A METHOD OF IDENTIFYING A MODULATOR FOR A SERINE/THERONINE KINASE
FIELD OF INVENTION
The following invention relates to a method of identifying a modulator for serine/threonine kinase.
BACKGROUND
Enzymes are large proteins that catalyze reactions in living cells. Enzymes build up or tear down other molecules. For example, enzymes catalyze the synthesis of fat from fatty acids, form complex sugars from glucose and fructose, and aid in the formation of other proteins from amino acids. Enzymes also reverse the build-up process by breaking down more complex structures. Enzymes are generally specific to certain substrates for their reactions. For example, an individual enzyme may catalyze the reaction where only one substrate is involved or it may act on a group of related substrates.
Protein kinases are enzymes which catalyze the transfer of phosphorous from adenosine triphosphate (ATP), or guanosine triphosphate (GTP) to the targeted protein to yield a phosphorylated protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively. ATP or GTP is first hydrolyzed to form ADP or GDP and inorganic phosphate. The inorganic phosphate is then attached to the targeted protein. The protein substrate which is targeted by kinases may be a structural protein, found in membrane material such as a cell wall, or another enzyme which is a functional protein.
It is estimated about three to four percent of the human genome contains transcription information for the formation of protein kinases. Currently, there are about up to 400 known different protein kinases. However, because three to four percent of the human genome is a code for the formation of protein kinases, there may be many thousands of distinct and separate kinases in the human body.
Due to their physiological relevance, variety and ubiquitousness, protein kinases have become one of the most important and widely studied family of enzymes in biochemical and medical research. Studies have shown that protein kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, and cell division. Several oncogenes have also been shown to encode protein kinases, suggesting that kinases play a role in oncogenesis.
oncogenesis.
Protein kinases are often divided into two groups based on the amino acid residue they phosphorylate. The first group, called serine/threonine kinases (PSTK), includes cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell division cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins. Aberrant protein serine/threonine kinase activity has been implicated or is suspected in a number of pathologies such as rheumatoid arthritis, psoriasis, septic shock, bone loss, many cancers and other proliferative disease. Accordingly, serine/threonine kinases and the signal transduction pathways which they are part of are important targets for drug design.
The second group of kinases, called tyrosine kinases, phosphorylate tyrosine residues. They are present in much smaller quantities but also play an equally important role in cell regulation. These kinases include several receptors for molecules such as growth factors and hormones, including epidermal growth factor receptor, insulin receptor, platelet derived growth factor receptor and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside. Much work is also under progress to identify modulators of tyrosine kinases as well.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a method of identifying a modulator of a specific serine/threonine kinase, comprising: (a) expressing a fusion protein between a substrate and a serine/threonine kinase within a cell, (b) incubating the cell with a candidate modulator, and (c) determining the level of phosphorylation of the substrate, whereby when a candidate modulator increases the level of phosphorylation, the modulator is an agonist, and when a modulator decreases or inhibits the phosphorylation level, the modulator is an antagonist.
In further embodiment, the present invention relates to a kit comprising a host cell comprising a fusion protein between a substrate and a serine/threonine kinase for identifying a modulator of a serine/threonine kinase.
In one preferred embodiment, the substrate within the fusion protein is
p53 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. A Schematic Diagram of a typical fusion protein between a kinase and p53.
Figure 2. Result of ELISA measuring the level of phosphorylation for pCI expression vector (Control) and pCI/p53-ChklKD fusion protein
DETAILED DESCRIPTION OF THE INVENTION
It is extremely difficult to determine whether an agent can specifically inhibit or activate one particular serine/threonine kinase in a cell system. When a cell is stimulated by stress or a growth factor, multiple kinases are often simultaneously activated. In looking at the phosphorylation level of one kinase substrate engendered by an agent, because such substrate is being phosphorylated by multiple kinases, it is not easy to determine precisely which kinase the agent is actually inhibiting or activating.
For receptor tyrosine kinases, which are located upstream of signaling pathways, there have been reports of assays that allow one to identify specific activators; however, due to the reasons cited above, there has been no report for a method of identifying specific activators or inhibitors of serine/threonine kinases.
Applicants have now discovered that by using a cell which expresses a fusion protein between a serine/threonine kinase and a substrate, a specific modulator of the serine/threonine kinase can be identified. Until now such fusion proteins (such as MEK-ERK fusion protein, JNK-JNKK fusion protein, etc.) have only been used to study the biological function of the kinases, and not in the drug discovery process.
DEFINITION OF TERMS
As used herein below "kinase" refers to serine/threonine kinase.
