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

Abstract

The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.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. 1A 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).Another region of T antigen required for transformation is the J domain (Fig. 1A) (for a review, see reference 2). J domains are a hallmark of a class of chaperones called J proteins or D...

Transforming functions of Simian Virus 40

“…One model is that T antigen ®rst binds to Rb/E2F complexes through its LXCXE motif and then these complexes are dissolved using energy derived from hsc70-mediated ATP hydrolysis (Srinivasan et al, 1997). In fact, puri®ed T antigen has been shown to bind p130/E2F-4 and pRb/E2F-4 complexes in vitro and the dissolution of these complexes requires ATP hydrolysis by hsc70 (Sullivan et al, 2000a). This explains why the J domain is required in addition to the LXCXE motif to e ect E2F transcriptional activation.…”

Interaction of retinoblastoma protein family members with large T-antigen of primate polyomaviruses

“…This dissociation requires Hsc70, ATP and a functional J domain (Sullivan et al, 2000a). The mechanism of T-antigen J-domain-mediated inhibition of pRb2/p130 phosphorylation has also been investigated.…”

Role of the interaction between large T antigen and Rb family members in the oncogenicity of JC virus

“…It has been demonstrated that JCV T-Ag, like its simian counterpart SV40, binds pRb family members and influences their phosphorylation status and stability (Dyson et al, 1990;Howard et al, 1998). On the contrary, JCV T-Ag interaction with Hsc70 has only been shown using chimeric JCV-SV40 T-Ag (Sullivan et al, 2000b). Large T antigen J domains of JCV and SV40 are highly homologous (Pipas, 1992).…”