J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen

Polyomaviruses (Pys) have provided valuable models because the cellular pathways they use are generally important for cell function. The multifunctional large T antigens (LTs) represent one example. Their most obvious role is in DNA replication. Murine PyLT functions in initiation (21) of viral DNA synthesis, but it is also responsible for integration (15) and excision (4) of the viral genome. Biochemical studies on simian virus 40 (SV40) LT have provided important clues about basic cellular processes such as DNA replication (9, 78). LT's broad effects on the host cell led to the discovery of p53 (40, 46) and to considerable insight into the Rb tumor suppressor and E2F transcription factor families (47). The effects of murine PyLT on the host cell can be viewed from a biological or a mechanistic perspective. LT induces cellular DNA synthesis (24, 66) and can immortalize primary cells (3, 58). LT can also block the differentiation of either myoblasts (51) or preadipocytes (14). It has the ability to promote apoptosis (20). Finally, LT is a transactivator of cellular genes (39, 54, 55). Much of what LT does to cells depends upon its interactions with the Rb tumor suppressor family through an LXCXE motif starting at residue 141 (17, 32). Immortalization (22, 34, 41), induction of cell DNA synthesis (70), overcoming p53-induced cell cycle arrest (16), and prevention of differentiation (52) can all depend on Rb family binding. Transactivation of the thymidine kinase, polymerase α, PCNA, dihydrofolate reductase, and thymidylate synthase genes (54) as well as regulation of interferon-inducible gene expression (80) also requires an intact pRb/p107 binding site on LT and is mediated via the cellular transcription factor E2F. A major shift in the perception of how LT regulates Rb family function began with the realization that the highly conserved N terminus (56) of all the Py T antigens looks like a DnaJ domain (12, 38). DnaJs bind to and stimulate the activity of DnaK proteins (44, 53). Subsequent biochemical studies (61, 72) and genetic domain swapping experiments (10, 37) confirmed the T antigen/J domain connection. J domains make multiple contributions to LT function. For SV40 they contribute, for example, to DNA replication (10). However, the most striking aspect of their function was that mutation of the J domain prevented its interaction with hsc70, a DnaK, thereby blocking LT functions related to Rb family member binding (27, 70, 72, 86). Since many of the effects of the Rb family are mediated through interactions with the E2F family, these results have been interpreted in terms of effects on protein complexes containing Rb and E2F family members. The Rb family is, however, clearly multifunctional and can work independently of E2F. Studies of transgenic mice point to additional roles in development (76). A mutant pRb that does not bind to E2F or oncoproteins can still induce a flat-cell phenotype in Saos-2 cells and retains the ability to collaborate with MyoD in the differentiation of myoblasts (68). The minimal growth suppression domain of pRb contains at least three independent protein-binding functions (79). In addition to the A/B “pocket” domain and the E2F binding site, Rb has a C pocket domain required for interaction with MDM2 and c-Abl (82, 83) that is not found in p130 or p107. An N-terminally truncated pRb fails to rescue pRb−/− mice from embryonic lethality, indicating that the N-terminal sequences of pRb may also be important for pRb function (60). The work presented here extends our analysis of the interaction between PyLT and the Rb family. Our previous work demonstrated that DnaJ was required for LT to affect Rb functions related to cell cycle progression or activation of promoters containing E2F sites. This work characterizes LT effects on the Rb family related to apoptosis and release of promoter repression that are largely independent of a functional DnaJ domain.