"Modulator" of a kinase is defined as an agent which affects the activity of the kinase. When a candidate modulator increases the kinase activity (phosphorylating ability), the modulator is an agonist; and when a modulator decreases or inhibits the kinase activity, the modulator is an antagonist. The agent is any agent such as a small or large organic molecule, natural product,
protein, external stress, oligonucletide, antibody, etc.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
It will be appreciated that "proteins" often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given protein, either by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in proteins are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and thus are well known to those of skill in the art. Known modifications which may be present in proteins of the present invention include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications including glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation are described in most basic texts such as PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993. Detailed reviews are also available on this subject.
See e.g., Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1- 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth. Enzymol., 1990, 182:626-646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann. N.Y. Acad. Sci., 1992, 663: 48-62.
It will be appreciated, as is well known, that "proteins" are not always entirely linear. For instance, proteins may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular proteins may be synthesized by non-translation natural processes and by entirely synthetic methods, as well.
The modifications that occur in a protein often will be a function of how it is made. For proteins made by expressing a cloned gene in a host, for instance, the nature and extent of the modifications in large part will be determined by the host cell's posttranslational modification capacity and the modification signals present in the protein amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacterial hosts such as E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given protein may contain many types of modifications. The term "protein" (or "polypeptides" used interchangeably with "proteins" herein) encompasses all such modifications, particularly those which result from expressing a polynucleotide in a host cell.
"Fusion protein" or "chimeric protein," as used herein, is a protein decribing a non-naturally occurring combination of two or more different protein sequences. For example, EP-A0464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. 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. The fusion protein of the invention can be prepared in any suitable manner. Means for preparing such proteins are
well understood in the art, and also described generally in some detail below.
The fusion protein of the present invention comprises combination of a serine/threonine kinase and a kinase substrate. The serine/threonine kinase portion of the fusion protein can either be a naturally occurring full protein (wild type) or a protein which varies from the naturally occurring full protein, but retains the essential kinase properties of the naturally occurring version (a variant or a mutant). For example, the variant may result from one or more amino acid being substituted, added, and/ or deleted in any combinations from the naturally occurring protein. For the sake of clarity, a naturally occurring protein includes an allelic variant. Non-naturally occurring variants of a protein may be made by mutagenesis techniques of polynucleotides which encode them or by direct synthesis.
Preferred variants in the kinase are those that vary from the naturally occuring version by conservative amino acid substitutions — i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination. The variants also include catalytically active fragments of the naturally occurring kinase, and further variants thereof.
In one preferred embodiment, preferred kinase partner of the fusion protein is the use of a protein which has kinase activity higher than the wild type or having constitutive kinase activity. Such constitutive kinase activity or higher kinase activity can be achieved through the use of hyper-active mutant or a catalytic domain of a kinase.
In more detailed aspect, a preferred kinase to be fused within the fusion protein can be derived from p38, JNK3, SGK, PLK1 , YAK3, MAPKAPK2, MYT1, CDK5, ROCK1 /2 and Chkl . The references for the above kinases can be found in:
Yak3:
GenBank Accession no. AF186773
Kenneth A. Lord, Caretha L. Creasy, Andrew G. King, Caroline King, Brian M. Burns,
John C. Lee, and Susan B. Dillon, "REDK, a novel human regulatory erythroid kinase,."
