Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.
The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.
Maxwell
S A
SA
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
Ames
S K
SK
Sawai
E T
ET
Decker
G L
GL
Cook
R G
RG
Butel
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000302:isolation & purification / Q000502:physiology*; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003078:Colchicine / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification*; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects; D008969:Molecular Sequence Data; D015398:Signal Transduction; D013539:Simian virus 40 / Q000502:physiology; D014404:Tubulin / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000302:isolation & purification / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D016159:Tumor Suppressor Protein p53; D003078:Colchicine