind or form a complex with a 14-3-3 protein therefore indicates that the protein is a key element linking external or internal signal transduction with other cell functions.

In the present invention that other function is the cell replication cycle, specifically passage from G2 to mitosis.

Summary of the Invention

The cDNA (SEQ ID NO: l) of human TcAKl kinase (SEQ ID NO:2), which phosphorylates Cdc25C on serine 216, has been cloned and sequenced. The invention provides isolated, purified and structurally defined DNA encoding human TcAKl , a method for making TcAK l protein by expressing the DNA encoding TcAKl , novel fusion proteins having TcAKl protein coupled to additional amino acids, and methods for measuring levels of TcAKl in RNA or of TcAKl protein in a cell sample. The invention also demonstrates that phosphorylation of human Cdc25C on serine 216 creates a 14-3-3 recognition motif and that TcAKl plays a role in mediating interactions between Cdc25C and 14-3-3 proteins. In addition, TcAK l also has a general function in mediating interactions between 14-3-3 proteins and other cellular proteins that play a key role in oncogenesis or key signalling events. Measurement of protein binding at TcAKl -mediated 14-3-3 recognition sites are therefore useful for detecting the presence of cancer or other disorder of cell proliferation.

Brief Description of the Figures

Figure 1 : Sequence obtained from rat p36TcAK 1

A: Sequence of 25 amino acids obtained from purified rat TcAKl (SEQ ID NO:3).

B: Schematic representation of human p78 (from database), TcAK l PCR product and TcAKl cDNA #6 (SEQ ID NO: l) clone.

Figure 2: Expression and activity of TcAK l

A: mRNA from the following human cancer cell lines was probed with radiolabeled human TcAKl cDNA: promyelocytic leukemia HL-60 (lane 1); HeLa cell S3

(lane 2); chronic myelogenous leukemia K-562 (lane 3); lymphoblastic leukemia MOLT-4 (lane 4); Burkitt's lymphoma Raji (lane 5); colorectal adenocarcinoma SW480 (lane 6); lung carcinoma A549 (lane 7); melanoma G361 (lane 8). The human TcAKl probe was stripped and the blot described above was hybridized with radiolabeled human B-actin probe (bottom).

B: Left Panel: Lysates prepared from HeLa (lane 1 ) and Jurkat cells (lanes 2) were resolved directly on an 8% SDS polyacrylamide gel. Proteins were transferred to nitrocellulose and incubated with TcAKl -specific antibodies. Proteins were visualized with an ECL Western blotting detection method. Right Panel: Lysates prepared from HeLa (lanes 1 and 2) and Jurkat cells (lane 3) were immunoprecipitated with either pre- immune sera (lane 1) or affinity purified TcAKl antibodies (lanes 2, 3) prior to SDS- PAGE. TcAK l was visualized as described in left panel.

C: TcAK l (lane 1) and a mutant of TcAKl (lane 2) where N183 was changed to alanine were produced in bacteria as His-tagged fusion proteins. Recombinant protein was isolated on Ni-NTA beads and kinase assays were performed in vitro (left). Radiolabeled

His-TcAKl was subjected to phosphoamino acid analysis (right).

Figure 3: TcAKl phosphorylates Cdc25C on serine 216 in vitro.

A. Kinase assays were performed in vitro in the presence of bacterially produced His-tagged TcAK l and the following GST-fusion proteins purified from overproducing bacteria: GST-Cdc25C (lane 1) GST-Cdc25C(S216A) (lane 2), GST-N258 (lane 3) GST-

N258(S216A) (lane 4). Reactions were resolved on a 7% gel and visualized by autoradiography.

B. Radiolabeled GST-Cdc25 fragment (SEQ ID NO:4) was digested with trypsin and the tryptic peptides were resolved by reverse phase HPLC. Column fractions were

collected and monitored for the presence of radioactivity (left panel). Manual Ed man degradation of tryptic phosphopeptides present in fraction 52 (right panel).

C. The tryptic phosphopeptide from HPLC fraction 52 (above) was further digested with proline specific endopeptidase and reaction products were resolved by reverse phase HPLC (left panel). Manual Edman degradation of phosphopeptides present in fractions 18 and 51 (right panels).

Figure 4: Coprecipitation of Cdc25C and a kinase with the same substrate specificity as TcAK 1.

A: Lysates prepared from HeLa cells were incubated with immobilized GST or GST-motif [Cdc25C(200-256)]. After washing, the beads were divided into four aliquots each and peptide kinase assays were performed in vitro. The peptides used correspond to amino acids 210-231 of human Cdc25C (SPS) or with substitutions of serine 214 with alanine (APS) or serine 216 with either alanine (SPA) or threonine (SPT). Phosphorylated peptides were resolved by SDS-PAGE and visualized by autoradiography. B. Lysates prepared from HeLa cells were incubated with pre-immune sera, affinity purified TcAKl antibodies (anti-TcAKl) or affinity purified Cdc25C antibodies (anti-Cdc25C). Immune complex kinase assays were performed in vitro in the presence of the Cdc25 peptides described in panel A above. Phosphorylated peptides were resolved by SDS-PAGE and visualized by autoradiography.

Figure 5: Binding specificity of recombinant TcAKl

Lysates prepared from bacteria overproducing CST (lane 1), GST-Cdc25C (lane 2), GST-25C(N2 8) (lane 3), GST-25C(C215) (lane 4) or GST-25C motif (lane 5) were incubated with GSH-agarose beads. A portion of the beads were resolved on a 10% SDS gel followed by staining with Coomassie Blue (left panel) the remainder of the beads were incubated with lysates prepared from insect cells overproducing TcAKl . Beads were pelleted, washed, resolved on a 10% SDS gel, transferred to nitrocellulose and probed with TcAKl antibody (right panel). TcAKl was visualized using an ECL Western blot detection method. 15 μg of lysate prepared- from TcAK l expressing insect cells were resolved and analyzed by blotting as described above (lane 6).

Figure 6: Binding of Cdc25C motif to endogenous TcAKl

Lysates prepared from bacteria overproducing GST (lanes 1 , 3) or GST-25C motif (lanes 2, 4) were incubated with GSH-agarose beads. Half of the beads were resolved directly on a 10% SDS gel (lanes 1 , 2), the other half was incubated with lysates prepared from either HeLa (left panel) or Jurkat (right panel) cells prior to SDS-PAGE. Beads were pelleted, washed, resolved on a 10% SDS gel, transferred to nitrocellulose and probed with TcAKl antibody. TcAKl was visualized using an ECL Western blot detection method. 50 μg of total cellular lysate prepared from either HeLa (lane 5, left panel) or Jurkat cells (lane 5, right panel) were resolved and analyzed as described above.

Figure 7: The majority of Cdc25C is phosphorylated on serine 216 in asynchronously growing HeLa cells.

A. Lysates were prepared from HeLa cells in the presence (lanes 1 , 2) or absence (lane 3) of microcystin. Lysates were incubated with pre-immune sera (lane 1) or sera specific for Cdc25C (lanes 3, 4). Immunoprecipitates were resolved by SDS-PAGE and Cdc25C was detected by immunoblotting.

B. HeLa cells were transfected with vector alone (lane 1) vector encoding myc- tagged Cdc25C (lane 2) or vector encoding myc-tagged Cdc25C(S216A) (lane 3). At 20 hrs. after transfection, lysates were prepared, resolved by SDS-PAGE and immunoblotted for Cdc25C using a monoclonal antibody specific for the myc epitope sequence.

Figure 8: Cell cycle regulation of serine 216 Phosphorylation, TcAkl levels and

TcAK l activity.

A. Asynchronous or elutriated populations of Jurkat cells were lysed, resolved by SDS-PAGE and immunoblotted for Cdc25C. Cdc25C in asynchronous cells (lane 1); cells enriched in Gl (lane 2); cells enriched in S (lane 3); cell enriched in G2/M (lane 4). B and C. Asynchronous (lanes 1 , 2) or elutriated populations (lanes 3, 4, 5) of

Jurkat cells were lysed and immunoprecipitated with either pre-immune sera (lane 1) or sera specific for TcAKl (lanes 2, 3, 4, 5). Immunoprecipitates were divided in half. One half was resolved by SDS-PAGE and immunoblotted for TcAK l (B). Immune complex kinase assays were performed with the second half of each reaction using the APS peptide as substrate (C). Reactions were resolved by SDS-PAGE and subjected to

autoradiography. Asynchronous cells (lanes 1 , 2); cells enriched in G l (lane 3); cells enriched in S (lane 4); cell enriched in G2/M (lane 5).

Figure 9: Association between Cdc25C and 14-3-3 in insect cells.

Insect cells infected with recombinant baculoviruses encoding GST-C215 (lane 1); GST-N258 (lane 2); GST-Cdc25C (lane 3); or GST-Cdc25C(C377S) (lane 4) were lysed and incubated with GSH agarose beads. Precipitates were washed, resolved by SDS- PAGE, transferred to nitrocellulose and probed with either GST antibodies to visualize recombinant Cdc25C proteins (A) or with antibodies specific for 14-3-3 (B). Proteins were visualized using an ECL Western blot detection method.

Figure 10: Association between Cdc25C and 14-3-3 in Hela cells requires serine

216.

HeLa cells were transfected with pcDNA3 lacking an insert (pcDNA3, lane 2) or with plasmids encoding either myc-epitope tagged Cdc25C (Myc-Cdc25C, lane 1) or myc- epitope tagged Cdc25CS216A [Myc-Cdc25C(S216A), lane 3]. Lysates were prepared and incubated with monoclonal antibodies to the myc epitope. One half of the immunoprecipitate was analyzed on an 8% polyacrylamide gel, transferred to nitrocellulose and blotted with the E10 monoclonal antibody to detect Cdc25C (A). Detection of bound secondary antibody (HRP-conjugated goat anti-mouse, Cappel) was achieved using Amersham's ECL kit. The other half of the sample was analyzed for activation of exoenzyme S activity (B).

Figure 1 1 : Association between endogenous Cdc25C and 14-3-3 in Hela Cells.

Immunoprecipitates were prepared from HeLa cell lysates using C20 antibody which was generated using a peptide derived from the carboxy-terminus of human Cdc25C (A, lanes 3, 4) or with antibody that was pre-incubated with the immunogenic peptide (A, lanes 1 , 2). Half of the immunoprecipitate was immunoblotted for the presence of

Cdc25C (A) and half was assayed for the activation of exoenzyme S activity (B).

Figure 12: Phosphorylation of Cdc25C on serine 216 creates a 14-3-3 recognition motif.

Insect cells overproducing Cdc25C were lysed and lysates were resolved directly by SDS-PAGE (lane 2). Alternatively, lysates were incubated with purified GST- 14-3-3 and bound proteins were precipitated using GSH agarose. Precipitates were washed and resolved by SDS-PAGE (lane 1). Cdc25C was visualized by immunoblotting.

Detailed Description of the Invention

Definitions The following terms are defined herein for convenience and clarity in describing and claiming the invention.

The term kinase as used herein refers to an enzyme which catalyzes transfer of a phosphate group from a high energy form of phosphate, such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to an acceptor group such as a hydroxyl. The acceptor is said to be phosphorylated. Protein kinases are enzymes which act to phosphorylate an acceptor group on a protein, notably a hydroxyl of serine, threonine or tyrosine. Many protein kinases are very specific in that the acceptor substrate can be one particular hydroxyl group on one particular protein. Some acceptor proteins become activated by phosphorylation, others inactivated. As noted for Cdc25C, phosphorylation at Ser 216 regulates its function, including providing a 14-3-3 binding site.

