Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1.
The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact.
Bogdan
J A
JA
DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, USA.
Adams-Burton
C
C
Pedicord
D L
DL
Sukovich
D A
DA
Benfield
P A
PA
Corjay
M H
MH
Stoltenborg
J K
JK
Dicker
I B
IB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D005095:Exoribonucleases; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D012260:Ribonucleases; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics
C489552:Btg1 protein, mouse; C489553:Btg1 protein, rat; C489780:CNOT8 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C074086:BTG1 protein, human; D012694:Serine; D016203:CDC2 Protein Kinase; C489778:Cnot7 protein, mouse; D005095:Exoribonucleases; D012260:Ribonucleases; C079009:POP2 protein, S cerevisiae