Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor
Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor
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DOI：
10.1091/mbc.02-05-0068
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发表时间：
2002-12-01
影响因子：
3.3
通讯作者：
Treisman, R
中科院分区：
文献类型：
--
作者：
Posern, G;Sotiropoulos, A;Treisman, R
信号诱导的转录因子血清反应因子（SRF）的激活需要肌动蛋白动力学的改变。SRF活性可以被P-肌动蛋白的异位表达所抑制，这是因为肌动蛋白本身参与SRF调节，或者是细胞骨架扰动的结果。为了区分这些可能性，我们研究了肌动蛋白突变体。三个突变肌动蛋白，G13 R，R62 D，和C-末端VP 16融合蛋白，被证明不在体内，判断双杂交，免疫荧光，和细胞分级分离研究。这些肌动蛋白有效地抑制SRF激活，野生型肌动蛋白，这增加了G-肌动蛋白水平，而不改变F：G-肌动蛋白的比例。SRF和肌动蛋白之间的物理相互作用是不可检测的哺乳动物或酵母双杂交试验，表明SRF调节涉及一个身份不明的辅因子。SRF活性不被阻断后，抑制CRM 1介导的核输出由来普霉素B。两个肌动蛋白突变体，V159 N和S14 C，其表达有利于F-肌动蛋白的形成，并强烈激活SRF在没有外部信号。这些突变体似乎无法抑制SRF活性，因为它们的表达并没有降低G-肌动蛋白的绝对水平，如通过DNA酶I结合所评估的。综上所述，这些结果提供了强有力的证据，G-肌动蛋白，或它的一个亚群，在信号转导SRF中起着直接的作用。
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 P-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.