The term 14-3-3 protein(s) or 14-3-3 as used herein refers to a highly conserved multigene family of small (approximately 30kDa) acidic proteins found in a wide variety of eukaryotic organisms. 14-3-3 proteins function as activators of tyrosine hydroxylase, as regulators of protein kinase C and as co-factors of the Pseudomonas toxin exoenzyme S.

Various 14-3-3 isoforms have been found in complexes with oncogenes, signaling molecules and cell cycle regulators. Genetic studies implicate 14-3-3 proteins as negative regulators of mitosis. This 14-3-3 term as used herein encompasses synthetic peptides comprising one or more 14-3-3 recognition motifs.

The term 14-3-3 recognition motif or 14-3-3 recognition site or 14-3-3 binding site as used herein refers to an amino acid sequence comprising the sequence RSXp_SXP where X is any amino acid and the "pS" designation is a phosphorylated serine residue.

The term 14-3-3 binding protein as used herein refers to a 14-3-3 protein capable of binding to a 14-3-3 binding site.

The term TcAK l substrate as used herein refers to a natural or synthetic peptide or protein comprising an amino acid sequence recognized by TcAKl as a site for phosphorylation.

The term phosphatase as used herein refers to a general term for an enzyme that catalyzes hydrolysis of a phosphate ester. Many highly specific protein phosphatases are known to function as regulators of activity, acting in concert with kinases to fine tune the activity of cellular processes.

The term DNA Sequence as used herein denotes a sequence of polydeoxynucleotides containing genetic information. Both cDNA (obtained by copying messenger RNA) and genomic DNA are included in the term "DNA sequence. " Many DNA sequences are originally cloned as cDNA. Once a cDNA clone is available, genomic DNA can be obtained without undue experimentation, by techniques known in the art. For most eukaryotic genes, genomic DNA includes sequences not found in cDNA.

These include introns, regulatory regions, polyadenylation signals and the like. By convention, DNA sequences are written in the direction from the 5' end to the 3' end of that strand whose sequence is that of the corresponding messenger RNA, sometimes called the "sense" strand.

The term expression as used herein is used to denote processes by which the information content of a DNA sequence is converted to an observable function. Typically, the term refers to transcription (mRNA synthesis) and/or translation (encoded protein synthesis) of coding regions. However, non-coding regions can also be expressed, usually resulting in a regulatory effect. When a cloned DNA sequence is expressed in a heterologous cell, the expression can either be direct (where only sequences contained within the cloned DNA sequence are translated) or through synthesis of a fusion protein (including translation of an additional coding sequence). A sequence is "expressible" when combined in appropriate orientation and position with respect to control sequences

(promoter, ribosome binding site, polyadenylation site, etc.) that are operative in the desired host cell as is well-known in the art.

The term coding as used herein refers generally to the relationship between the nucleotide sequence of a DNA segment, and the amino acid sequence to which it corresponds, according to the known relationship of the genetic code. As is well known, a sequence of three nucleotides (triplet) encodes a single amino acid. Each of the twenty principal amino acids is encoded by at least one triplet, and most are encoded by more than one. Consequently, a single amino acid sequence can be encoded by a large number of different triplets. All the DNA sequences that encode the same amino acid sequence (synonymous codings) are therefore equivalent, although certain individual sequences may prove advantageous in certain types of host cells. Once a single coding sequence has been cloned, a person of ordinary skill in the art can readily make equivalent synonymous sequences by known methods, without undue experimentation. As used herein, a DNA sequence is said to encode a given amino acid sequence if it includes a nucleotide sequence that is translatable to the corresponding amino acid sequence. The coding nucleotide sequence may also include one or more introns, as well as untranslated nucleotide sequences, and sequences encoding other amino acid sequences.

The term chimeric as used herein is a term describing a non-naturally occurring combination of two or more different DNA sequences expressible as a single amino acid sequence, a chimeric protein. The source of the different sequences can be from the same or different species or from synthetic, non-naturally occurring sequences. A chimeric protein can have separate functions attributable to the different sequences, or the different sequences can contribute to a single function. [An example of the former is given herein for synthesis of cDNA encoding a myc epitope at the N-terminus of TcAKl . The expressed chimeric myc-TcAKl protein combines the functions of TcAKl with ability to bind to an anti-myc antibody.] An example of a chimeric protein where the different parts contribute a single function would be a Xenop .s-human hybrid TcAKl or other inter- species hybrid, or even a combination of a non-naturally-occurring sequence substituted for a portion of TcAKl . Typical chimeric amino acid sequences of the invention will include the TcAKl sequence combined with an added sequence that provides an additional

function, e.g. , where the added amino acid sequence is an epitope, has catalytic activity, is a cellular localization signal or confers a specific binding property. It will be understood that the foregoing functions need not be mutually exclusive. A specific binding property can, for example, be the specific binding of a substrate associated with catalytic (enzyme) activity or it could be the property of binding an antibody or it could simply be the ability to bind to an affinity chromatography ligand. A cellular localization signal can result in concentration of the chimeric amino acid sequence in the cell nucleus, on the endoplasmic reticulum, into an organelle or in transport to the cell exterior. Amino acid sequences conferring such properties are known in the art.

The term heterologous as used herein is a term applied to an in vivo expression system where the host cells are of a different species than the DNA sequence expressed. An example herein is given by the expression of TcAK 1 in cultured insect cells. Typically, use of a heterologous expression system requires combining the coding sequence with a control sequence known to function in the heterologous host cell (a heterologous control sequence). Where TcAK l is expressed in insect cells, a baculovirus promoter (from an insect-pathogenic virus) is provided as the control region.

The term control sequence as used herein is the term used for an untranslated DNA sequence that can function to insure expression, affect rate of expression, lifetime of mRNA and the like. Examples of control sequences include promoters, operators, enhancers, ribosome binding sites, polyadenylation signals and the like, as known in the art.

The term non-human sequence as used herein is used to denote a sequence known to have a source from a species other than human, including a synthetic source.

The term antibody as used herein is used herein to include both monoclonal and polyclonal antibodies as well as antibody fragments. An antibody is "specifically reactive" with a protein or epitope if it binds with the protein or epitope preferentially compared to other proteins or epitopes which may be present in the same mixture. An antibody can be "cross-reactive" with another (usually similar) protein or epitope. However, under

equilibrium conditions, antibody will be more strongly bound to a protein or epitope to which it is specifically reactive than to one to which it is merely cross-reactive.

The term an internal C-terminal peptide as used herein refers to a stretch of amino acids comprised in the amino acid sequence of a TcAKl kinase, e.g. , a stretch of approximately thirteen or greater amino acids located at the C-terminus of the TcAKl protein or a stretch of approximately thirteen amino acids located internally.

The term TcAKl as used herein refers to a protein kinase which catalyzes the phosphorylation of the Ser 216 of Cdc25C. The phosphorylation exerts a regulatory effect on Cdc25C activity, permitting the latter to associate with a 14-3-3 protein. Association of Cdc25C with a 14-3-3 protein provides a linkage between cell cycle regulation and external or internal cell signals which increase or decrease the cell replication rate.

The term hybridize under stringent conditions as used herein refers to hybridization carried out under optimal reaction conditions of temperature, ionic strength and time of reaction which permit selective hybridization between oligomers and eliminate nondiscriminate hybridization. In a preferred embodiment of the invention, hybridization was carried out at a temperature of 42 °C for 16-20 hours in 2 x PIPES buffer (0.8 M NaCl, 20 mM PIPES buffer, pH 6.5), 50% of formamide, 0.5 % SDS, 100 mg/ml denatured salmon sperm DNA, followed by a sequence of fifteen-minute washes at 42 °C- 55°C in 2x or less SSC medium with 0. 1 % SDS.

The term a level of interaction significantly different from that obtained for the corresponding . . . as used herein refers to the condition wherein the level of protein inter-action using a mutated protein is detectably greater than or less than the level of protein interaction using a corresponding nonmutated protein.

TcAKl protein was purified 8000-fold from rat liver and enzyme activity was identified with a 36-38 kD doublet by SDS-polyacrylamide gel electrophoresis [Ogg et al. ,

(1994) supra]. An amino terminal amino acid segment of the p36 was determined and the sequence is presented in Figure 1A (SEQ ID NO: 3). A database scan yielded a sequence

match for a protein denoted "p78. " Although a sequence of p78 was present in GenBank (Accession M80359), no information was presented other than it was a 78kD marker protein lost in chemically induced transplantable carcinoma and primary carcinoma of human pancreas. Despite the lack of characterization and the great difference of molecular weights, PCR primers were constructed from the 5' and 3' terminal sequences of p78 and used to obtain a full-length cDNA from a human B-cell library. The sequence of the PCR product revealed (see Figure IB) several differences from the p78 sequence: seven single nucleotide differences resulting in seven amino acid substitutions and an additional 48 nucleotides encoding 16 amino acids inserted between residues 370 and 371 of p78. The seven amino acid differences were Q125E; K 139E; G409A; T440S; D500A;

T587N and K605E. (The number is the position in the sequence, the preceding letter is the p78-encoded amino acid, the letter following is the TcAK l-encoded amino acid). The PCR product was used to probe a HeLa cell cDNA library from which several cDNAs were obtained and sequenced. A full length cDNA (#6) was found to have the nucleotide sequence (SEQ ID NO: l), presented in Figure IB, which was shown to encode TcAKl protein. The sequence of the cDNA#6 (SEQ ID NO: l) was identical to that of the PCR product except for the substitution of glycine for serine at position 443 and a silent substitution of A for C at the third position of the threonine 438 codon.

The predicted open reading frame of TcAKl (SEQ ID NO: l) encodes a protein of 729 amino acids (SEQ ID NO:2). The kinase domain is found in the N-terminal portion of the molecule encompassing amino acids 54-310 (Figure IB).

TcAKl was expressed in several human cancer cell lines as revealed (see Figure 2A) by the presence of mRNAs of approximately 3.8 and 3. 1 in all lines tested. Northern analysis performed using mRNAs derived from a variety of human tissues (brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) revealed the same size transcripts. An additional mRNA of -3.0 was observed in heart tissue.

TcAKl expression in a cell or tissue can be detected and/or quantified with a TcAKl DNA probe comprising a DNA sequence encoding a TcAKl kinase or a characteristic portion thereof, e.g. , the kinase domain, etc. , to form a nucleic acid hybrid

under stringent conditions between the TcAKl DNA and any corresponding nucleic acid present in the cell or tissue. After separating the nucleic acid hybrid with art known methods, e.g. , electrophoresis, etc. , the amount of TcAK l DNA present in the nucleic acid hybrid can be quantitated and related to total TcAKl expression in the cell or tissue.

TcAKl protein expression in HeLa and Jurkat cells was detected with affinity purified TcAKl -specific antibodies (Figure 2B). In HeLa cells, TcAKl resolved as a doublet of approximately 78 and 80 kDa whereas in Jurkat cells the 80 kDa form predominated. Also, recombinant TcAKl was expressed as a histidine tagged-fusion protein in bacteria and was found to be catalytically active with autophosphorylation occurring on serine residues as illustrated in Figure 2C.

