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. 1998 Dec 1;336 ( Pt 2)(Pt 2):471-81. doi: 10.1042/bj3360471.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • CDC2 Protein Kinase / metabolism
  • Cell Division / drug effects
  • Cloning, Molecular
  • Exoribonucleases
  • HeLa Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / metabolism
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Proteins*
  • Rats
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Repressor Proteins
  • Ribonucleases*
  • Saccharomyces cerevisiae Proteins*
  • Serine / metabolism
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism*
  • Yeasts / genetics

Substances

  • Btg1 protein, mouse
  • Btg1 protein, rat
  • CNOT8 protein, human
  • Neoplasm Proteins
  • Nuclear Proteins
  • Proteins
  • Recombinant Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • BTG1 protein, human
  • Serine
  • CDC2 Protein Kinase
  • Cnot7 protein, mouse
  • Exoribonucleases
  • Ribonucleases
  • POP2 protein, S cerevisiae

Associated data

  • GENBANK/L46722