Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.The retinoblastoma tumor suppressor gene (Rb-1) is mutated in all cases of retinoblastoma and is also frequently mutated in a variety of other cancers (55,91,92). Loss of the antiproliferative activity of the RB protein (pRB) is thought to play a critical role in oncogenic transformation. There are two other members of the RB family of proteins, p107 and p130. These proteins have a high degree of sequence homology to pRB (31,38,56,62). pRB, p107, and p130 share a number of functional properties, including the ability to associate with and negatively regulate members of the E2F family of transcription factors (3,6,8,36,42,78,89) and the ability to induce a G 1 arrest in certain sensitive cell types (16,43,103). The presence of a p130-E2F complex has been proposed to define a quiescent, or G 0 , state of a cell (80). pRB, p107, and p130 are phosphorylated in a cell cycle-dependent manner (2,4,10,21,61,96). Phosphorylation in the mid/late G 1 phase inactivates the growth-suppressive properties of pRB and possibly also of p107 and p130 (15,43,103). pRB, p107, and p130 associate with cyclins (28,30,32,38,47,54,56,78) and are likely to be phosphorylated by cyclin-dependent kinases (cdks). Despite all these similarities, no mutations in p107 or p130 have yet been described in human tumors, and the role of these two proteins in oncogenic transformation remains unclear. pRB appears to be an essential protein in mouse embryonic development, as mice with homozygous deletions of the Rb-1 gene die before birth (14, 46, 52). Moreover, mice heterozygous for Rb-1 develop pituitary tumors with a penetrance of almost 100% (40,45,46). In contrast, mice lacking either p107 or p130 apparently develop norm...

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage g in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G 1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.Simian virus 40 (SV40) large T antigen (TAg) can transform a variety of cell types. Manifestations of the transformed phenotype include cell immortalization, growth to a high density, reduced requirement for serum, anchorage independence, and the ability to form tumors in various animal models. TAg achieves this transformation by targeting negative regulators of cell growth, including p53 and the RB family (pRB, p107, and p130). p53 and pRB are well-established tumor suppressor proteins, and their corresponding genes are commonly lost or mutated in human cancer. While p107 and p130 inactivation likely contributes to TAg-mediated transformation (see below), there is little evidence that these proteins are tumor suppressors. However, loss of p130 was recently reported in a human lung cancer cell line (12).TAg contains at least three transforming domains. A Cterminal domain extending from approximately residue 350 to residue 550 binds to and inactivates p53 (22,31,42,52). The LXCXE domain (residues 103 to 107) mediates binding to the retinoblastoma family proteins pRB, p107, and p130 (5,6,9,19,51). Mutations within TAg that disrupt binding to p53 or pRB render it unable to fully transform cells (46). An intact LXCXE domain is required for TAg to transform fibroblasts derived from Rb-1 knockout mice (3, 51). This result implies that p130 and p107, in addition to pRB, are likely to be relevant targets of TAg during the transforming process. In addition to the LXCXE and p53 binding domains, the N-terminal 82 residues of TAg, encoded by the first exon and shared with small t antigen, are required for transformation (28). Until recently, the mechanism by which the N terminus contributes to transformation was unknown.The N terminus of TAg shares sequence homology with the J ...