TcAK l was originally purified based on its ability to bind to Cdc25C (within a domain bordered by amino acids 200 to 256) and to phosphorylate Cdc25C on serine 216 (Ogg et al. , 1994). Recombinant TcAKl was determined to be capable of phosphorylating Cdc25C on serine 216. As shown in Figure 3A, bacterial TcAKl phosphorylated full length Cdc25C (Figure 3A, lane 1) as well as a deletion mutant containing the N-terminal

258 amino acids of Cdc25C [(N258), lane 3]. Substitution of alanine for serine at position 216 ablated the phosphorylation of N258 and greatly reduced the phosphorylation of Cdc25C by TcAK l (Figure 3A, lanes 2, 4). Manual Edman degradation identified serine 216 as the major site of TcAKl phosphorylation (Figures 3B and 3C). A minor site of phosphorylation was observed within the C-terminus of Cdc25C.

Substrate specificity of TcAK 1 was evaluated with a series of peptides synthesized to reflect a region of Cdc25C surrounding serine 216. These peptides were synthesized to correspond to amino acids 210-231 of human Cdc25C (Ogg et al. 1994, supra) containing serines at positions 214 and 216 (SPS) or containing substitutions of serine 214 with alanine (APS) or serine 216 with either alanine (SPA) or threonine (SPT). Substrate specificity was evaluated for the HeLa cell Cdc25C-associated kinase capable of binding to GST-Cdc25C in vitro (GST, glutathione S-transferase; Ogg et al. , 1994). Bacterially produced GST and GST-motif were purified on GSH beads and immobilized proteins were incubated with HeLa cell extracts, washed, and assayed for kinase activity in the presence

of each of the peptides. As shown in Figure 4, the Cdc25C-associated kinase preferentially phosphorylated peptides containing serine 216 (upper panel). Peptides containing either alanine or threonine in place of serine at position 216 were not efficiently phosphorylated in vitro. Substitution of serine 214 with alanine had no apparent effect on peptide phosphorylation. Substrate specificity of endogenous TcAKl was also determined.

Immunoprecipitates of TcAKl from HeLa cells showed the same selectivity for a serine at position 216 (lower panel) as did bacterially produced TcAKl . Also evaluated was whether a kinase having the same specificity as TcAKl could be detected in Cdc25C immunoprecipitates. As shown in Figure 4, immunoprecipitates of Cdc25C contained a protein kinase with the same selectivity for serine at position 216 (lower panel).

The binding specificity of recombinant TcAKl is presented in Figure 5. Cdc25C and various deletion mutants were expressed in bacteria as GST-fusion proteins and subsequently purified on GSH agarose (Figure 5, left panel). Each protein was tested for its ability to bind to TcAK l that had been overproduced in insect cells. As shown in Figure 5 (right panel) neither GST (lane 1) nor the C-terminus of Cdc25C (lane 4) bound to TcAK l . However, both full length Cdc25C (lane 2) and the N-terminal 258 amino acids of Cdc25 bound to TcAKl (lane 3). Also, the Cdc25C motif region (bordered by amino acids 200 to 256) bound to recombinant TcAK l (lane 5).

The Cdc25C motif was also able to bind to endogenous HeLa and Jurkat cell TcAKl . GST and GST-motif proteins were expressed in bacteria and bound to GSH agarose. Recombinant proteins were incubated with lysates prepared from either HeLa or Jurkat cells. Bound proteins were incubated with lysates prepared from either HeLa or Jurkat cells. Bound proteins were resolved by SDS-PAGE and the presence of TcAKl was monitored by immunoblotting. As shown in Figure 6, the Cdc25C motif bound to endogenous TcAK l present in both HeLa (left panel, lane 4) and Jurkat cells (right panel, lane 4).

Serine 216 phosphorylation and TcAKl activity were determined throughout the cell cycle. As shown previously the predominant site of Cdc25C phosphorylation in asynchronously growing HeLa cells is serine 216. Phosphorylation of Cdc25C on serine

216 retards the electrophoretic mobility of Cdc25C in SDS gels (see Figure 7). This property can be used to monitor and/or quantitate the levels of serine 216-phosphorylated Cdc25C in vivo. As shown in Figure 7A, the majority of Cdc25C is retarded in its electrophoretic mobility (lane 2) indicative of serine 216 phosphorylation. When cell lysis and immunoprecipitation reactions were carried out in the absence of microcystin, the faster electrophoretic form of Cdc25C predominated (Figure 7A, lane 3). Transient transfection of HeLa cells with plasmids encoding Myc-epitope tagged Cdc25C resulted in approximately 40% of the Cdc25C being converted to the slower electrophoretic form indicative of serine 216 phosphorylation (Figure 7B, lane 2), indicating that the serine 216 kinase is active under these conditions. Substitution of serine 216 with alanine prevented the phosphorylation of Cdc25C on serine 216 and, therefore, only the faster electrophoretic form of Cdc25C was detected (Figure 7B, lane 3).

To determine whether phosphorylation of Cdc25C on serine 216 was cell cycle regulated, Jurkat cells were elutriated and fractions were analyzed for Cdc25C by immunoblotting. As seen in Figure 8A (top panel), the majority of Cdc25C was phosphorylated on serine 216 throughout the G l-(lane 2);S-(lane 3) and G2-phases of the cell cycle (lane 4), indicating that serine 216 phosphorylation was constitutive throughout most of the cell cycle. During mitosis, Cdc25C undergoes major shifts in its electrophoretic mobility due to N-terminal phosphorylations (Izumi et al. , 1992; Kumagai and Dunphy, 1992; Hoffmann et al. , 1993; Villa-Moruzzi, 1993) and, thus, it was not possible to determine the state of serine 216 phosphorylation in mitosis by immunoblotting. If, however, TcAKl is the serine 216 kinase, it might be predicted that the activity of TcAKl would also be constitutive throughout the G1-, S- and G2-phases of the cell cycle. Indeed, as shown with immunoblotting experiments (Figure 8B, middle panel) the levels of TcAKl remained constant throughout these phases of the cell cycle.

In addition, immune complex kinase assays performed in the presence of the APS peptide (corresponds to amino acids 210-231 of human Cdc25C with a substitution of alanine for serine at position 214) demonstrated that TcAK l activity was also constant (Figure 8C, lower panels).

Cdc25C was shown to interact directly with 14-3-3 protein and this interaction was shown to be dependent upon Cdc25C being phosphorylated on serine 216. The "recognition motif" for 14-3-3 binding consists of the sequence RSXpSXP where X is any amino acid and the underlined serine is phosphorylated [Muslin et al, (1996) Cell 84: 889- 897). This consensus motif fits perfectly the sequence bordering and inclusive of serine

216 in Cdc25C (RSPS ₂₁₆ MP). Various forms of Cdc25C were tested for binding to 14-3- 3 in insect cells. Figure 9 illustrates the interaction between 14-3-3 and Cdc25C (lane 3) as well as the N-terminal 258 amino acids of Cdc25C(N258, lane 2) was detected. The C-terminus of Cdc25C (lane 1) was negative in this assay. Furthermore, 14-3-3 binding was independent of phosphatase activity, since a catalytically inactive form of Cdc25C also bound to 14-3-3 (lane 4), localizing the 14-3-3 interaction to the N-terminus of Cdc25C.

The association between Cdc25C and 14-3-3 was also examined in HeLa cells by transiently expressing Cdc25C and the serine 216 mutant of Cdc25C as myc-epitope tagged proteins. At 31 hours after transfection, Cdc25C and Cdc25CS216A were immunoprecipitated with monoclonal antibodies to the myc-epitope tag. One half of each immunoprecipitate was assayed for Cdc25C my immunoblotting (Figure 10A), the second half was assayed for the presence of 14-3-3 using an enzymatic assay that measures exoenzyme S activity (Figure 10B). Because 14-3-3 migrates in the same region of the gel as the light chain of IgG, its presence is difficult to detect using standard one dimensional SDS-PAGE and immunoblotting. Therefore, an enzymatic assay based on the fact that 14-3-3 serves as cofactor in vitro for an adenosine 5'-diphosphate (ADP)- ribosyltransferase enzyme (exoenzyme S) from Pseudowonas aeruginisa was used. As shown in Figure 10A, Cdc25C resolved as a doublet in SDS gels (lane 1) whereas Cdc25CS216A migrated as a single electrophoretic form (lane 3). Cdc25C was not detected in control immunoprecipitates (lane 2). The slower electrophoretic form of Cdc25C represents serine 216 phosphorylated Cdc25C. As shown in Figure 10B, Cdc25C immunoprecipitates activated ADP-ribosylation by exoenzyme S. Immunoprecipitates from control cells or cells overproducing Cdc25S216 were significantly lower in this assay. These results indicate a specific interaction between Cdc25C and 14-3-3 and illustrate the importance of serine 216 in this interaction.

To assay for an interaction between 14-3-3 and endogenous Cdc25C, immunoprecipitates were prepared from HeLa cell lysates using an antibody against the carboxy-terminal peptide of human Cdc25C (Figure 1 1). Half of the immunoprecipitate was immunoblotted for the presence of Cdc25C (Figure 1 1 A) and half was assayed for activation of exoenzyme S activity (Figure 1 IB). As shown in Figure 1 1 , exoenzyme S activity specifically immunoprecipitated with Cdc25C. Cdc25C was not detectably precipitated when the antibody was pre-incubated with the iminunogenic peptide (Figure 1 1 A, lanes 1 , 2) nor were there significant levels of exoenzyme S activity in the precipitate (Figure 1 IB). These results indicate a functional interaction between Cdc25C and 14-3-3 in vivo.

The interaction between Cdc25C and 14-3-3 was shown by phosphorylation of serine 216. Insect cells were infected with recombinant baculoviruses encoding Cdc25C. When produced in insect cells, the majority of Cdc256C was not phosphorylated and migrated in SDS gels as the faster electrophoretic form (Figure 12, lane 2). However, a minor population of Cdc25C was phosphorylated on serine 216 and migrated as the slower electrophoretic form (Figure 12, lane 2). A binding assay was performed in vitro to determine which form of Cdc25C preferentially interacted with 14-3-3. Recombinant 14- 3-3 fused to GST was purified from bacteria and was incubated with lysates prepared from Cdc25C-overproducing insect cells. Bound proteins were washed, resolved by SDS- PAGE and Cdc25C was detected by immunoblotting. As seen in Figure 12 (lane 1), only the serine 216-phosphorylated form of Cdc25C (slower electrophoretic form) bound to GST-14-3-3 in this assay, demonstrating the importance of phosphorylated serine 216 for this interaction.

TcAKl functions not only to mediate the interaction between 14-3-3 and Cdc25C, but also plays a more general role in mediating the interaction between 14-3-3 and other cellular proteins that have been shown to either mediate oncogenesis or key signalling events. Various 14-3-3 isoforms have been found in complexes with oncogenes (the middle T antigen of polyoma virus and Bcr-Abl), and signaling molecules (c-Rafl , c-Bcr and PI3K). Mutations in these proteins and in proteins present in pathways involving these proteins are associated with human cancers. Participation of TcAKl in these

pathways was shown by the demonstration that TcAK l phosphorylates RAF1 on serine 259 and 621 in vitro (the two sites that regulate RAF 1/ 14-3-3 binding in vivo).

Mutations in 14-3-3 proteins or TcAKl substrates that prevent or interfere with the TcAKl phosphorylation-specific interaction between a 14-3-3 protein and a TcAKl substrate are detected by the methods of this invention. Such mutations are recognized and/or determined by measuring the extent of TcAKl phosphorylation-specific interaction between a mutated 14-3-3 protein and a TcAKl substrate or a mutated TcAKl substrate and a 14-3-3 protein in a cell or tissue transformed with a DNA sequence encoding a TcAKl protein and comparing it to the extent of interaction obtained with the corresponding non-mutated protein.

