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 becau se its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this stu dy, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-a ssociative factor 1 (hCAF-1), a homologue of mouse CAF-I (mCAF-1) and Sacch aromyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34(cdc2) kinase site o n 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 t he cell cycle kinases p34(CDK2)/cyclin E and p34(CDK2)/cyclin A, but not fo r p34(CDK4)/cyclin D1 Or P34(cdc2)/cyclin B. Cell synchrony experiments wit h primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated t hat message and protein levels of rat CAF-I (rCAF-1) were up-regulated unde r conditions of cell contact, as previously reported for BTG1 [Wilcox, Scot t, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulati on 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was ph ysically 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 divi sion that lead to changes in cellular proliferation associated with cell-ce ll contact.