Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics. SRF activity can be inhibited by ectopic expression of -actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations. To distinguish between these possibilities, we studied actin mutants. Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies. These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio. Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor. SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B. Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals. These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding. Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF.
INTRODUCTIONSerum response factor (SRF) is a transcription factor that regulates many immediate-early and muscle-specific genes. Deletion of SRF in ES cells leads to alterations in cellular morphology and adhesion, and is lethal in mice at gastrulation owing to defects in mesoderm formation (Arsenian et al., 1998;Weinhold et al., 2000;Schratt et al., 2002). SRF activity is controlled by the Rho family of small GTPases (Hill et al., 1995), and recent studies have revealed a close connection between SRF activation and actin polymerization. Downstream of RhoA, both the ROCK-LIMK-cofilin and the mDia effector pathways can promote both F-actin accumulation and SRF activity (Sotiropoulos et al., 1999;Tominaga et al., 2000;Copeland and Treisman, 2002;Geneste et al., 2002). The ability of LIMK and mDia mutants to activate SRF correlates with their ability to promote F-actin accumulation, and interfering derivatives of these proteins can inhibit the activation of SRF by extracellular signals (Sotiropoulos et al., 1999;Tominaga et al., 2000;Copeland and Treisman, 2002;Geneste et al., 2002). Alterations in actin dynamics are required for RhoA-mediated SRF activation, which is inhibited upon treatment of cells with the G-actin binding drug latrunculin or C2 toxin (Sotiropoulos et al., 1999). The RhoA-actin pathway controls a subset of SRF target genes, including the immediate-early genes -actin, vinculin, and srf, and the muscle-specific SM22 and SM ␣-actin genes (Sotiropoulos et al., 1999;Gineitis and Treisman, 2001;Mack et al., 2001).Several lines of evidence suggest that actin itself is intimately involved in the control o...
