ATP-dependent simian virus 40 T-antigen-Hsc70 complex formation.
Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen--Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 microM), but T-antigen--Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen--Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.
Sullivan
C S
CS
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Gilbert
S P
SP
Pipas
J M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002499:Centrifugation, Density Gradient; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate