Phosphorylation of human fascin inhibits its actin binding and bundling activities
Phosphorylation of human fascin inhibits its actin binding and bundling activities
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DOI：
10.1074/jbc.271.21.12632
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发表时间：
1996-05-24
影响因子：
4.8
通讯作者：
Yamashiro, S
中科院分区：
文献类型：
--
作者：
Yamakita, Y;Ono, S;Yamashiro, S
人肌成束蛋白是一种肌动蛋白成束蛋白，被认为参与存在于微刺以及膜皱褶和应力纤维中的肌动蛋白丝束的组装。我们已经发现，在用12-O-十四酰基佛波醇-13-乙酸酯（一种肿瘤促进剂）处理后，人肌成束蛋白在体内被磷酸化。在体内磷酸化逐渐增加，从0.13至0.30 mol/mol，在2小时的治疗过程中，伴随着人肌成束蛋白从应力纤维，膜皱褶，和microspikes消失。人肌成束蛋白也可以在体外被蛋白激酶C磷酸化，其位点与通过磷酸肽图谱判断的体内观察到的相同。磷酸化的程度取决于pH：在pH 7.0、6.0和5.0下，化学计量分别为0.05、0.38和0.6 mol磷酸盐/mol蛋白质。磷酸化大大降低了人肌成束蛋白的肌动蛋白结合，而单独将pH降低至6.0不影响肌成束蛋白-肌动蛋白结合。当每摩尔蛋白质掺入0.25摩尔磷酸盐时，肌动蛋白结合亲和力从6.7 × 10（6）m（-1）降低到1.5 × 10（6）m（-1）。肌动蛋白成束活性也降低。这些结果表明，肌成束蛋白的磷酸化在用12-O-十四酰基佛波醇-13-乙酸酯处理后的肌动蛋白重组中起作用。
Human fascin is an actin-bundling protein that is thought to be involved in the assembly of actin filament bundles present in microspikes as well as in membrane ruffles and stress fibers. We have found that human fascin is phosphorylated in vivo upon treatment with 12-O-tetradecanoylphorbol-13-acetate, a tumor promoter. The in vivo phosphorylation is gradually increased from 0.13 to 0.30 mol/mol during 2 h of treatment, concomitant with the disappearance of human fascin from stress fibers, membrane ruffles, and microspikes. Human fascin can also be phosphorylated in vitro by protein kinase C at the same sites as observed in vivo as judged by phosphopeptide mapping. The extent of phosphorylation depends on pH: the stoichiometries are 0.05, 0.38, and 0.6 mol of phosphate/mol of protein at pH 7.0, 6.0, and 5.0, respectively. Phosphorylation greatly reduces actin binding of human fascin, while lowering the pH to 6.0 alone does not affect fascin-actin binding. With the incorporation of 0.25 mol of phosphate/mol of protein, the actin binding affinity is reduced from 6.7 x 10(6) to 1.5 x 10(6) m(-1). The actin bundling activity is also decreased. These results suggest that phosphorylation of fascin plays a role in actin reorganization after treatment with 12-O-tetradecanoylphorbol-13-acetate.