Human cyclin-dependent kinase (hPFTAIRE)

Abstract

A human gene encoding a novel cyclin-dependent kinase termed hPFTAIRE and its expression products can be used to provide reagents and methods for detecting migrating or metastasizing cells. Compositions and methods for treating proliferative disorders and neoplasia are also provided.

1 . An isolated protein comprising an amino acid sequence which is at least 94% identical to the amino acid sequence shown in SEQ ID NO:2, wherein percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.  
     
     
         2 . The isolated protein of  claim 1  which has the amino acid sequence shown in SEQ ID NO:2.  
     
     
         3 . The isolated protein of  claim 1  which cannot be phosphorylated.  
     
     
         4 . The isolated protein of  claim 3  which comprises an amino acid substitution relative to the amino acid sequence shown in SEQ ID NO:2, wherein the amino acid substitution is selected from the group consisting of a histidine for the serine at position 99, a phenylalanine for the serine at position 99, a glutamine for the threonine at position 98, and an alanine for the threonine at position 98.  
     
     
         5 . The isolated protein of  claim 1  wherein a kinase activity of the protein is reduced relative to kinase activity of a protein having the amino acid sequence shown in SEQ ID NO:2.  
     
     
         6 . The isolated protein of  claim 5  wherein an aspartic acid at position 227 is replaced by an asparagine.  
     
     
         7 . The isolated protein of  claim 1  wherein a cyclin binding domain comprising the sequence PFTAIRE (SEQ ID NO:5) is replaced by a domain selected from the group consisting of NRTALRE (SEQ ID NO:6), PISSLRE (SEQ ID NO:7), PITALRE (SEQ ID NO:8), PLSTIRE (SEQ ID NO:9), PISTVRE (SEQ ID NO:10), PSTAIRE (SEQ ID NO:11), and PNQALRE (SEQ ID NO:12).  
     
     
         8 . An isolated polypeptide comprising at least 218 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2.  
     
     
         9 . The isolated polypeptide of  claim 8  wherein the 218 contiguous amino acids of SEQ ID NO:2 comprise amino acids 139-141 of SEQ ID NO:2.  
     
     
         10 . The isolated polypeptide of  claim 8  which comprises an amino acid substitution relative to the amino acid sequence shown in SEQ ID NO:2, wherein the amino acid substitution is selected from the group consisting of a histidine for the serine at position 99, a phenylalanine for the serine at position 99, a glutamine for the threonine at position 98, an alanine for the threonine at position 98, and an arginine at position 227.  
     
     
         11 . A fusion protein comprising a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment consists of at least 218 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2.  
     
     
         12 . The fusion protein of  claim 11  wherein the first protein segment comprises amino acids 44-51 of SEQ ID NO:2.  
     
     
         13 . The fusion protein of  claim 11  wherein the first protein segment comprises an amino acid substitution relative to the amino acid sequence shown in SEQ ID NO:2, wherein the amino acid substitution is selected from the group consisting of a histidine for the serine at position 99, a phenylalanine for the serine at position 99, a glutamine for the threonine at position 98, an alanine for the threonine at position 98, and an asparagine at position 227.  
     
     
         14 . A cDNA molecule which encodes a protein comprising an amino acid sequence which is at least 94% identical to the amino acid sequence shown in SEQ ID NO:2, wherein percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.  
     
     
         15 . A cDNA molecule which encodes at least 218 contiguous amino acids of SEQ ID NO:2.  
     
     
         16 . The cDNA molecule of  claim 15  which encodes SEQ ID NO:2.  
     
     
         17 . The cDNA molecule of  claim 15  which comprises SEQ ID NO:1.  
     
     
         18 . A cDNA molecule which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:1, wherein percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.  
     
     
         19 . An isolated and purified subgenomic polynucleotide comprising a nucleotide sequence which hybridizes to SEQ ID NO:1 after washing with 0.2×SSC at 65° C., wherein the nucleotide sequence encodes an hPFTAIRE protein having the amino acid sequence of SEQ ID NO:2.  
     
     
         20 . A construct comprising: 
 a promoter; and    a polynucleotide segment encoding at least 218 contiguous amino acids of an hPFTAIRE protein as shown in SEQ ID NO:2, wherein the polynucleotide segment is located downstream from the promoter, wherein transcription of the polynucleotide segment initiates at the promoter.    
     
     
         21 . A host cell comprising a construct which comprises: 
 a promoter and:    a polynucleotide segment encoding at least 218 contiguous amino acids of an hPFTAIRE protein having an amino acid sequence as shown in SEQ ID NO:2.    
     
     
         22 . A homologously recombinant cell having incorporated therein a new transcription initiation unit, wherein the new transcription initiation unit comprises: 
 (a) an exogenous regulatory sequence;    (b) an exogenous exon; and    (c) a splice donor site, wherein the new transcription initiation unit is located upstream of a coding sequence of an hPFTAIRE gene, wherein the hPFTAIRE gene comprises the coding sequence shown in SEQ ID NO:1, wherein the exogenous regulatory sequence directs transcription of the coding sequence of the hPFTAIRE gene.    
     
     
         23 . The homologously recombinant cell of  claim 22  wherein the homologously recombinant cell is a neoplastic cell.  
     
     
         24 . A polynucleotide probe comprising at least 12 contiguous nucleotides of SEQ ID NO:1.  
     
     
         25 . The polynucleotide probe of  claim 24  which comprises a detectable label.  
     
     
         26 . A method of detecting migrating cells in a body sample of a human, comprising the step of assaying the body sample for the presence of an expression product of a gene comprising the coding sequence shown in SEQ ID NO:1, wherein the presence of the expression product indicates that the body sample comprises migrating cells.  
     
     
         27 . The method of  claim 26  wherein the body sample is a tumor.  
     
     
         28 . The method of  claim 27  wherein the body sample is a metastatic lesion.  
     
     
         29 . The method of  claim 26  wherein the expression product is protein.  
     
     
         30 . The method of  claim 26  wherein the expression product is mRNA.  
     
     
         31 . A method of diagnosing or prognosing neoplasia, comprising the step of: 
 comparing expression of a first hPFTAIRE gene in a first tissue suspected of being neoplastic with expression of a second hPFTAIRE gene in a second tissue which is normal, wherein the second hPFTAIRE gene comprises the coding sequence shown in SEQ ID NO:1, wherein over-expression of the first hPFTAIRE gene relative to the second hPFTAIRE gene indicates neoplasia in the first tissue.    
     
     
         32 . The method of  claim 31  wherein levels of hPFTAIRE mRNA in the first and second tissues are compared.  
     
     
         33 . The method of  claim 31  wherein levels of hPFTAIRE protein in the first and second tissues are compared.  
     
     
         34 . A method of identifying an agent which alters mitosis, comprising the steps of: 
 contacting a cell with a test compound; and    measuring expression of an hPFTAIRE gene, wherein the hPFTAIRE gene comprises the coding sequence shown in SEQ ID NO:1, wherein a test compound which increases expression of the hPFTAIRE gene is identified as a potential agent for inducing mitosis, and wherein a test compound which decreases expression of the hPFTAIRE gene is identified as a potential agent for decreasing mitosis.