It will be apparent to those of ordinary skill in the art that alternative methods, reagents, procedures and techniques other than those specifically detailed herein can be employed or readily adapted to practice the detection methods of this invention. Such alternative methods, reagents, procedures and techniques are within the spirit and scope of this invention.

The methods of this invention are further illustrated in the following non-limiting Examples. All abbreviations used herein are standard abbreviations in the art. Specific procedures not described in detail in the Examples are well-known in the art.

Example 1 : Purification and sequencing of rat liver Cdc25C-associated kinase (rat TcAKl)

To obtain protein sequence information, 202 g of rat liver was used to purify TcAKl . Purification of the kinase was performed essentially as described in Ogg et al. , 1994 supra. Briefly, the homogenate was divided into two parts and each half was individually fractionated over a S-Sepharose Fast Flow column (Pharmacia). Fractions from each separation that contained peak Cdc25C associated kinase activity were pooled and the purification was continued through a buffer exchange column, a Q-Sepharose column (Pharmacia), an ATP-agarose column (Sigma), another buffer exchange column, a Resource S column (Pharmacia) and finally a Superose-12 10/30 column (Pharmacia).

Fractions from the Superose-12 10/30 that contained the peak of the activity were concentrated using Centricon- 10 concentrators (Amicon). Proteins were resolved on an 1 1 % SDS-polyacrylamide gel and visualized by staining with 1 % Coomassie brilliant blue (w/v). The protein band corresponding to TcAK l was excised from the gel and placed in the well of a 10%-T, 3% C gel with a 4% T, 3% C stacking gel as described (Schagger and von Jagow, 1987). The gel slice was overlaid with 200 μl of sample buffer containing 0. 1 μg Staphylococcal V-8 protease. Samples were electrophoresed until the proteins were stacked up at the stacking/resolving gel interface and then the electrophoresis was stopped. After a 1 h incubation, the electrophoresis was continued to completion. The gel was then transferred to PolyScreen PVDF Transfer Membrane (NEN

Research Products). Proteins were visualized by staining for 1 min in 0. 1 % Coomassie blue/50% methanol (w/v) and destaining in 10% acetic acid/40% methanol (v/v/v). Protein sequence was obtained by automated Edman degradation and analysis, using an Applied Biosystems 477 protein sequencer and Applied Biosystems 120.

Example 2: Constructs of Cdc25C and mutants thereof

A: Construction of pUC19-Cdc25C. A 1.4 Bam Hl-Pfl MI fragment encoding Cdc25C was excised from pML25 (Lee et al. , ( 1992) treated with Klenow and ligated to phosphorylated Xba I Lingers. The fragment was inserted into the polylinker of pUC19 to generate pUC 19-Cdc25C. B. Construction of pGC52-N258 and pGC52-N258(S216A). Site directed mutagenesis of the codon for serine 216 of Cdc25C was performed using an oligonucleotide directed mutagenesis kit (Amersham). A 774 pb Bam HI-Eco RI fragment encoding the N-terminal 258 amino acids of Cdc25C was excised from pGEX2T-N258 (Lee et al. , 1992) and inserted into the polylinker of pGC52 (obtained by Bam HI-Eco RI digestion of pSAFlO (Parker et al. , 1991) to excise the 1 .2 kb cdc2 fragment) to generate pGC52-N258. Single-stranded pGC52-N258 was mutagenized using the oligo 5' ATATCGCTCCCCGGCGATGCCAGAGAACTT3' (SEQ ID NO:5), where underlined codon, residue 216, encoded Ala rather than Ser, generating pGC52-N258(S216A). C. Construction of pGEX2T-N258(S216A). The 774 bp Bam HI-Eco RI fragment encoding N258(S216A) was excised from pGC52-N258(S216A) and inserted into the polylinker of pGEX2T to generate pGEX2T-N258(S216A).

D. Construction of pUC19-Cdc25C(S216A). A 330 pb Bal I fragment encoding the S216A mutation was excised from pGC52-N258(S216A) and inserted into the corresponding Bal I site in pUC19-Cdc25C to generate pUC 19-Cdc25C(S216A).

E. Construction of pGEX2T-Cdc25C(S216A). A 1.2 kb Ppu MI-Nsi I fragment encoding the S216A mutation was excised from pUC19-Cdc25c(S216A) and inserted into the corresponding sites of pML25 to generate pGEX2T-Cdc25C(S216A).

F. Construction of pFASTBACl-Cdc25C(S216A) and generation of recombinant baculovirus. A 1.9 kb Bam HI fragment encoding Cdc25C(S216A) was excised from pGEX2T-Cdc25C(S216A) and inserted into the polylinker of pFASTBACl , generating pFASTBAC l -Cdc25C(S216A). Recombinant baculovirus encoding

Cdc25S216A was generated according to the manufacturer's recommendations (Gibco BRL).

G. N-terminal myc-epitope Cdc25C fusion constructs. The first 300 nucleotidase of Cdc25C were amplified by PCR using wild type Cdc25C as template with a 5' primer coding for the myc epitope EQKLISEEDL (SEQ ID NO: 17) and containing a

Hind III site, and a 3' primer containing a Bst EII site [5' primer,

CAGCATAAGCTTACCATGGCAGAACAGAAGCTCATTTCTGAAGAAGACTTGTCT ACGGAACTCTTCTCA (SEQ ID NO:6); 3" primer, AATGCACTTCCTGAAGTCCTGAAGA (SEQ ID NO: 7)]. The conditions for the PCR were as follows: 20 ng of template, 0.3 mM each deoxynucleoside triphosphate, lμM each primer, 4 mM MgSO ₄ , and 2U of Vent DNA polymerase (New England Biolabs, Beverly, MA); thermal cycles: 1 at 94°C for 10 min; 3 at 94°C for 90s, 45°C for 30s, 50°C for 30s, 72°C for 20s; and 32 at 94°C for 90s, 60°C for 60s, 72°C for 20s. The amplified DNA was digested with Bst EII and Hind III, followed by purification of the 350 pb fragment by agarose gel electrophoresis and Qiaquick Gel Extraction (Qiagen,

Chatsworth, CΛ). The resulting fragment was ligated into the purified 6.5 kb Bst EII/Hind III backbone-fragment of both pRc/CMV-Cdc25C wild type and S216A mutant, followed by transformation of E. coli JM109. Plasmids were then isolated from single bacterial clones and checked both by restriction digest and sequencing. H. Construction of pET15b-Cdc25C. A 2.0 kb Bam HI fragment encoding

Cdc25C was excised from pML25 (Lee et al. , 1992), blunt ended with DNA polymerase I

large (Klenow) fragment and ligated to Nde I digested and Klenow-treated pET15b (Novagen).

I. Construction of pFASTBACl-His ₆ Cdc25C. ρET15b-Cdc25C was digested with Nco I and Bam HI, Klenow-treated and the 2kb His ₆ Cdc25C fragment was subcloned into pFASTBACl (Gibco BRL) that had been digested with Bam HI and Klenow-treated.

Recombinant baculovirus encoding His ₆ -Cdc25C was generated according to the manufacturer's recommendations (Gibco BRL).

J. Construction of pET-15b(mod)Cdc25C(S216A). The pET-15b vector was modified by addition of a 10-mer oligonucleotide 5'-TCGAGGTACC-3'(SEQ ID NO: 8) into the Xho I site of the polylinker to generate pET-15b(mod). This creates a Kpn I site and changes the reading frame of the Bam HI site in the polylinker. A 1.9 kb Bam HI fragment encoding Cdc25c(S216A) was excised from pGEX-2T and inserted into the polylinker of pET- 15b(mod).

K. Construction of pFASTBACl-His ₆ Cdc25C(S216A). A 1.9 kb Xba I-Sma I fragment encoding His ₆ Cdc25C(S216A) was excised from pET-15b(mod), the Xba I overhang was filled in with DNA polymerase I large (Klenow) fragment, and inserted into the Stu I site of pFASTBACl .

L. Construction of a catalytically inactive form of Cdc25C(C377S). To create a phosphatase-dead version of Cdc25C, serine was substituted for cysteine at position 377. The full-length Cdc25C cDNA was excised as an Xba I fragment from pUC19-Cdc25C and subcloned into the Xba I site of pGC52. The resulting plasmid was then digested with S a I and Dra I and the 3.5 kb backbone containing the C-terminal 215 amino acids of Cdc25C was isolated, religated and used to transform the CJ236 bacterial strain. Single- stranded DNA was prepared from the bacterial culture siiperinfected with helper phage M13K07. Next, the oligonucleotide

CGTGTTCCACTCTGAATTCTCCTCAGAGAGGGGCCCCCGGATGTGCCGC (SEQ ID NO:9) (encoding serine for cysteine at position 377 and containing a silent mutation which creates a Hpa II site for screening of mutants) was annealed to the single stranded DNA and the complementary strand was synthesized. The reaction mixture was used to transform MV1 190 to recover the double stranded DNA. Mutants were screened by performing a Hpa II digest of the miniprep DNA. The detailed procedures of the mutagenesis was essentially as described in the instruction manual of Muta-gene phagemid

in vitro mutagenesis kit (BIO-RAD). The Bgl II to Nsi I fragment of pUC19-Cdc25C was replaced with the corresponding fragment containing the C377S mutation to generate the full length Cdc25C(C377S) mutant. The Ppu MI to Nsi I fragment of Cdc25C in the pGEX2TCdc25C was in turn replaced with the corresponding fragment from the pUC19 Cdc25C(C377S) to generate pGEX2TCdc25C(C377S) construct.

M. Construction of pFASTBACl-Cdc25C(C377S). A 1.9 kb Bam HI fragment encoding Cdc25C(S216A) was excised from pGEX-2T and inserted into the polylinker of pFASTBACl .

N. Construction of pGEX2TB-Cdc25C(C377S). A 1.9 kb Bam HI fragment encoding Cdc25C(C377S) was excised from pGEX-2T and inserted into the polylinker of pGEX-2TB.

O. Construction of pFASTBACl-GST-Cdc25C(C377S). pGEX2TB- Cdc25C(C377S) was digested with Xba I and Sma I and the fragment containing GST- Cdc25C(C377S) was subcloned into the Stu I site of pFASTBAC l . Recombinant baculoviruses were generated as described below.

Example 3: Constructs of TcAKl and mutants thereof

3A. Creation of pGEX-2TN(TcAKl) and pGEX-2TN[TcAKl(l-412)]. TcAKl proteins: Human TcAK l and TcAKl(l-412) were obtained by polymerase chain reaction (PCR) amplification from a B lymphocyte cDNA library [gift of S. J. Elledge, (Durfee et al. , 1993)] using the oligonucleotides: 5 '-CG AGTC ATATGTCC ACTAGG ACCCC-3 '

(SEQ ID NO: 10) and either 5 ^* -

CCAGTCATATGTTAACTTACAGCTTTAGCTCATTTGGC-3' (SEQ ID NO: 1 1) for TcAKl or 5'-CCAGTCATATGTTAACTTAGCTTGAAGAAACA CTTCTCTGC-3'(SEQ ID NO: 12) for TcAK l(l-412). To obtain enough TcAKl DNA for subcloning it was necessary to reamplify the initial PCR product. The regions of the primers that are underlined are Nde I restriction sites which were used to ligate the PCR products into linearized pGEX-2TN [gift of Dr. Giulio Draetta) to generate pGEX-2TN(TcAK l) and pGEX-2TN(TcAK 1 (1-412)].

