Ubiquitin ligases, and uses related thereto

Abstract

The present invention relates to the discovery in eukaryotic cells of a ubiquitin ligases. These proteins are referred to herein collectively as “pub” proteins for Protein UBiquitin ligase, and individually as h-pub1, h-pub2 and s-pub1 for the human pub1 and pub2 and Schizosaccharomyces pombe pub1 clones, respectively. Pub1 proteins apparently play a role in the ubiquitination of the mitotic activating tyrosine phosphatase cdc25, and thus they may regulate the progression of proliferation in eukaryotic cells by activating the cyclin dependent kinase complexes. In S. pombe , disruption of s-pub1 elevates the level of cdc25 protein in vivo increasing the activity of the tyrosine kinases, wee1 and mik1, required to arrest the cell-cycle. Loss of weel function in an S. pombe cell carrying a disruption in the s-pub1 gene results in a lethal premature entry into mitosis; such lethal phenotype can be rescued by the loss of cdc25 function. An ubiquitin thioester adduct of s-pub1 can be isolated from S. pombe and disruption of s-pub1 dramatically reduces ubiquitination of cdc25.

We claim:  
     
         1 . An isolated and/or recombinant pub polypeptide, which polypeptide comprises an amino acid sequence identical or homologous to the amino acid sequence designated by one or more of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6, or a fragment thereof.  
     
     
         2 . The pub polypeptide of  claim 1 , which polypeptide affects cell-cycle progression of eukaryotic cells.  
     
     
         3 . The pub polypeptide of  claim 1 , which polypeptide possesses a ubiquitin ligase activity.  
     
     
         4 . The pub polypeptide of  claim 3 , which polypeptide ubiquitinates cdc25.  
     
     
         5 . The pub polypeptide of  claim 1 , which polypeptide is at least 75% homologous to amino acid sequence designated by SEQ ID NO. 2.  
     
     
         6 . The pub polypeptide of  claim 1 , which polypeptide is at least 75% homologous to amino acid sequence designated by SEQ ID NO. 4.  
     
     
         7 . The pub polypeptide of  claim 1 , which polypeptide is at least 75% homologous to amino acid sequence designated by SEQ ID NO. 6.  
     
     
         8 . The pub polypeptide of  claim 1 , which polypeptide is a fusion protein.  
     
     
         9 . The pub polypeptide of  claim 1 , which polypeptide is of mammalian origin.  
     
     
         10 . The pub polypeptide of  claim 9 , which polypeptide is of human origin.  
     
     
         11 . The pub polypeptide of  claim 1 , which polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions to at least a portion of the nucleic acid designated by SEQ ID No. 1 corresponding to a catalytic domain, a calcium lipid binding domain, or both.  
     
     
         12 . The pub polypeptide of  claim 1 , which polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions to at least a portion of the nucleic acid designated by SEQ ID No. 3 corresponding to a catalytic domain, a calcium lipid binding domain, or both.  
     
     
         13 . The pub polypeptide of  claim 1 , which polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions to at least a portion of the nucleic acid designated by SEQ ID No. 5 corresponding to a catalytic domain.  
     
     
         14 . A isolated nucleic acid comprising a nucleotide sequence encoding a pub polypeptide, or a nucleotide sequence complementary thereto, said pub polypeptide including an amino acid sequence identical or homologous to the amino acid sequence designated by one or more of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6, or a portion thereof.  
     
     
         15 . The nucleic acid of  claim 14 , wherein said pub polypeptide possesses a ubiquitin ligase activity.  
     
     
         16 . The nucleic acid of  claim 14 , wherein said pub polypeptide possesses a calcium binding motif.  
     
     
         17 . The nucleic acid of  claim 14 , wherein said pub polypeptide ubiquitinates cdc25.  
     
     
         18 . The nucleic acid of  claim 14 , wherein said pub polypeptide encoding nucleotide sequence is at least 75% homologous to amino acid sequence designated by SEQ ID NO. 1.  
     
     
         19 . The nucleic acid of  claim 14 , wherein said pub polypeptide encoding nucleotide sequence is at least 75% homologous to amino acid sequence designated by SEQ ID NO. 3.  
     
     
         20 . The nucleic acid of  claim 14 , wherein said pub polypeptide encoding nucleotide sequence is at least 75% homologous to amino acid sequence designated by SEQ ID NO. 5.  
     
     
         21 . The nucleic acid of  claim 14 , wherein said pub polypeptide is a fusion protein.  
     
