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.[1]References
- Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1. Bogdan, J.A., Adams-Burton, C., Pedicord, D.L., Sukovich, D.A., Benfield, P.A., Corjay, M.H., Stoltenborg, J.K., Dicker, I.B. Biochem. J. (1998) [Pubmed]
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