Blood. 95, pp2838-2846 (2000)
Myt1 :
GenBank Accession no. U56816
Liu F, Stanton JJ, Wu Z, Piwnica-Worms H., The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex. Mol Cell Biol 17, pp571-83 (1997)
SGK:
GenBank Accession no. XM_037046
Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL, Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum, Mol Cell Biol 13, pp2031-40 (1993)
Chk1:
GenBank Accession no. NM_001274
Sanchez, Y, Wong, C, Thoma, R., Richiman, R., Wu, Z., Piwnica-Worms, H. and Elledge, S.J., "Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25, " Science 277, pp 1497-1501 (1997)
CDK5:
GenBank Accession no. AY049778
Philpott, A., Porro, EB., Kirschner, MW., and Tsai, LH, tje role of cyclin-dependent kinase 5 and a novel regulatory subunit in regulating muscle differentiation and patterning," Genes Dev. 11 , pp1409-1421 (1997)
P38:
GenBank Accession no. L35253
Han,J., Lee.J.D., Bibbs, L and Ulevitch.R.J., "A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells," Science 265, pp808-811 (1994)
JNK3:
GenBank Accession no. U34820
A. A. Mohit , J. H. Martin , and C. A. Miller, "p493F12 Kinase: a Novel MAP Kinase
Expressed In a Subset of Neurons In the Human Nervous System," Neuron 14, pp67-78 (1995)
Plk1 :
GenBank Accession no. NM_005030
Holtrich.U., Wolf.G, Brauninger.A., Karn.T, Bohme, B., Rubsamen-Waigmann.H. and
Strebhardt, K., Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors, Proc. Natl. Acad. Sci. U.S.A. 91 , pp1736-
1740 (1994)
MAPKAPK2:
GenBank Accession no. NM_004759
Zu.Y.L., Wu,F., Gilchrist.A., Ai,Y, Labadia.M.E. and Huang.C.K., The primary structure of a human MAP kinase activated protein kinase 2, Biochem. Biophys. Res. Commun. 200 (2), pp1118-1124 (1994)
ROCK1/2:
GenBank Accession no. D87931
Takahashi.N., Tuiki.H., Saya.H. and Kaibuchi.K., Localization of the gene coding for
ROCK ll/Rho kinase on human chromosome 2p24, Genomics 55, pp235-237 (1999)
Vectors, Host Cells, Expression
The present invention also relates to vectors (expression systems) comprising a polynucleotide or polynucleotides which encode fusion proteins of the present invention, and host cells which are genetically engineered with such vectors and to the production of fusion proteins of the invention by recombinant techniques.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
A great variety of expression vectors can be used to express a fusion protein of the invention. Such vectors include chromosomal, episomal and virus- derived vectors e.g., vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses such as baculoviruses, papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids. Generally, any vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used for expression in this regard.
Preferred vectors, in certain respects, for expression of polynucleotides and proteins of the present invention comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors are either supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
The following vectors, which are commercially available, are provided by way of example. Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
In certain preferred embodiments, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific expression. Particularly preferred among inducible vectors are vectors that can be induced to express a protein by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.
In one aspect, a preferred inducible expression vector is Ecdysone system (see: Proc. Natl. Acad. Sci. USA, Vol 93, pp3346-3351, April 1996). By using such inducible expression system, an assay system with a low background noise can be achieved.
The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques. In general, a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase. Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skilled in the art, are set forth in great detail in Sambrook et al.
The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription. Large numbers of suitable promoters are known to those of skill in the art.
Among known bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.
Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virusf'RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.
Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for construction of expression vectors, introduction of the vector into the host and expression in the host are routine skills in the art.
In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
In addition, the constructs may contain control regions that regulate, as well as engender, expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription. Examples include repressor binding sites and enhancers, among others.
Vectors for propagation and expression generally will include selectable markers. Selectable marker genes provide a phenotypic trait for selection of transformed host cells. Preferred markers include, but are not limited to, dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria. Such markers may also be suitable for amplification. Alternatively, the vectors may contain additional markers for this purpose.
Transcription of DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp, that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early
promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
SPECIFIC EMBODIMENTS
More preferred aspect of the present invention is to employ p53 protein as a substrate within the fusion protein construct . The choice for the p53 lies in the fact that (i) p53 can be phosphorylated by multiple kinases such as Chkl/2, PKA, and JNK; (ii) within the p53 there are several phosphorylation sites, such as Ser 15 and Ser 20; and (iii) antibodies are commercially available which recognize a particular phosphorylation site within the p53.
As in the case of a kinase partner, the p53 portion of the fusion protein can either be a naturally occurring full protein (wild type) or a protein which varies from the naturally occurring full protein, but retains the essential p53 properties of the naturally occurring version (a variant or a mutant). For example, the variant may result from one or more amino acid being substituted, added, and/or deleted in any combinations from the naturally occurring protein. For the sake of clarity, a naturally occurring protein includes an allelic variant. Non-naturally occurring variants of a protein may be made by mutagenesis techniques of polynucleotides which encode them or by direct synthesis.
The kinase and the substrate within the fusion protein can be linked through either of their C or N terminus. In forming a fusion, it may be desirable to link the substrate portion and the kinase portion with a linking moiety (a linker) comprising between 1 to 100 amino acids. The purpose of a linker is to confer the flexibility within the fusion protein to allow the kinase portion be proximal to the substrate to allow phosphorylation to occur on the substrate. A preferred linker moiety comprises a polypeptide with an amino acid sequence GGGGS x3 + GGGG or a polypeptide which contains many glycines to allow high flexibility.
Screening Asssay and Specific Examples:
The present invention relates to a method of identifying a mfodulator of a specific serine/threonine kinase, comprising: (a) expressing a fusion protein between a substrate and a serine/threonine kinase within a cell, (b) incubating the cell with a candidate modulator, and (c) determining the level of phosphorylation of the substrate.
