The Molecular Chaperone Activity of Simian Virus 40 Large T Antigen Is Required To Disrupt Rb-E2F Family Complexes by an ATP-Dependent Mechanism

The tumor suppressor proteins retinoblastoma protein (pRb) and p53 have been the focus of intense study due to their crucial role in the regulation of normal cellular growth (reviewed in references 14 and 28). T antigen can inactivate both p53 and pRb and has been used as a tool to disable these cellular signaling pathways (3, 10, 17, 22). The Rb family is composed of at least three proteins, pRb, p107, and p130, which are critical for cell cycle regulation and are thought to have overlapping functions in different stages of the cell cycle (31). pRb, p107, and p130 proteins bind to the E2F family of transcription factors. When an Rb family member binds to E2F, E2F-mediated transactivation is inhibited, and expression of E2F-responsive genes, such as cyclins A and E and dihydrofolate reductase, is decreased (1, 12, 24). When phosphorylated, pRb dissociates from E2F, inducing DNA synthesis and E2F-mediated gene expression, leading to advancement of the cell cycle (9, 46). T antigen binds to the pRb proteins through an LXCXE motif (15) (see Fig. ​Fig.1A1A for T-antigen domain map). This sequence is conserved in many pRb binding proteins, including cellular proteins such as BRG1 (13) and those expressed by other small DNA tumor viruses, such as E1A (adenovirus) and E7 (papillomavirus) (11, 15, 25). Mutation of the LXCXE motif renders T antigen defective for transformation (36, 42, 48). This suggests that T antigen's affinity for Rb family members displaces Rb from binding E2F, thus inducing transformation. However, the binding of pRb family members by T antigen is not sufficient to induce transformation (42). FIG. 1 Structure of SV40 large T antigen. (A) T antigen comprises multiple domains, some of which are represented here as shaded boxes. The first 82 amino acids of T antigen have been shown to be a functional J domain that can bind to the DnaK homologue Hsc70. ... Another region of T antigen required for transformation is the J domain (Fig. ​(Fig.1A)1A) (for a review, see reference 2). J domains are a hallmark of a class of chaperones called J proteins or DnaJ chaperones that bear homology to Escherichia coli DnaJ. J proteins interact directly with DnaK-like proteins (such as Hsc70) and stimulate their ATPase activity. Stimulation of the ATPase activity of DnaK regulates its interaction with bound substrates (20). J proteins and DnaK-like chaperones are involved in numerous biological activities, including protein folding, protein transport across cellular membranes, protein degradation, and rearrangement of multiprotein complexes (4). We have previously shown that T antigen requires a functional J domain and Rb binding motif in cis to transform REF52 and C3H10T1/2 cells (42). Both the J domain and pRb binding motif are also required for T-antigen-mediated inhibition of apoptosis (40). Furthermore, both the J domain and pRb binding motif are required for T antigen to alleviate pRb-mediated inhibition of E2F transactivation (19, 39, 47). Finally, cells expressing a mutant of T antigen defective for J domain or pRb binding functions contain a p130/E2F DNA-binding complex that is absent in cells expressing wild-type T antigen (47). Thus, it is likely that the J domain activity cooperates with the LXCXE motif of T antigen to disable Rb function, although the mechanism for how this occurs remains undetermined. This study seeks to better understand the mechanism by which T antigen inactivates the function of the Rb family of proteins. Specifically we show that T antigen requires its J domain function to cooperate with Hsc70 to release “free” E2F from association with Rb family members. The implications of these findings are discussed.