Src family tyrosine kinases are involved in modulating various signal transduction pathways leading to the induction of DNA synthesis and cytoskeletal reorganization in response to cell-cell or cell-matrix adhesion. The critical role of these kinases in regulating cellular signaling pathways requires that their activity be tightly controlled. Src family proteins are regulated through reversible phosphorylation and dephosphorylation events that alter the conformation of the kinase. We have found evidence that Src also is regulated by ubiquitination. Activated forms of Src are less stable than either wild-type or kinase-inactive Src mutants and can be stabilized by proteasome inhibitors. In addition, poly-ubiquitinated forms of active Src have been detected in vivo. Taken together, our results establish ubiquitin-mediated proteolysis as a previously unidentified mechanism for irreversibly attenuating the effects of active Src kinase.

E2F activity is regulated in part by the retinoblastoma family of tumor suppressor proteins. Viral oncoproteins, such as simian virus 40 (SV40) large-T antigen (TAg), adenovirus E1A, and human papillomavirus E7, can disrupt the regulation of cellular proliferation by binding to pRb family members and dissociating E2F-pRb family protein complexes. BK virus (BKV), which infects a large percentage of the human population and has been associated with a variety of human tumors, encodes a TAg homologous to SV40 TAg. It has been shown that BKV TAg, when expressed at low levels, does not detectably bind to pRb family members, yet it induces a serum-independent phenotype and causes a decrease in the overall levels of pRb family proteins. The experiments presented in this report show that, despite the lack of TAg-pRb interactions, BKV TAg can induce transcriptionally active E2F and that this induction does in fact require an intact pRb-binding domain as well as an intact J domain. In addition, E2F-pRb family member complexes can be detected in both BKV and SV40 TAg-expressing cells. These results suggest the presence of alternate cellular mechanisms for the release of E2F in addition to the well-established model for TAg-pRb interactions. These results also emphasize a role for BKV TAg in the deregulation of cellular proliferation, which may ultimately contribute to neoplasia.Cell cycle progression is a tightly controlled process that involves the interactions of a complex network of proteins, including the members of the retinoblastoma family of tumor suppressor proteins and the E2F family of transcription factors. The retinoblastoma family includes the retinoblastoma susceptibility protein, pRb, and the related proteins p107 and p130. Overexpression of any of these three proteins can induce growth arrest, implicating this family of proteins as negative regulators of cell growth (32,70,92,96,99). Overexpression of E2F can induce quiescent cells to enter the S phase, implicating this family of proteins as positive regulators of cell growth (48). Further experiments have shown that ectopic expression of E2F in cells arrested by overexpression of pRb is sufficient to relieve the growth arrest phenotype (78, 99). In addition, E2F-pRb complexes have been shown to bind to and repress E2F-responsive promoters (see reference 94 for a review). The cell must therefore maintain a delicate balance between the active forms of these and other cell cycle regulators in order to maintain proliferative control. When pRb, E2F, and other key regulatory proteins are mutated or when their expression is altered, regulation is lost and cell proliferation can proceed unchecked. Evidence supporting the importance of these proteins in maintaining cell growth control comes from the discovery that many human cancers in addition to retinoblastoma have inactivating mutations in the retinoblastoma susceptibility gene, RB1, or in other genes whose products are involved in the pRb pathway (36,43,44,56,57,59,91).The current model for pRb regulation of the...

BK virus (BKV) is a polyomavirus which infects a large percentage of the human population. It is a potent transforming agent and is tumorigenic in rodents. BKV DNA has also been found in human brain, pancreatic islet, and urinary tract tumors, implicating this virus in neoplastic processes. BKV T antigen (TAg) is highly homologous to simian virus 40 TAg, particularly in regions required for mitogenic stimulation and binding to tumor suppressor proteins. The experiments presented in this report show that BKV TAg can bind the tumor suppressor protein p53. BKV TAg also has the ability to bind to members of the retinoblastoma (pRb) family of tumor suppressor proteins both in vivo and in vitro. However, these interactions are detected only when large amounts of total protein are used, because the levels of BKV TAg normally produced from viral promoterenhancer elements are too low to bind a significant amount of the pRb family proteins in the cell. The low levels of BKV TAg produced by the viral promoter elements are sufficient to affect the levels and the phosphorylation patterns of these proteins and to induce serum-independent growth in these cells. Additional events, however, are required for full transformation. These data further support the notion that BKV TAg can affect cellular growth control mechanisms and may in fact be involved in neoplastic processes.