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 beta-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.
Posern
Guido
G
Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, London WC2A 3PX, United Kingdom.
Sotiropoulos
Athanassia
A
Treisman
Richard
R
D016475:3T3 Cells; D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002469:Cell Separation; D003850:Deoxyribonuclease I / Q000378:metabolism; D005434:Flow Cytometry; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D026362:Serum Response Factor / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014981:Xenopus
D000199:Actins; D017405:Herpes Simplex Virus Protein Vmw65; D026362:Serum Response Factor; D003850:Deoxyribonuclease I