The Diaphanous-related Formin mDia1 Controls Serum Response Factor Activity through its Effects on Actin Polymerization

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin

Mutant Actins Demonstrate a Role for Unpolymerized Actin in Control of Transcription by Serum Response Factor

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 β-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