Zhou Guo-Lei, Zhang Haitao, Wu Huhehasi, Ghai Pooja, Field Jeffrey
Department of Biological Sciences, Arkansas State University, State University, AR 72467, USA Molecular Biosciences Program, Arkansas State University, State University, AR 72467, USA
Department of Biological Sciences, Arkansas State University, State University, AR 72467, USA Molecular Biosciences Program, Arkansas State University, State University, AR 72467, USA.
J Cell Sci. 2014 Dec 1;127(Pt 23):5052-65. doi: 10.1242/jcs.156059. Epub 2014 Oct 14.
Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.
细胞信号传导可通过调节肌动蛋白调节蛋白来控制丝状肌动蛋白和单体肌动蛋白之间的动态平衡。一类控制肌动蛋白动态变化的肌动蛋白调节蛋白包括环化酶相关蛋白1和2（分别为CAP1和CAP2）。然而，调节CAPs的细胞信号仍然未知。我们绘制了小鼠CAP1上的磷酸化位点，发现S307和S309是调节位点。我们进一步确定糖原合酶激酶3是磷酸化S309的激酶。模拟磷酸化突变体S307D/S309D失去了与其伴侣cofilin的结合，并且在细胞中表达时，会导致肌动蛋白应激纤维的积累，类似于CAP表达降低的细胞中的情况。相反，不可磷酸化的S307A/S309A突变体显示cofilin结合大幅增加，而与肌动蛋白的结合减少。这些结果表明，磷酸化有助于促进cofilin的释放，以便进行后续的肌动蛋白丝切断循环。此外，我们的结果表明S307和S309协同发挥作用；在S307或S309的点突变体中，未观察到与cofilin和/或肌动蛋白结合的改变，也未观察到在CAP1敲低细胞中挽救细胞大小增大表型的缺陷。总之，我们通过磷酸化确定了一种新的CAP1调节机制。