Creation of a kinase-deficient form of TcAKl. To create a catalytically-inactive form of TcAKl , alanine was substituted for asparagine at position 183 using a PCR based site-directed mutagenesis strategy. To achieve this, the following two mutagenic primers:

5'-CAAGGCTGAAGCTCTATTGTTAGATGC-3'(SEQ ID NO: 13) and 5 '- GCATCTAACAATAGAGCTTCAGCCTTG-3' (SEQ ID NO: 14) were used in conjunction with the two TcAKl primers described above to amplify a 1 187 pb product in which the codon A AT was changed to GCT (underlined). The PCR product was digested with Bgl II and Xba I, and 427 bp fragment was isolated and used to replace the same region of wild-type TcAKl in pGEX-2TN (to generate ρGEX-2TN[TcAKl(N183A)]. The mutation was confirmed by sequencing.

3B. Construction of pET.TcAKl and pET.TcAKl (N183A). Wild-type and kinase-deficient TcAK l were engineered for expression in Escherichia coli with NH ₂ - terminal hexahistidine tags. To achieve this, pGEX-2TN(TcAKl) and pGEX-

2TN[TcAKl(N183A)] were digested with Ndel and ligated to the pET15b vector (Novagen) which had been previously linearized with Nde I. The resulting plasmids are referred to as pET.TcAK l or pET.TcAK l(N 183A) for wild-type TcAKl and kinase- deficient TcAKl , respectively.

Example 4: Library Screening for TcAKl cDNAs

To obtain a full-length human TcAKl cDNA, a lambda ZAP II HeLa cDNA library was screened according to the manufacturer's instructions (Stragene). A total of 1 x 10 ⁶ phage were plated and then lifted onto nylon membrane filters (NEN). The DNA was denatured and filters were UV-irradiated to crosslink the DNA. To make the probe, the Nde I-Xba I fragment of TcAKl ( 1-899) was isolated from pGEX 2TN p78 and labeled with dCTP(α- ³² P) using the Megaprime DNA Labeling System (Amersham). The filters were prehybridized with 2X PIPES buffer (0.8 M NaCl,20 mM PIPES buffer, pH 6.5), 50% formamide, 0.5 % SDS, 100 mg/ ml denatured salmon sperm DNA at 42°C for 6 hours, then hybridized with the labeled probe in the same buffer at 42 °C for 16-20 hours. The filters were washed with 2X SSC, 0. 1 % SDS at room temperature for 15 mins, with

0.1X SSC, 0.1 % SDS twice at 42°C for 15 mins each time, three times at 50°C for 15 min, and three times at 55 °C for 15 min. They were then subjected to autoradiography at -80°C overnight. Positive clones were picked, plated and subjected to two additional rounds of plaque purification. pBluescript phagemids containing the cDNA inserts were excised in vivo from the parental lambda ZAP II vector. The cDNA sequences were determined by direct automated DNA sequencing.

Northern Analysis. pGEX-2TN[TcAK l( l -412)] was digested with Nde I and the 1.1 kb fragment encoding the kinase domain of TcAKl was labeled with ( ³² P)α-dCTP using the Megaprime DNA Labeling System (Amersham). A human cancer cell line MTN blot (Clontech) was probed and processed according to the manufacturer's instructions.

Example 5: Expression and purification of TcAKl and mutants thereof

5A. Expression and purification of Hexahistidine-tagged TcAKl . The E. coli strain BL21 (DE3) was transformed with either the pET.TcAK l or pET.TcAKl(N183A). Cultures were grown at 37°C until reaching an A ₆₀₀ of -0.6 at which time the T7RNA polymerase was induced with 100 μM isopropyl-1-thio-β-D-galactopyranoside (IPTG).

After induction, cells were grown at 28°C for 1 1 h, collected by centrifugation at 5000 X g for 10 min, and resuspended in IMAC-5 buffer (20 mM Tris (pH 7.5), 500 M NaCl and 50 mM imidazole) supplemented with 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 20 μM leupeptin, and 5 μg/ml pepstatin. Cells were lysed by one treatment with a French press at 1000 p.s.i. and lysates were clarified by centrifugation at 50,000 X g for 30 min. The supernatant was batch-absorbed to Ni ²⁺ -NTA-agarose (Qiagen) for 1 h at 4°C and the resin collected by centrifugation at 5000 x g for 10 min. Following washing with 5 column volumes of IMAC-5 buffer, the resin was washed with 2 column volumes of IMAC-50 buffer [20 mM Tris (pH 7.5), 500 mM NaCl and 50 mM imidazole] and TcAK l was eluted with IMAC- 100 (20 M Tris (pH 7.5), 500 mM NaCl and 100 mM imidazole).

5B. Autophosphorylation of TcAKl. Wild-type TcAKl or TcAKl (N 183 A) was incubated for 1 h at 30°C in buffer D (75 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , and 1 mM DTT) supplemented with 200 μM ATP and 30 μCi of γ- ³² P ATP. Reactions were terminated by the addition of 3X SDS-sample buffer followed by heating at 100°C for 10 min. Samples were resolved by electrophoresis on an 8% SDS gel and visualized by staining with Coomassie Blue and autoradiography.

5C. Detection of TcAKl in HeLa and Jurkat cells. HeLa and Jurkat cells (-1 x 10 ⁷ ) were washed twice in PBS and then lysed in 1 ml of mammalian cell lysis buffer consisting of 50 mM Tris pH 8.0, 2 mM DTT, 5 mM EDTA (pH 8.0), 0. 1 % NP-40, 100 mM NaCl, 1 mM microcystin, 50 mM NaF, 1 mM sodium orthovanadate, 2 mM PMSF,

0.15 U/ml aprotinin, 20 mM leupeptin, and 20 mM pepstatin for 30 min. at 4°C. Lysates were clarified and 2 mg of protein were incubated with 5 μl of affinity-purified TcAKl antibody at 4°C for 2 h followed by incubation for an additional 1 h at 4°C in the presence of 30 μl of a 50% slurry of Sepharose CL-4B protein A beads. Beads were washed four times in lysis buffer, boiled in protein sample buffer for 10 min. , and then resolved on an 8% SDS-gel. Alternatively clarified lysates containing 200 μg of total cellular protein were boiled directly in sample buffer and resolved on an 8% SDS gel. Proteins were transferred to nitrocellulose membranes. Membranes were incubated in blocking buffer (5 % milk in 10 mM Tris (pH 8.0), 150 mM NaCl, and 0.3 % Tween 20) at room temperature for Ih; followed by incubation with affinity-purified TcAKl antibody

(at 1 : 10,000 dilution) in washing buffer ( 10 mM Tris, pH 8.0), 150 mM NaCl, and 0.3% Tween 20) at room temperature for 1 h. Membranes were subjected to four 15 min. washes in wash buffer and then processed to visualize TcAK l . HRP-conjugated protein A (Amersham) was used as secondary antibody (at a 1 :5000 dilution). 5D. HPLC analysis and manual sequencing. Proteins were separated by SDS-

PAGE, transferred to nitrocellulose and visualized by autoradiography. The nitrocellulose containing radiolabeled GST-Cdc25C was excised, blocked with 0.5 % polyvinylpyrrolidone (PVP-40) in 100 mM acetic acid for 30 min. at 37°C, washed three times with water and digested with TPCK trypsin (Worthington) at a final concentration of 0.03 μg/ml in 0.1 M NH ₄ CO ₃ (pH8.0) for 2 h at 37°C. Two additional 2 h incubations were carried out at 37°C in the presence of fresh trypsin (15 μg each). Further digestion on selected HPLC fractions was performed with 2 units of proline specific endopeptidase (1CN) in 0. 1M sodium phosphate, 5mM EDTA (pH 7.4) at 37°C for 16 hr.

Reactions were acidified in 1 % trifluoroacetic acid (TFA) and loaded onto a Vydac C18 column (25 cm x 0.46 cm I.D.). Reverse phase HPLC was performed at 37°C.

Reactions were loaded in 0. 1 % TFA (Buffer A) and eluted with a gradient from 0 to 60% Buffer B (90 acetonitrile, 0.95 % TFA). Fractions were collected at 0.5 min intervals and counted for radioactivity. Selected fractions were immobilized on Sequenlon-AA membrane discs (Millipore) for amino-terminal sequencing. Manual Edman degradation was performed as described (Bodwell et al., 1991 ; Sullivan and Wong, 1991) with a coupling and cleavage temperature of 55 °C.

Example 6: Antibodies

6A. Preparation of Antibodies. Antibodies to TcAK l were prepared using bacterial GST-TcAK l as antigen or using peptide antigens composed of either the C- terminal 13 amino acids of TcAKl (KNIASKIANELKL) (SEQ ID NO: 15) or a stretch of 13 amino acids (KQKDENKEAKPRS) (SEQ ID NO: 16) located internally. Both peptides contained a cysteine residue at their N-termini and were coupled to keyhole limpet hemocyanin (KLH). Two antibodies were used for the detection of Cdc25C: an affinity purified polyclonal antibody (see below) and a polyclonal antibody directed against a C- terminal peptide of Cdc25C (C-20, Santa Cruz Biotechnology). In all cases, bound primary antibodies were detected using horseradish-peroxidase conjugated anti-rabbit or anti-mouse secondary antibodies (Cappel) and an ECL detection system (Amersham).

6B. Preparation of affinity columns. Two liters of induced bacterial culture expressing hexahistidine (His ₆ )-tagged TcAKl were pelleted and resuspended in 180 ml of homogenization buffer consisting of 50 mM Tris (pH 7.5), 500 mM NaCl supplemented with protease inhibitors (2 mM PMSF, 0.15 U/ml aprotinin, 20 mM leupeptin, and 20 mM pepstatin). Cells were lysed with one pass through a French Press at 1000 psi. Clarified lysates were incubated with 4 ml prepared Ni-NTA agarose beads (Qiagen) at 4°C for 1 h. Beads were washed with IMAC 50 buffer and eluted with IMAC 100 buffer. Eluted TcAK l was first concentrated by dialysis against a solution consisting of 20% polyethylene glycol (MW 15,000 to 20,000) in 50 mM Tris (pH 7.5), 200 mM

NaCl, followed by dialyzed against 0. 1 M NaHCO ₃ , 0.5 M NaCl (pH 8.3) at 4°C overnight. TcAK l was covalently coupled to 1.5 gram of activated CH Sepharose 4B at room temperature for 3 h. Blocking and washings were performed according to the manufacturer's instructions. Preparation of the affinity column for Cdc25C antibodies was performed as described previously (Ogg et al. , 1994).

6C. Affinity purification of TcAKl and Cdc25C(N258) antibodies. CST- TcAKl antisera was passed over the His-TcAKl affinity column at 4°C overnight. The column was washed with RIPA buffer (20 mM Tris pH 7.4, 137 mM NaCl, 10% glycerol, 0. 1 % SDS, 0.5% sodium deoxycholate, 1 % Triton X- 100, 2 mM EDTA) followed by NETN buffer (20 mM Tris pH 8, 1 mM EDTA, 0.5 % NP-40) containing 0.5

M NaCl. Antibodies specific for TcAKl were eluted first with 100 mM glycine (pH 2.5) and then with 0. 1 M triethylamine. All fractions containing antibodies were neutralized

with 1 M Tris (pH 8.0), pooled and adjusted to 0. 1 mg/ml Bovine serum albumin. Proteins were precipitated with 50% ammonium sulfate (w/v) and centrifugation. Pellets were resuspended in and dialyzed against Tris buffered saline (10 mM Tris pH 8.0, 0.5 M NaCl). Antibodies were aliquoted and stored at -80°C. The purification of anti-N258 Cdc25C antibodies was essentially the same except that polyclonal GST-25C antisera was first passed over a GST-Sepharose column to remove antibodies specific for GST. Unbound antisera was loaded onto a GST-25(N258)-Sepharose column.