There are many ways to detect the phosphorylation level of a kinase- substrate fusion protein, which are well within the skilled person in the art. For example, the phosphorylation can be detected using the Western blotting or ELISA. It is immaterial how the phosphorylation level is detected. By mesauring the phosphorylation level, IC50 level of a candidate inhibitor agent can be ascertained. The following examples are illustrative embodiments of the present invention, and by no means limit the invention in any manner.
Example 1. A general method for constructing a fusion protein between p53 and a kinase
The cDNAs encoding full-length p53 and full-length or truncated kinases were amplified using pfu DNA porymerase (Stratagene, Inc., La Jolla, CA). p53 was amplified with a pair of primers: 5' incorporated with a Xbal recognition sequence and Kozac plus start codon and 3' with a Hindlll sequence. The stop codon of p53 was deleted from its C-terminus to make an open reading frame with a linker and the kinase. The full-length kinase or truncated kinase were also amplified with a pair of primers: 5' incorporated with Nhel or Xbal recognition sequences and 3' with Nhel or Xbal sequence. The linker was prepared using DNA synthesizer (Nihon Bioservice K.K., Tokyo, Japan) with 5' Nhel recognition sequence and 3' Xbal sequence. Tow PCR amplified products, p53 and full-length- truncated kinase, and linker flagment were ligated at Xbal or Nhel sites and the fusion was cloned into pCI expression vector (Promega).
Using these methods, fusion proteins of substrate-kinase fusion proteins were constructed. See Figure I
The following provides examples of some of the fusion protein polypeptide and polynucleotide sequences.
An example of an amino acid sequence of the p53-Yak3 fusion protein (SEQ ID NO: l) and its correspoinding polynucleotide sequence (SEQ ID NO: 2) are as follows:
SEQ ID NO: l
MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTED
PGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLH
SGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEV
VRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVWPYEPPEVGSDC
TTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEE
NLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELN
EALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDSSGGGGS
GGGGSGGGGSGGGGSSTPEQALKQYKHHLTAYEKLEIINYPEIYFNGPNAKKRHGVIG
GPNNGGYDDADGAYIHVPRDHLAYRYEVLKIIGKGSFGQVARVYDHKLRQYVALKMV
RNEKRFHRQAAEEIRILEHLKKQDKTGSMNVIHMLESFTFRNHVCMAFELLSIDLYELI
KKNKFQGFSVQLVRKFAQSILQSLDALHKNKIIHCDLKPENILLKHHGRSSTKVIDFGS
SCFEYQKLYTYIQSRFYRAPEIILGSRYSTPIDIWSFGCILAELLTGQPLFPGEDEGDQLA
CMMELLGMPPPKLLEQSKRAKYFINSKGIPRYCSVTTQADGRWLVGGRSRRGKKRG
PPGSKDWGTALKGCDDYLFIEFLKRCLHWDPSARLTPAQALRHPWISKSV
SEQ ID NO: 2 atggaggagccgcagtcagatcctagcgtcgagccccctctgagtcaggaaacattttcagacctatggaaactactt cctgaaaacaacgttctgtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaacaat ggttcactgaagacccaggtccagatgaagctcccagaatgccagaggctgctccccgcgtggcccctgcaccagca gctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagg gcagctacggtttccgtctgggcttcttgcattctgggacagccaagtctgtgacttgcacgtactcccctgccctcaaca agatgttttgccaactggccaagacctgccctgtgcagctgtgggttgattccacacccccgcccggcacccgcgtccg cgccatggccatctacaagcagtcacagcacatgacggaggttgtgaggcgctgcccccaccatgagcgctgctcag atagcgatggtctggcccctcctcagcatcttatccgagtggaaggaaatttgcgtgtggagtatttggatgacagaaa cacttttcgacatagtgtggtggtgccctatgagccgcctgaggttggctctgactgtaccaccatccactacaactaca tgtgtaacagttcctgcatgggcggcatgaaccggaggcccatcctcaccatcatcacactggaagactccagtggta atctactgggacggaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcgcacagaggaagagaatc tccgcaagaaaggggagcctcaccacgagctgcccccagggagcactaagcgagcactgcccaacaacaccagct cctctccccagccaaagaagaaaccactggatggagaatatttcacccttcagatccgtgggcgtgagcgcttcgaga tgttccgagagctgaatgaggccttggaactcaaggatgcccaggctgggaaggagccaggggggagcagggctcac tccagccacctgaagtccaaaaagggtcagtctacctcccgccataaaaaactcatgttcaagacagaagggcctga ctcagactctagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcgggaggaggtggttctagcact ccagaacaagccctgaagcaatataaacaccacctcactgcctatgagaaactggaaataattaattatccagaaat ttactttgtaggtccaaatgccaagaaaagacatggagttattggtggtcccaataatggagggtatgatgatgcagat ggggcctatattcatgtacctcgagaccatctagcttatcgatatgaggtgctgaaaattattggcaaggggagttttgg gcaggtggccagggtctatgatcacaaacttcgacagtacgtggccctaaaaatggtgcgcaatgagaagcgctttca tcgtcaagcagctgaggagatccggattttggagcatcttaagaaacaggataaaactggtagtatgaacgttatcca catgctggaaagtttcacattccggaaccatgtttgcatggcctttgaattgctgagcatagacctttatgagctgattaa aaaaaataagtttcagggttttagcgtccagttggtacgcaagtttgcccagtccatcttgcaatctttggatgccctcca caaaaataagattattcactgcgatctgaagccagaaaacattctcctgaaacaccacgggcgcagttcaaccaagg tcattgactttgggtccagctgtttcgagtaccagaagctctacacatatatccagtctcggttctacagagctccagaa atcatcttaggaagccgctacagcacaccaattgacatatggagttttggctgcatccttgcagaacttttaacaggac agcctctcttccctggagaggatgaaggagaccagttggcctgcatgatggagcttctagggatgccaccaccaaaac ttctggagcaatccaaacgtgccaagtactttattaattccaagggcataccccgctactgctctgtgactacccaggc agatgggagggttgtgcttgtggggggtcgctcacgtaggggtaaaaagcggggtcccccaggcagcaaagactggg ggacagcactgaaagggtgtgatgactacttgtttatagagttcttgaaaaggtgtcttcactgggacccctctgcccgc ttgaccccagctcaagcattaagacacccttggattagcaagtctgtctga
An example of an amino acid sequence of the p53-Mytl ι-3 s protein (SEQ ID NO:3), and its corresponding polynucleotide sequence (SEQ ID NO: 4) are as follows:
SEQ ID NO: 3
MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTED
PGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLH
SGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEV
VRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVWPYEPPEVGSDC
TTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEE
NLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELN
EALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDSSGGGGS
GGGGSGGGGSGGGGSRMLERPPALAMPMPTEGTPPPLSGTPIPVPAYFRHAEPGFSL
KRPRGLSRSLPPPPPAKGSIPISRLFPPRTPGWHQLQPRRVSFRGEASETLQSPGYDPS
RPESFFQQSFQRLSRLGHGSYGEVFKVRSKEDGRLYAVKRSMSPFRGPKDRARKLAE
VGSHEKVGQHPCCVRLEQAWEEGGILYLQTELCGPSLQQHCEAWGASLPEAQVWG
YLRDTLLALAHLHSQGLVHLDVKPANIFLGPRGRCKLGDFGLLVELGTAGAGEVQEG
DPRYMAPELLQGSYGTAADVFSLGLTILEVACNMELPHGGEGWQQLRQGYLPPEFTA
GLSSELRSVLVMMLEPDPKLRATAEALLALPVLRQPRAWGVLWCMAAEALSR
SEQ ID NO: 4 atggaggagccgcagtcagatcctagcgtcgagccccctctgagtcaggaaacattttcagacctatggaaactactt cctgaaaacaacgttctgtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaacaat ggttcactgaagacccaggtccagatgaagctcccagaatgccagaggctgctccccgcgtggcccctgcaccagca gctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagg gcagctacggtttccgtctgggcttcttgcattctgggacagccaagtctgtgacttgcacgtactcccctgccctcaaca agatgttttgccaactggccaagacctgccctgtgcagctgtgggttgattccacacccccgcccggcacccgcgtccg cgccatggccatctacaagcagtcacagcacatgacggaggttgtgaggcgctgcccccaccatgagcgctgctcag atagcgatggtctggcccctcctcagcatcttatccgagtggaaggaaatttgcgtgtggagtatttggatgacagaaa cacttttcgacatagtgtggtggtgccctatgagccgcctgaggttggctctgactgtaccaccatccactacaactaca tgtgtaacagttcctgcatgggcggcatgaaccggaggcccatcctcaccatcatcacactggaagactccagtggta atctactgggacggaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcgcacagaggaagagaatc