SV40 large T antigen promotes dephosphorylation of p130

SV40 large T antigen (Ag) binds to all members of the retinoblastoma (RB) tumor suppressor family including pRb, p107, and p130. Although the LXCXE motif of T Ag binds directly to the RB proteins, it is not sufficient to fully inactivate their function. The N-terminal DNA J domain of T Ag cooperates with the LXCXE motif to override RB-mediated repression of E2F-dependent transcription. In addition, T Ag can reduce the overall phosphorylation state of p107 and p130 that is dependent on an intact J domain and LXCXE motif. However, the mechanism of this activity has not been described. Here we describe the use of a cell-free system to characterize the effect of T Ag on p130 phosphorylation. When incubated in extracts prepared from S phase cells, p130 undergoes specific phosphorylation. Addition of T Ag to S phase extracts leads to a reduction of p130 phosphorylation in vitro. The ability of T Ag to reduce the phosphorylation of p130 in vitro is dependent on an intact DNA J domain and can be inhibited by okadaic acid and PP2A-specific inhibitors. These results suggest that T Ag recruits a phosphatase activity in a DNA J domain-dependent manner to reduce the phosphorylation of p130.