Example 7: Interaction of Cdc25C with TcAKl

7A. Phosphorylation of Cdc25C by TcAKl in vitro: Induction and purification of GST-fusion proteins: JM109 cells were transformed with plasmids encoding GST-

Cdc25C, GST-CdcC(S216A), GST-N258, and GST-N258(S216A). 20 mL of overnight culture was used to inoculate 2L of Luria Broth (containing 100 ug/mL ampicillin). The culture was grown at 37°C to an O.D. ₆₀₀ of 0.6. IPTG was added to a final concentration of 0.5 mM and the culture was incubated for 3 hours at 30°C. The cells were collected by centrifugation at 3800 x g for 10 min.

Bacterial pellets were washed with PBS and then resuspended in 120 mL of STE (100 mM NaCl, 10 mM Tris HCl, 1 mM EDTA, pH 8.0) supplemented with 2 mM PMSF, 0.15 U/mL aprotinin, 20 μM leupeptin, 20 μM pepstatin, and 0.5 mg/mL lysozyme, and rocked at 4°C for 20 min. 21.2 mL of 10% sarkosyl (N-lauroylsarcosine, Sigma, L-5125) in STE was added ( 1.5 % final concentration) and lysed by sonication.

Lysates were clarified by centrifugation at 15,000 x g for 10 min. , and 35 mL 10% Triton X-100 (Sigma, X- 100) in STE was added (2% final concentration). 20 mL packed glutathione (GSH) agarose beads (Sigma, G-4510) was incubated with the clarified lysates for two hours at 4°C. Pelleted beads were washed twice in STE, twice in LiCl Buffer (0.5 M LiCl, 50 mM Tris HCl, pH 8.0), and twice in 50 mM Tris HCl, pH 7.4. Washed beads were packed into two 10 mL reusable columns (Econo-Column Chromatography Columns, Biorad), and GST-fusion proteins were eluted with 20 mM glutathione (Sigma, G-4251 ) in 50 mM Tris pH 7.4. 0.5 L fractions were collected and peak fractions were pooled, dialyzed overnight in 25 mM Tris-HCl, pH 7.4, and frozen at -80°C. The

concentration of each GST-fusion construct was estimated by comparison to known BSA standards after SDS-PAGE and Coomassie blue staining.

Kinase assays: Kinase reactions consisted of 1 μg of purified His-tagged TcAKl , 5 μg of GST-fusion protein (GST-Cdc25C, GST-Cdc25C(S216A), GST-N258, or GST- N258(S216A) in 40 μL of complete kinase buffer (50 mM Tris pH 7.4, 10 mM MgCl ₂ ,2 mM DTT, 10 μM ATP, and 10 uCiγ- ³² P ATP ( > 4000 Ci/mmol). Reactions were incubated at 30°C for 20 minutes, and then boiled in SDS-sample buffer, resolved by SDS-PAGE (7% gel), proteins were visualized by Coomassie blue staining and by autoradiography. 7B. Peptide kinase assays. Preparation of HeLa cell lysates: HeLa cells (~1 x

10 ⁷ ) were washed twice in PBS and then lysed in 1 ml of mammalian cell lysis buffer consisting of 50 mM Tris pH 8.0, 2 mM DTT, 5mM EDTA pH 8.0, 0. 1 % NP-40, 100 mM NaCl, 1 mM microcystin, 50 mM NaF, 1 mM sodium orthovanadate, 2 mM PMSF, 0. 15 U/ml aprotinin, 20 mM leupeptin, and 20 mM pepstatin for 30 min. at 4°C. Lysates were clarified and incubated with either GST-fusion proteins (as outlined below) or with affinity purified antibodies. Lysates containing -750 μg of protein were incubated with 5 μl of either N258 antibody or TcAK l antibody at 4°C for 1 h followed by an additional 1 h at 4°C in the presence of 30 μl of a 50% slurry of sepharose CL-4B protein A beads. Beads were pelleted and washed four times with lysis buffer and twice with incomplete kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl ₂ , and 2 mM DTT).

Preparation of recombinant GST-fusion protein: Bacteria expressing GST or GST- Cdc25C(motif) were suspended in STE (100 mM NaCl, 10 mM Tris HCl, 1 mM EDTA, pH 8.0) supplemented with 2 mM PMSF, 0.15 U/mL aprotinin, 20 μM leupeptin, 20 μM pepstatin, and 1.0 mg/mL lysozyme, and rocked at 4°C for 20 min. The solution was adjusted to 1.5 % sarkosyl (N-lauroylsarcosine, Sigma, L-5125) and bacteria were lysed by sonication. The lysate was clarified by centrifugation at 3200 x g for 20 min and Triton X-100 (Sigma) was added to a final concentration for 2% . Approximately 1 μg of GST- fusion protein was bound to 15 μl packed glutathione (GSH) agarose beads (Sigma) for 45 min at 4°C. Pelleted beads were washed twice with NETN buffer containing 1 M NaCl, followed by twice with NETN buffer and then incubated with HeLa cell lysate (1.5 mg) at

4°C for lh. Beads were pelleted and washed four times with mammalian cell lysis buffer

and twice with incomplete kinase buffer twice (50 mM Tris (pH 7.5), 10 mM MgCl ₂ , and 2 mM DTT).

Kinase reactions: 40 μl kinase reactions containing approximately 1 μg of GST- fusion protein bound to 15 μl of GSH agarose beads, 5 μM peptide, 50 mM Tris (pH 7.5), 10 mM MgCl ₂ , 2 mM DTT, 20 μM ATP, 10 uCi γ- ³² P ATP ( > 4000 Ci/mmol), were incubated at 30°C for 10 minutes. Immune complex kinase assays were incubated at 30°C for 20 min. Reactions were resolved on a 20% SDS polyacrylamide gel, dried and subjected to autoradiography.

7C. Binding specificity of Cdc25C and TcAKl . Preparation of insect cell lysate; Sf9 cells were infected with recombinant baculovirus encoding TcAKl . Atd 40 h post infection, cells (-2 x 10 ⁷ ) were rinsed twice in PBS and then lysed in 2 ml of NETN buffer (20 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5 % NP-40) supplemented with 1 mM DTT, I mM Na ₃ VO ₄ , 10 mM NaF, 1 μM microcystin, 0. 15 unit/ml aprotinin, 20 μM leupeptin, 20 μM pepstatin, and 2 mM PMSF. Preparation of GST-proteins and binding assay: Induced bacterial pellets expressing GST, GST-Cdc25C,GST-Cdc25C(N258),GST-25C(C215) and GST-25C motif were suspended in STE supplemented with 2 mM PMSF, 0. 15 U/mL aprotinin, 20 μM leupeptin, 20 μM pepstatin, and 1.0 mg/mL lysozyme, and rocked at 4°C for 20 min. The solution was adjusted to 1.5 % sarkosyl (N-Lauroyl Sarcosine, Sigma, L-5125) and bacteria were lysed by sonication. The lysate was clarified by centrifugation at 3200 x g for 20 min and Triton X-100 (Sigma) was added to a final concentration of 2% . 4 μg of each protein were bound to 50 μl of packed GSH agarose beads by incubation at 4°C for 45 min. Beads were washed 3 times with NETN. Half of the beads were resolved directly on a 10% SDS gel and proteins were visualized by staining the gel with Coomassie blue. The second half was incubated with insect cell lysate ( -400 ug). Bound proteins were washed 4 times with NETN, resolved on a 7% SDS gel and transferred to nitrocellulose. Membranes were incubated with antibodies raised against an internal TcAK l peptide (KQKDENKEAKPRS) at a 1 : 1 ,000 dilution.

7D. Binding of Cdc25C-motif to endogenous TcAKl in vitro. Preparation of cell lysate: HeLa and Jurkat cells (-6 x 10 ⁷ ) were rinsed twice in PBS and then lysed in

6 ml of NETN Buffer (20 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40)

supplemented with 1 mM DTT, 1 mM Na ₃ VO ₄ , 10 mM NaF, 1 μM microcystin, 0. 15 unit/ml aprotinin, 20 μM leupeptin, 20 μM pepstatin, and 2 mM PMSF.

Preparation of GST-proteins and binding assay: Induced bacterial pellets expressing GST and GST-25C(motif) were lysed by sonication in STE (25 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA) supplemented with 1.5% sarcosyl. 4 μg of each protein were bound to 50 μl of packed GSH agarose beads by incubation at 4°C for 45 minutes. Beads were washed 3 times with NETN (20 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5 % NP-40). Half of the beads were resolved directly on a 7% SDS gel, the other half was incubated with either HeLa (~2 mg) or Jurkat (-1 mg) lysates at 4°C for 1 h. Bound proteins were washed 4 times with NETN, resolved on a 7% SDS gel and transferred to nitrocellulose. Membranes were incubated with affinity purified TcAKl antibody at 1 : 10,000 dilution.

Example 8: Interaction of Cdc25c with 14-3-3 protein

8A. Association of Cdc25C with 14-3-3 in insect cells. Sf9 cells were infected with viruses encoding GST-Cdc25C, GST-N258, GST-Cdc25C(C377S), and GST-C215.

In other experiments, cells were infected with viruses encoding hexahistidine tagged forms of Cdc25C or Cdc25C(S216A) in the absence of presence of viruses encoding 14-3-3 (zeta isoform). For GST-fusion proteins, cells were lysed in buffer A. For hexahistidine tagged proteins, cells were lysed in buffer B. Lysates were clarified by centrifugation at 13,000 x g for 15 min. To - 1.5 mg of protein was added 50 μL of a 50% suspension of

GSH agarose (Sigma) or 50 μL Ni ^{2 +} -NTA-agarose (Qiagen) for cells expressing GST- fusion or hexahistidine tagged forms of Cdc25C, respectively. Following incubation for 30 min at 4°C, GSH agarose or Ni ^{2 +} -NTA-agarose was collected by centrifugation in a microfuge at maximum speed for 10 sec. Beads were washed 3 times with 1 ml buffer A (GSH-agarose) or buffer B (Ni ^{2 +} -NTA-agarose). Ni ²⁺ -NTA-agarose beads were washed once more with buffer B containing 50 mM imidazole. SDS sample buffer was added and samples were heated for 10 min at 100°C. Samples were subjected to 12.5% SDS-PAGE and transferred to nitrocellulose membrane. Nitrocellulose membranes were cut in half and the top portion immunoblotted for either GST using a GST rabbit polyclonal antibody or for Cdc25C using the E10 antibody. The lower half was blotted for 14-3-3 using 14-3-

3 β (K- 19, Santa Cruz).