tccgcaagaaaggggagcctcaccacgagctgcccccagggagcactaagcgagcactgcccaacaacaccagct cctctccccagccaaagaagaaaccactggatggagaatatttcacccttcagatccgtgggcgtgagcgcttcgaga tgttccgagagctgaatgaggccttggaactcaaggatgcccaggctgggaaggagccaggggggagcagggctcac tccagccacctgaagtccaaaaagggtcagtctacctcccgccataaaaaactcatgttcaagacagaagggcctga ctcagactctagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcgggaggaggtggttctagaat gctagaacggcctcctgcactggccatgcccatgcccacggagggcaccccgccacctctgagtggcacccccatcc cagtcccagcctacttccgccacgcagaacctggattctccctcaagaggcccagggggctcagccggagcctccca cctccgccccctgccaagggcagcattcccatcagccgcctcttccctcctcggaccccaggctggcaccagctgcag ccccggcgggtgtcattccggggcgaggcctcagagactctgcagagccctgggtatgacccaagccggccagagtc cttcttccagcagagcttccagaggctcagccgcctgggccatggctcctacggagaggtcttcaaggtgcgctccaag gaggacggccggctctatgcggtaaagcgttccatgtcaccattccggggccccaaggaccgggcccgcaagttggc cgaggtgggcagccacgagaaggtggggcagcacccatgctgcgtgcggctggagcaggcctgggaggagggcggc atcctgtacctgcagacggagctgtgcgggcccagcctgcagcaacactgtgaggcctggggtgccagcctgcctgag gcccaggtctggggctacctgcgggacacgctgcttgccctggcccatctgcacagccagggcctggtgcaccttgatg tcaagcctgccaacatcttcctggggccccggggccgctgcaagctgggtgacttcggactgctggtggagctgggtac agcaggagctggtgaggtccaggagggagacccccgctacatggcccccgagctgctgcagggctcctatgggacag cagcggatgtgttcagtctgggcctcaccatcctggaagtggcatgcaacatggagctgccccacggtggggagggct ggcagcagctgcgccagggctacctgccccctgagttcactgccggtctgtcttccgagctgcgttctgtccttgtcatga tgctggagccagaccccaagctgcgggccacggccgaggccctgctggcactgcctgtgttgaggcagccgcgggcct ggggtgtgctgtggtgcatggcagcggaggccctgagccgatga
An example of an amino acid sequence of the p53-SGK6o- 3i protein (SEQ ID NO: 5), and its corresponding polynucleotide sequence (SEQ ID NO: 6) are as follows:
SEQ ID NO: 5
MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTED
PGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLH
SGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEV
VRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVWPYEPPEVGSDC
TTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEE
NLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELN
EALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDSSGGGGS
GGGGSGGGGSGGGGSSISQPQEPELMNANPSPPPSPSQQINLGPSSNPHAKPSDFHF
LKVIGKGSFGKVLLARHKAEEVFYAVKVLQKKAILKKKEEKHIMSERNVLLKNVKHPF
LVGLHFSFQTADKLYFVLDYINGGELFYHLQRERCFLEPRARFYAAEIASALGYLHSLN
IVYRDLKPENILLDSQGHIVLTDFGLCKENIEHNSTTSTFCGTPEYLAPEVLHKQPYDRT
VDWWCLGAVLYEMLYGLPPFYSRNTAEMYDNILNKPLQLKPNITNSARHLLEGLLQKD
RTKRLGAKDDFMEIKSHVFFSLINWDDLINKKITPPFNPNVSGPNDLRHFDPEFTEEPV
PNSIGKSPDSVLVTASVKEAAEAFLGFSYAPPTDSFL
SEQ ID NO: 6 atggaggagccgcagtcagatcctagcgtcgagccccctctgagtcaggaaacattttcagacctatggaaactactt cctgaaaacaacgttctgtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaacaat ggttcactgaagacccaggtccagatgaagctcccagaatgccagaggctgctccccgcgtggcccctgcaccagca gctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagg gcagctacggtttccgtctgggcttcttgcattctgggacagccaagtctgtgacttgcacgtactcccctgccctcaaca agatgttttgccaactggccaagacctgccctgtgcagctgtgggttgattccacacccccgcccggcacccgcgtccg cgccatggccatctacaagcagtcacagcacatgacggaggttgtgaggcgctgcccccaccatgagcgctgctcag atagcgatggtctggcccctcctcagcatcttatccgagtggaaggaaatttgcgtgtggagtatttggatgacagaaa cacttttcgacatagtgtggtggtgccctatgagccgcctgaggttggctctgactgtaccaccatccactacaactaca tgtgtaacagttcctgcatgggcggcatgaaccggaggcccatcctcaccatcatcacactggaagactccagtggta atctactgggacggaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcgcacagaggaagagaatc tccgcaagaaaggggagcctcaccacgagctgcccccagggagcactaagcgagcactgcccaacaacaccagct