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.
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December 1998
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Research Article|
December 01 1998
Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1
John A. BOGDAN;
John A. BOGDAN
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Catherine ADAMS-BURTON;
Catherine ADAMS-BURTON
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Donna L. PEDICORD;
Donna L. PEDICORD
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Drew A. SUKOVICH;
Drew A. SUKOVICH
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Pamela A. BENFIELD;
Pamela A. BENFIELD
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Martha H. CORJAY;
Martha H. CORJAY
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Janet K. STOLTENBORG;
Janet K. STOLTENBORG
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
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Ira B. DICKER
Ira B. DICKER
1
1DuPont Pharmaceuticals Company, Experimental Station E400-3231, Wilmington, DE 19880-0400, U.S.A.
1To whom correspondence should be addressed (e-mail Dickerib@a1.lldmpc.umc.dupont.com).
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Publisher: Portland Press Ltd
Received:
February 20 1998
Revision Received:
July 16 1998
Accepted:
September 17 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 336 (2): 471–481.
Article history
Received:
February 20 1998
Revision Received:
July 16 1998
Accepted:
September 17 1998
Citation
John A. BOGDAN, Catherine ADAMS-BURTON, Donna L. PEDICORD, Drew A. SUKOVICH, Pamela A. BENFIELD, Martha H. CORJAY, Janet K. STOLTENBORG, Ira B. DICKER; Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1. Biochem J 1 December 1998; 336 (2): 471–481. doi: https://doi.org/10.1042/bj3360471
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