8B. Expression of Cdc25C and Cdc25C (S216A) in HeLa cells and Immunoprecipitations from HeLa and Jurkat cells. Approximately 3 x 10 ⁵ HeLa cells were seeded per 35 cm tissue culture dish and allowed to grow for 24 hrs in DMEM (high glucose; Gibco-BRL) containing 10% fetal calf serum. Cells were transfected with vectors encoding myc-Cdc25C or myc-Cdc25C (S216A) in pcDNA ₃ (Invitrogen) using lipfectamine (Gibco-BRL) according to the manufacturer's instructions. Cells were also transfected with the empty vector (pcDNA ₃ ) as a control. At 31 hrs post transfection, cells were washed twice in ice-cold PBS, and then lysed in buffer A. Cells were then snap-frozen and stored in liquid nitrogen until future use. Frozen lysates were thawed and then rocked on a nutator for 10 min at 4°C. Lysates were clarified by centrifugation at

13,000 X g for 15 min and pre-cleared by incubation with Sepharose CL-4B protein A beads (Sigma). Jurkat cell lysates (prepared from untransfected Jurkat cells grown to sub- confluency) were prepared in a similar manner. For immunoprecipitations of endogenous Cdc25C from Jurkat cells, either the E10 monoclonal antibody or the C-20 antibody (Santa Cruz) were used. To -3.5 mg of protein was added either 155 μL of the E10 supernatant or 155 μL of supernatant containing monoclonal antibody specific for GST (as a control). As a control for the C-20 immunoprecipitations, the C20 antibody was pre- incubated with a synthetic peptide consisting of the C-terminal 20 amino acids of Cdc25C for 20 min at 4°C. Immunoprecipitations were performed for 2 hrs at 4°C followed by the addition of 50 μL of a 50% solution of Sepharose CL-4B protein A beads (Sigma) for an additional 30 min. For immunoprecipitations of myc-epitope tagged proteins, 20 μL of myc-agarose (Santa Cruz) was added to -1 mg of transfected HeLa cell lysate and allowed to incubate at 4°C for 3 hrs. The Sepharose CL-4B protein A beads or the myc-agarose was isolated by centrifugation in a microfuge at maximum speed for 1 sec, and washed four times with 1 ml of buffer A. One half of the sample was resolved directly by SDS-

PAGE and processed for immunoblotting, the other half assayed for the presence of 14-3- 3 as described below.

8C. ADP-ribosylation assays. ADP-ribosylation assays were performed as described previously (Coburn et al. , 1991 ; Fu et al. , 1993). Immunoprecipitates from either Jurkat or HeLa cells were incubated with reaction mixtures containing 5 μM

(adenylate- ³² P)NAD(NEN), 100 μg/ml soybean trypsin inhibitor (Sigma), and Exoenzyme

S (UBI) at 1 μg/ml in a total volume of 20 μL. Reactions were carried out for 20 min at 25°C and then terminated by either spotting 10 μL onto p-81 paper, or mixing with SDS sample buffer. P81 papers were washed in 0.75% phosphoric acid three times and once with acetone prior to scintillation counting. Specific incorporation of ( ³² P)NAD into soybean trypsin inhibitor was confirmed by analysis of the reaction on 12.5% SDS-PAGE.

Radiolabeled species were excised and ³ P-incorporation quantitated by scintillation counting.

Example 9: Buffers

Buffer A consisted of: phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na ₂ HPO4, 1.5 mM KH ₂ PO ₄ , pH 7.4) supplemented with 1 % NP-40, 5 mM EDTA, 1 mM sodium vanadate, 1 μM microcystin-LR (Gibco-BRL), 2 mM dithiothreitol (DTT), 10 μg/ml aprotinin, 20 μM leupeptin, and 5 μg/ml pepstatin. Buffer B is identical to buffer A except it lacks EDTA and DTT. Buffer C consisted of 20 mM Tris (pH 7.5), 500 mM NaCl, 5 mM imidazole, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 20 μM leupeptin, and 5 μg/ml pepstatin. Buffer D consisted of: 75 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , and 1 mM DTT.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Pi nica-Worms, Helen

(ii) TITLE OF INVENTION: DNA SEQUENCES ENCODING HUMAN TcAK-1 KINASE

(iii) NUMBER OF SEQUENCES: 17

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Greenlee, Winner and Sullivan, P.C. (B) STREET: 5370 Manhattan Circle, Suite 201

(C) CITY: Boulder

(D) STATE: CO

(E) COUNTRY: USA

(F) ZIP: 80303

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US Unassigned

(B) FILING DATE: 09-JUL-1996

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Caruthers, Jennie M.

(B) REGISTRATION NUMBER: 34,464

(C) REFERENCE/DOCKET NUMBER: 9-96

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (303) 499-8080 (B) TELEFAX: (303) 499-8089

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2698 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 376..2565

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAGCTGAAAT TCGCGGTGCG ACGGGAGGGA GTGGAGAAGG AGGTGAGGGG GCCCAGGATC 60

GCGGGGCGCC CTGAGGCAAG GGGACGCCGG TGGGTCGAAG CGCAGCCCGC CGCCCGCAGG 120

CTCGGCTCCG CCACTGCCGC CCTCCCGGTC TCCTCGCCTC GGGCGCCGAG GCAGGGAGAG 180

AATGAGCCCC GGGACCCGCC GGGGGACGGC CCGGGCCAGG CCCGGGATCT AGAACGGCCG 240

TAGGGGGAAG GGAGCCGCCC TCCCCACGGC GCCTTTTCGG AACTGCCGTG GACTCGAGGA 300

CGCTGGTCGC CGGCCTCCTA GGGCTGTGCT GTTTTGTTTT GACCCTCGCA TTGTGCAGAA 360

TTAAAGTGCA GTAAA ATG TCC ACT AGG ACC CCA TTG CCA ACG GTG AAT GAA 411

Met Ser Thr Arg Thr Pro Leu Pro Thr Val Asn Glu 1 5 10

CGA GAC ACT GAA AAC CAC ACG TCA CAT GGA GAT GGG CGT CAA GAA GTT 459 Arg Asp Thr Glu Asn His Thr Ser His Gly Asp Gly Arg Gin Glu Val 15 20 25

ACC TCT CGT ACC AGC CGC TCA GGA GCT CGG TGT AGA AAC TCT ATA GCC 507 Thr Ser Arg Thr Ser Arg Ser Gly Ala Arg Cys Arg Asn Ser lie Ala 30 35 40

TCC TGT GCA GAT GAA CAA CCT CAC ATC GGA AAC TAC AGA CTG TTG AAA 555 Ser Cys Ala Asp Glu Gin Pro His lie Gly Asn Tyr Arg Leu Leu Lys 45 50 55 60

ACA ATC GGC AAG GGG AAT TTT GCA AAA GTA AAA TTG GCA AGA CAT ATC 603 Thr lie Gly Lys Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His lie 65 70 75

CTT ACA GGC AGA GAG GTT GCA ATA AAA ATA ATT GAC AAA ACT CAG TTG 651

Leu Thr Gly Arg Glu Val Ala lie Lys lie lie Asp Lys Thr Gin Leu 80 85 90

AAT CCA ACA AGT CTA CAA AAG CTC TTC AGA GAA GTA AGA ATA ATG AAG 699 Asn Pro Thr Ser Leu Gin Lys Leu Phe Arg Glu Val Arg lie Met Lys 95 100 105

ATT TTA AAT CAT CCC AAT ATA GTG AAG TTA TTC GAA GTC ATT GAA ACT 747 lie Leu Asn His Pro Asn lie Val Lys Leu Phe Glu Val lie Glu Thr 110 115 120

GAA AAA ACA CTC TAC CTA ATC ATG GAA TAT GCA AGT GGA GGT GAA GTA 795

Glu Lys Thr Leu Tyr Leu lie Met Glu Tyr Ala Ser Gly Gly Glu Val 125 130 135 140

TTT GAC TAT TTG GTT GCA CAT GGC AGG ATG AAG GAA AAA GAA GCA AGA 843

Phe Asp Tyr Leu Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg 145 150 155

TCT AAA TTT AGA CAG ATT GTG TCT GCA GTT CAA TAC TGC CAT CAG AAA 891

Ser Lys Phe Arg Gin lie Val Ser Ala Val Gin Tyr Cys His Gin Lys 160 165 170

CGG ATC GTA CAT CGA GAC CTC AAG GCT GAA AAT CTA TTG TTA GAT GCC 939 Arg lie Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala 175 180 185

GAT ATG AAC ATT AAA ATA GCA GAT TTC GGT TTT AGC AAT GAA TTT ACT 987

Asp Met Asn lie Lys lie Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr 190 195 200

GTT GGC GGT AAA CTC GAC ACG TTT TGT GGC AGT CCT CCA TAC GCA GCA 1035

Val Gly Gly Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala 205 210 215 220

CCT GAG CTC TTC CAG GGC AAG AAA TAT GAC GGG CCA GAA GTG GAT GTG 1083

Pro Glu Leu Phe Gin Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Val 225 230 235

TGG AGT CTG GGG GTC ATT TTA TAC ACA CTA GTC AGT GGC TCA CTT CCC 1131

Trp Ser Leu Gly Val lie Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro 240 245 250

TTT GAT GGG CAA AAC CTA AAG GAA CTG AGA GAG AGA GTA TTA AGA GGG 1179 Phe Asp Gly Gin Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly 255 260 265

AAA TAC AGA ATT CCC TTC TAC ATG TCT ACA GAC TGT GAA AAC CTT CTC 1227 Lys Tyr Arg He Pro Phe Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu 270 275 280

AAA CGT TTC CTG GTG CTA AAT CCA ATT AAA CGC GGC ACT CTA GAG CAA 1275 Lys Arg Phe Leu Val Leu Asn Pro He Lys Arg Gly Thr Leu Glu Gin 285 290 295 300

ATC ATG AAG GAC AGG TGG ATC AAT GCA GGG CAT GAA GAA GAT GAA CTC 1323 He Met Lys Asp Arg Trp He Asn Ala Gly His Glu Glu Asp Glu Leu 305 310 315

AAA CCA TTT GTT GAA CCA GAG CTA GAC ATC TCA GAC CAA AAA AGA ATA 1371 Lys Pro Phe Val Glu Pro Glu Leu Asp He Ser Asp Gin Lys Arg He 320 325 330

GAT ATT ATG GTG GGA ATG GGA TAT TCA CAA GAA GAA ATT CAA GAA TCT 1419 Asp He Met Val Gly Met Gly Tyr Ser Gin Glu Glu He Gin Glu Ser 335 340 345

CTT AGT AAG ATG AAA TAC GAT GAA ATC ACA GCT ACA TAT TTG TTA TTG 1467 Leu Ser Lys Met Lys Tyr Asp Glu He Thr Ala Thr Tyr Leu Leu Leu 350 355 360

GGG AGA AAA TCT TCA GAG CTG GAT GCT AGT GAT TCC AGT TCT AGC AGC 1515 Gly Arg Lys Ser Ser Glu Leu Asp Ala Ser Asp Ser Ser Ser Ser Ser 365 370 375 380

AAT CTT TCA CTT GCT AAG GTT AGG CCG AGC AGT GAT CTC AAC AAC AGT 1563 Asn Leu Ser Leu Ala Lys Val Arg Pro Ser Ser Asp Leu Asn Asn Ser 385 390 395

ACT GGC CAG TCT CCT CAC CAC AAA GTG CAG AGA AGT GTT TCT TCA AGC 1611 Thr Gly Gin Ser Pro His His Lys Val Gin Arg Ser Val Ser Ser Ser 400 405 410

CAA AAG CAA AGA CGC TAC AGT GAC CAT GCT GGA CCA GCT ATT CCT TCT 1659 Gin Lys Gin Arg Arg Tyr Ser Asp His Ala Gly Pro Ala He Pro Ser 415 420 425

GTT GTG GCG TAT CCG AAA AGG AGT CAG ACA AGC ACT GCA GAT GGT GAC 1707

Val Val Ala Tyr Pro Lys Arg Ser Gin Thr Ser Thr Ala Asp Gly Asp 430 435 440

CTC AAA GAA GAT GGA ATT TCC TCC CGG AAA TCA AGT GGC AGT GCT GTT 1755 Leu Lys Glu Asp Gly He Ser Ser Arg Lys Ser Ser Gly Ser Ala Val