cctctccccagccaaagaagaaaccactggatggagaatatttcacccttcagatccgtgggcgtgagcgcttcgaga tgttccgagagctgaatgaggccttggaactcaaggatgcccaggctgggaaggagccaggggggagcagggctcac tccagccacctgaagtccaaaaagggtcagtctacctcccgccataaaaaactcatgttcaagacagaagggcctga ctcagactctagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcgggaggaggtggttctagcatc tcccaacctcaggagcctgagcttatgaatgccaacccttctcctccaccaagtccttctcagcaaatcaaccttggcc cgtcgtccaatcctcatgctaaaccatctgactttcacttcttgaaagtgatcggaaagggcagttttggaaaggttcttc tagcaagacacaaggcagaagaagtgttctatgcagtcaaagttttacagaagaaagcaatcctgaaaaagaaaga ggagaagcatattatgtcggagcggaatgttctgttgaagaatgtgaagcaccctttcctggtgggccttcacttctcttt ccagactgctgacaaattgtactttgtcctagactacattaatggtggagagttgttctaccatctccagagggaacgct gcttcctggaaccacgggctcgtttctatgctgctgaaatagccagtgccttgggctacctgcattcactgaacatcgttt atagagacttaaaaccagagaatattttgctagattcacagggacacattgtccttactgacttcggactctgcaagga gaacattgaacacaacagcacaacatccaccttctgtggcacgccggagtatctcgcacctgaggtgcttcataagca gccttatgacaggactgtggactggtggtgcctgggagctgtcttgtatgagatgctgtatggcctgccgcctttttatag ccgaaacacagctgaaatgtacgacaacattctgaacaagcctctccagctgaaaccaaatattacaaattccgcaa gacacctcctggagggcctcctgcagaaggacaggacaaagcggctcggggccaaggatgacttcatggagattaa gagtcatgtcttcttctccttaattaactgggatgatctcattaataagaagattactcccccttttaacccaaatgtgagt gggcccaacgacctacggcactttgaccccgagtttaccgaagagcctgtccccaactccattggcaagtcccctgac agcgtcctcgtcacagccagcgtcaaggaagctgccgaggctttcctaggcttttcctatgcgcctcccacggactcttt cctctga
An example of amino acid sequence of the p53-Chkl ι-2 o protein (SEQ ID NO:7), and its corresponding polynucleotide sequence (SEQ ID NO:8) are as follows:
SEQ ID NO:7
MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTED
PGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLH
SGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEV
VRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVWPYEPPEVGSDC
TTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEE
NLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELN
EALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDSSGGGGS
GGGGSGGGGSGGGGSRMAVPFVEDWDLVQTLGEGAYGEVQLAVNRVTEEAVAVKI
VDMKRAVDCPENIKKEICINKMLNHENWKFYGHRREGNIQYLFLEYCSGGELFDRIE
PDIGMPEPDAQRFFHQLMAGWYLHGIGITHRDIKPENLLLDERDNLKISDFGLATVFR
YNNRERLLNKMCGTLPYVAPELLKRREFHAEPVDVWSCGIVLTAMLAGELPWDQPSD
SCQEYSDWKEKKTYLNPWKKIDSAPLALLHKILVENPSARITIPDIKKDRWYNKPLK
SEQ ID NO:8 atggaggagccgcagtcagatcctagcgtcgagccccctctgagtcaggaaacattttcagacctatggaaactactt cctgaaaacaacgttctgtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaacaat ggttcactgaagacccaggtccagatgaagctcccagaatgccagaggctgctccccgcgtggcccctgcaccagca gctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagg gcagctacggtttccgtctgggcttcttgcattctgggacagccaagtctgtgacttgcacgtactcccctgccctcaaca agatgttttgccaactggccaagacctgccctgtgcagctgtgggttgattccacacccccgcccggcacccgcgtccg cgccatggccatctacaagcagtcacagcacatgacggaggttgtgaggcgctgcccccaccatgagcgctgctcag atagcgatggtctggcccctcctcagcatcttatccgagtggaaggaaatttgcgtgtggagtatttggatgacagaaa cacttttcgacatagtgtggtggtgccctatgagccgcctgaggttggctctgactgtaccaccatccactacaactaca tgtgtaacagttcctgcatgggcggcatgaaccggaggcccatcctcaccatcatcacactggaagactccagtggta atctactgggacggaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcgcacagaggaagagaatc tccgcaagaaaggggagcctcaccacgagctgcccccagggagcactaagcgagcactgcccaacaacaccagct cctctccccagccaaagaagaaaccactggatggagaatatttcacccttcagatccgtgggcgtgagcgcttcgaga tgttccgagagctgaatgaggccttggaactcaaggatgcccaggctgggaaggagccaggggggagcagggctcac tccagccacctgaagtccaaaaagggtcagtctacctcccgccataaaaaactcatgttcaagacagaagggcctga ctcagactctagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcgggaggaggtggttctagaat ggcagtgccctttgtggaagactgggacttggtgcaaaccctgggagaaggtgcctatggagaagttcaacttgctgtg aatagagtaactgaagaagcagtcgcagtgaagattgtagatatgaagcgtgccgtagactgtccagaaaatattaa gaaagagatctgtatcaataaaatgctaaatcatgaaaatgtagtaaaattctatggtcacaggagagaaggcaata tccaatatttatttctggagtactgtagtggaggagagctttttgacagaatagagccagacataggcatgcctgaacc agatgctcagagattcttccatcaactcatggcaggggtggtttatctgcatggtattggaataactcacagggatatta aaccagaaaatcttctgttggatgaaagggataacctcaaaatctcagactttggcttggcaacagtatttcggtataa taatcgtgagcgtttgttgaacaagatgtgtggtactttaccatatgttgctccagaacttctgaagagaagagaatttc atgcagaaccagttgatgtttggtcctgtggaatagtacttactgcaatgctcgctggagaattgccatgggaccaacc cagtgacagctgtcaggagtattctgactggaaagaaaaaaaaacatacctcaacccttggaaaaaaatcgattctg ctcctctagctctgctgcataaaatcttagttgagaatccatcagcaagaattaccattccagacatcaaaaaagata gatggtacaacaaacccctcaagtga
Example 2. Use of P53-kinase dead mutant fusion protein to demonstrate that p53 is specifically phosphorylated by the kinase within the fusion protein, and use of Western Blotting to measure phosphorylation
The construct DNAs including pCI/p53 - wt ChklKD, pCI/p53 - D148A Chkl and pCI/p53 - D130A Chkl* were purified and transfected into COS-7 cells using Fugeneό (Roche diagnostics) for determination of phosphorylation on fusion protein and its specificity. After transfection, cells were incubated at 37 degrees/ 5% C02 for 24 hours then lysed in SDS sample buffer. Proteins were separated by SDS-PAGE then transferred onto a Nylon membrane. Phosphorylated p53 proteins were detected by anti-phospho Ser 15 p53 antibody
(Cell Signaling). The signal was visualized using a chemiluminescent detection kit (Amersha Bioscience). Constructs with kinase-dead mutant and mock (cells with no vectors added) showed no phosphorylation, however, constructs with the wild type showed phosphorylation showing that p53 is indeed specifically phosphorylated by the fused kinase partner. This experiment demonstrates that p53 is specifically phosphorylated by the kinase within the fusion protein.
(*)Chkl (D148A) and Chkl (D130A) are kinase-dead mutant (inactive kinase) of Chkl
Example 3. Expression of the p53-kinase fusion protein in the cell, and using p53 ELISA to measure the level of phosphorylation.
pCI expression vector (Control) or pCI/p53-Chkl 1-270 were transfected into COS-7 cells using Fugeneδ. Cells were incubated at 37 degrees in a 5% C02 atmosphere for 24 hours then lysed in a lisys buffer (137 mM NaCl with 2mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor cocktail (Roche), 0.18mg/ml sodium vanadate, and 20mM Tris-Cl (pH8.0)) on ice for 30mins. Cell lysates were transferred into anti-p53 monoclonal antibody (BD Transduction Laboratories) coated microtiter plate and incubated at 4 degrees over night. Phosphorylation on p53 protein was quantitatively measured by Rabit anti- phospho Ser 15 p53 polyclonal antibody (Cell signaling). Signal was detected using a HRP color detection kit (Pierce) after incubation with HRP-labeled anti-Rabit IgG antibody (Cell signaling). The absorbance was measured at 450 nm using a Victor multilabel counter (Wallac). See Figure 2 for result of ELISA.
Typical Compound assay
To evaluate an effect of an agent, cells transiently or stably transfected with the substrate-kinase fusion protein are incubated for few hours with a candidate agent. After incubation, the cells are lysed, and phosphorylation level of the fusion protein measured, preferably by ELISA or Western blotting.