     
         22 . The nucleic acid of  claim 14 , which nucleic acid hybridizes under stringent conditions to a nucleic acid probe having a sequence represented by at least 60 consecutive nucleotides of SEQ ID No. 1, or a sequence complementary thereto.  
     
     
         23 . The nucleic acid of  claim 14 , wherein said pub polypeptide encoding nucleotide sequence hybridizes under stringent conditions to a nucleic acid probe having a sequence represented by at least 60 consecutive nucleotides of SEQ ID No. 1, 3 or 5; or a sequence complementary thereto.  
     
     
         24 . The nucleic acid of  claim 14 , further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector.  
     
     
         25 . An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of  claim 24 .  
     
     
         26 . A host cell transfected with the expression vector of  claim 25  and expressing said recombinant polypeptide.  
     
     
         27 . A method of producing a recombinant pub polypeptide comprising culturing the cell of  claim 26  in a cell culture medium to express said recombinant polypeptide and isolating said recombinant polypeptide from said cell culture.  
     
     
         28 . A transgenic animal having cells which harbor a transgene comprising the nucleic acid of  claim 14 , or in which a gene comprising said nucleic acid is disrupted.  
     
     
         29 . Isolated nucleic acid which selectively hybridizes under high stringency conditions to at least ten nucleotides of a nucleic acid sequence represented by one of SEQ ID Nos. 1, 3 or 5, or complementary sequences thereof, which nucleic acid can specifically detect or amplify a nucleic acid sequence of a pub gene.  
     
     
         30 . The nucleic acid of  claim 29 , which is nucleic acid is labelled.  
     
     
         31 . An assay for identifying an agent which modulates ubiquitination of a protein, comprising: 
 (i) providing a ubiquitin-conjugating system which comprises a pub ligase, a substrate protein which undergoes ubiquitination by the pub ligase, and ubiquitin, under conditions which promote pub-dependent ubiquitination of the substrate protein;    (ii) contacting the ubiquitin-conjugating system with a candidate agent;    (iii) measuring a level of ubiquitination of the substrate protein in the presence of the candidate agent; and    (iv) comparing the measured level of ubiquitination in the presence of the candidate agent with a level of ubiquitination of the substrate protein in the absence of the candidate agent,    wherein a statistically significant change in ubiquitination of the substrate protein in the presence of the candidate agent is indicative of an agent which modulates ubiquitination of the substrate protein.    
     
     
         32 . The assay of  claim 31 , wherein the ubiquitin-conjugating system is a reconstituted protein mixture.  
     
     
         33 . The assay of  claim 31 , wherein the ubiquitin-conjugating system is a cell lysate.  
     
     
         34 . The assay of  claim 31 , wherein the ubiquitin-conjugating system is a cell expressing a recombinant pub ligase.  
     
     
         35 . The assay of  claim 31 , wherein the pub ligase is a mammalian pub protein.  
     
     
         36 . The assay of  claim 31 , wherein the pub ligase is a recombinant polypeptide.  
     
     
         37 . The assay of  claim 31 , wherein the substrate protein is a cdc25 phosphatase.  
     
     
         38 . The assay of  claim 31 , wherein the ubiquitin is provided in a form selected from a group consisting of: 
 (i) an unconjugated ubiquitin, in which case the ubiquitin-conjugating system further comprises an E1 ubiquitin-activating enzyme (E1), an E2 ubiquitin-conjugating enzyme (E2), and adenosine triphosphate;    (ii) an activated E1:ubiquitin complex, in which case the ubiquitin-conjugating system further comprises an E2;    (iii) an activated E2:ubiquitin complex; and    (iv) an activated pub:ubiquitin complex.    
     
     
         39 . An assay for identifying an agent which competitively inhibits binding of a pub ubiquitinating complex with a protein, comprising: 
 (i) forming a mixture comprising a pub polypeptide, a substrate protein which undergoes ubiquitination by the pub polypeptide, and a candidate agent;    (ii) measuring a level of binding between the substrate protein and the pub polypeptide in the presence of the candidate agent; and    (iv) comparing the measured level of binding in the presence of the candidate agent with a level of binding of the substrate protein to the pub polypeptide in the absence of the candidate agent,    wherein a statistically significant decrease in binding of the substrate protein to the pub polypeptide in the presence of the candidate agent is indicative of an agent which competitively inhibits binding of a pub polypeptide with the substrate protein.