445 450 455 460

GGA GGA AAG GGA ATT GCT CCA GCC AGT CCC ATG CTT GGG AAT GCA AGT 1803

Gly Gly Lys Gly He Ala Pro Ala Ser Pro Met Leu Gly Asn Ala Ser 465 470 475

AAT CCT AAT AAG GCG GAT ATT CCT GAA CGC AAG AAA AGC TCC ACT GTC 1851

Asn Pro Asn Lys Ala Asp He Pro Glu Arg Lys Lys Ser Ser Thr Val 480 485 490

CCT AGT AGT AAC ACA GCA TCT GGT GGA ATG ACA CGA CGA AAT ACT TAT 1899

Pro Ser Ser Asn Thr Ala Ser Gly Gly Met Thr Arg Arg Asn Thr Tyr 495 500 505

GTT TGC AGT GAG AGA ACT ACA GCT GAT AGA CAC TCA GTG ATT CAG AAT 1947

Val Cys Ser Glu Arg Thr Thr Ala Asp Arg His Ser Val He Gin Asn 510 515 520

GGC AAA GAA AAC AGC ACT ATT CCT GAT CAG AGA ACT CCA GTT GCT TCA 1995 Gly Lys Glu Asn Ser Thr He Pro Asp Gin Arg Thr Pro Val Ala Ser

525 530 535 540

ACA CAC AGT ATC AGT AGT GCA GCC ACC CCA GAT CGA ATC CGC TTC CCA 2043

Thr His Ser He Ser Ser Ala Ala Thr Pro Asp Arg He Arg Phe Pro 545 550 555

AGA GGC ACT GCC AGT CGT AGC ACT TTC CAC GGC CAG CCC CGG GAA CGG 2091

Arg Gly Thr Ala Ser Arg Ser Thr Phe His Gly Gin Pro Arg Glu Arg 560 565 570

CGA ACC GCA ACA TAT AAT GGC CCT CCT GCC TCT CCC AGC CTG TCC CAT 2139

Arg Thr Ala Thr Tyr Asn Gly Pro Pro Ala Ser Pro Ser Leu Ser His 575 580 585

GAA GCC ACA CCA TTG TCC CAG ACT CGA AGC CGA GGC TCC ACT AAT CTC 2187

Glu Ala Thr Pro Leu Ser Gin Thr Arg Ser Arg Gly Ser Thr Asn Leu 590 595 600

TTT AGT AAA TTA ACT TCA AAA CTC ACA AGG AGT CGC AAT GTA TCT GCT 2235 Phe Ser Lys Leu Thr Ser Lys Leu Thr Arg Ser Arg Asn Val Ser Ala 605 610 615 620

GAG CAA AAA GAT GAA AAC AAA GAA GCA AAG CCT CGA TCC CTA CGC TTC 2283 Glu Gin Lys Asp Glu Asn Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe

625 630 635

ACC TGG AGC ATG AAA ACC ACT AGT TCA ATG GAT CCC GGG GAC ATG ATG 2331 Thr Trp Ser Met Lys Thr Thr Ser Ser Met Asp Pro Gly Asp Met Met 640 645 650

CGG GAA ATC CGC AAA GTG TTG GAC GCC AAT AAC TGC GAC TAT GAG CAG 2379 Arg Glu He Arg Lys Val Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gin 655 660 665

AGG GAG CGC TTC TTG CTC TTC TGC GTC CAC GGA GAT GGG CAC GCG GAG 2427 Arg Glu Arg Phe Leu Leu Phe Cys Val His Gly Asp Gly His Ala Glu 670 675 680

AAC CTC GTG CAG TGG GAA ATG GAA GTG TGC AAG CTG CCA AGA CTG TCT 2475 Asn Leu Val Gin Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser 685 690 695 700

CTG AAC GGG GTC CGG TTT AAG CGG ATA TCG GGG ACA TCC ATA GCC TTC 2523 Leu Asn Gly Val Arg Phe Lys Arg He Ser Gly Thr Ser He Ala Phe

705 710 715

AAA AAT ATT GCT TCC AAA ATT GCC AAT GAG CTA AAG CTG TAA 2565

Lys Asn He Ala Ser Lys He Ala Asn Glu Leu Lys Leu * 720 725 730

CCCAGTGATT ATGATGTAAA TTAAGTAGCA AGTAAAGTGT TTTCCTGAAC ACTGATGGAA 2625

ATGTATAGAA TAATATTTAG GCAATAACGT CTGCATCTTC TAAATCATGA AATTAAAGTC 2685

TGAGGACGAG AGC 2698

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 730 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

( i i ) MOLECULE TYPE : protein

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

Met Ser Thr Arg Thr Pro Leu Pro Thr Val Asn Glu Arg Asp Thr Glu

1 5 10 15

Asn His Thr Ser His Gly Asp Gly Arg Gin Glu Val Thr Ser Arg Thr

20 25 30

Ser Arg Ser Gly Ala Arg Cys Arg Asn Ser He Ala Ser Cys Ala Asp 35 40 45

Glu Gin Pro His He Gly Asn Tyr Arg Leu Leu Lys Thr He Gly Lys 50 55 60

Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His He Leu Thr Gly Arg 65 70 75 80

Glu Val Ala He Lys He He Asp Lys Thr Gin Leu Asn Pro Thr Ser 85 90 95

Leu Gin Lys Leu Phe Arg Glu Val Arg He Met Lys He Leu Asn His

100 105 110

Pro Asn He Val Lys Leu Phe Glu Val He Glu Thr Glu Lys Thr Leu 115 120 125

Tyr Leu He Met Glu Tyr Ala Ser Gly Gly Glu Val Phe Asp Tyr Leu 130 135 140

Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg Ser Lys Phe Arg 145 150 155 160

Gin He Val Ser Ala Val Gin Tyr Cys His Gin Lys Arg He Val His 165 170 175

Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Asp Met Asn He

180 185 190

Lys He Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Val Gly Gly Lys 195 200 205

Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu Leu Phe 210 215 220

Gin Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Val Trp Ser Leu Gly 225 230 235 240

Val He Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe Asp Gly Gin 245 250 255

Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys Tyr Arg He 260 265 270

Pro Phe Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu Lys Arg Phe Leu 275 280 285

Val Leu Asn Pro He Lys Arg Gly Thr Leu Glu Gin He Met Lys Asp 290 295 300

Arg Trp He Asn Ala Gly His Glu Glu Asp Glu Leu Lys Pro Phe Val 305 310 315 320

Glu Pro Glu Leu Asp He Ser Asp Gin Lys Arg He Asp He Met Val 325 330 335

Gly Met Gly Tyr Ser Gin Glu Glu He Gin Glu Ser Leu Ser Lys Met

340 345 350

Lys Tyr Asp Glu He Thr Ala Thr Tyr Leu Leu Leu Gly Arg Lys Ser 355 360 365

Ser Glu Leu Asp Ala Ser Asp Ser Ser Ser Ser Ser Asn Leu Ser Leu 370 375 380

Ala Lys Val Arg Pro Ser Ser Asp Leu Asn Asn Ser Thr Gly Gin Ser 385 390 395 400

Pro His His Lys Val Gin Arg Ser Val Ser Ser Ser Gin Lys Gin Arg 405 410 415

Arg Tyr Ser Asp His Ala Gly Pro Ala He Pro Ser Val Val Ala Tyr

420 425 430

Pro Lys Arg Ser Gin Thr Ser Thr Ala Asp Gly Asp Leu Lys Glu Asp 435 440 445

Gly He Ser Ser Arg Lys Ser Ser Gly Ser Ala Val Gly Gly Lys Gly 450 455 460

He Ala Pro Ala Ser Pro Met Leu Gly Asn Ala Ser Asn Pro Asn Lys 465 470 475 480

Ala Asp He Pro Glu Arg Lys Lys Ser Ser Thr Val Pro Ser Ser Asn 485 490 495

Thr Ala Ser Gly Gly Met Thr Arg Arg Asn Thr Tyr Val Cys Ser Glu

500 505 510

Arg Thr Thr Ala Asp Arg His Ser Val He Gin Asn Gly Lys Glu Asn 515 520 525

Ser Thr He Pro Asp Gin Arg Thr Pro Val Ala Ser Thr His Ser He 530 535 540

Ser Ser Ala Ala Thr Pro Asp Arg He Arg Phe Pro Arg Gly Thr Ala 545 550 555 560

Ser Arg Ser Thr Phe His Gly Gin Pro Arg Glu Arg Arg Thr Ala Thr 565 570 575

Tyr Asn Gly Pro Pro Ala Ser Pro Ser Leu Ser His Glu Ala Thr Pro

580 585 590

Leu Ser Gin Thr Arg Ser Arg Gly Ser Thr Asn Leu Phe Ser Lys Leu 595 600 605

Thr Ser Lys Leu Thr Arg Ser Arg Asn Val Ser Ala Glu Gin Lys Asp 610 615 620

Glu Asn Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe Thr Trp Ser Met 625 630 635 640

Lys Thr Thr Ser Ser Met Asp Pro Gly Asp Met Met Arg Glu He Arg 645 650 655

Lys Val Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gin Arg Glu Arg Phe

660 665 670

Leu Leu Phe Cys Val His Gly Asp Gly His Ala Glu Asn Leu Val Gin 675 680 685

Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly Val 690 695 700

Arg Phe Lys Arg He Ser Gly Thr Ser He Ala Phe Lys Asn He Ala 705 710 715 720

Ser Lys He Ala Asn Glu Leu Lys Leu * 725 730

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

Glu Gin Pro His He Gly Asn Tyr Arg Leu Leu Lys Thr He Gly Lys

1 5 10 15

Gly Asn Phe Ala Lys Val Lys Leu Ala 20 25

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro Arg Leu Lys 1 5 10 15

( 2 ) INFORMATION FOR SEQ ID NO : 5 :

< i ) SEQUENCE CHARACTERISTI CS : ( A ) LENGTH : 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide for site-directed mutagenesis"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATATCGCTCC CCGGCGATGC CAGAGAACTT 30

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 69 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CAGCATAAGC TTACCATGGC AGAACAGAAG CTCATTTCTG AAGAAGACTT GTCTACGGAA 60

CTCTTCTCA 69

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

AATGCACTTC CTGAAGTCCT GAAGA 25

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TCGAGGTACC 10

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iϋ) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CGTGTTCCAC TCTGAATTCT CCTCAGAGAG GGGCCCCCGG ATGTGCCGC 49

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide primer"

(in) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

CGAGTCATAT GTCCACTAGG ACCCC 25

(2) INFORMATION FOR SEQ ID NO: 11:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(in) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CCAGTCATAT GTTAACTTAC AGCTTTAGCT CATTTGGC 38

(2) INFORMATION FOR SEQ ID NO: 12:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CCAGTCATAT GTTAACTTAG CTTGAAGAAA CACTTCTCTG C 41

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CAAGGCTGAA GCTCTATTGT TAGATGC 27

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide primer"

(iϋ) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GCATCTAACA ATAGAGCTTC AGCCTTG 27

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: C-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Lys Asn He Ala Ser Lys He Ala Asn Glu Leu Lys Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Lys Gin Lys Asp Glu Asn Lys Glu Ala Lys Pro Arg Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Glu Gin Lys Leu He Ser Glu Glu Asp Leu

1 5 10