Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation
Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation
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DOI：
10.1083/jcb.200110063
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发表时间：
2002-02-18
影响因子：
7.8
通讯作者：
Cooley, L
中科院分区：
文献类型：
--
作者：
Kelso, RJ;Hudson, AM;Cooley, L
果蝇kelch基因编码由kelch重复序列的存在定义的蛋白质超家族的成员。在果蝇中，Kelch是维持卵巢环管肌动蛋白组织所必需的。我们着手研究Kelch的肌动蛋白交联活性以及Kelch功能是如何调节的。使用纯化的，重组Kelch蛋白的生化研究表明，全长Kelch束肌动蛋白丝，和Kelch重复5含有肌动蛋白结合位点。双向电泳表明，Kelch是酪氨酸磷酸化的src64依赖性途径。定点突变确定酪氨酸残基627被磷酸化。一个Kelch突变体与酪氨酸627改变为丙氨酸（KelY627A）拯救肌动蛋白解体的Kelch突变体环管表型，但未能产生野生型环管。电子显微镜表明，Kelch的磷酸化是关键的适当的肌动蛋白的形态发生在环管生长，和存在的非磷酸化KelY627A蛋白phenocopied src 64环管。KelY627A蛋白在环管也显着降低肌动蛋白单体交换率。由Src64突变体和KelY627A表达引起的表型表明Src64信号在环管中的主要功能是Kelch对肌动蛋白交联的负调节。
The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the non-phosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.