Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation.
Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation.
复制标题
SRC对ELMO1的Tyr724磷酸化参与了Rac1激活的细胞扩散和迁移。
DOI：
10.1186/s12964-015-0113-y
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发表时间：
2015-07-25
期刊：
影响因子：
--
通讯作者：
Tanaka S
中科院分区：
文献类型：
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作者：
Makino Y;Tsuda M;Ohba Y;Nishihara H;Sawa H;Nagashima K;Tanaka S
Dock180/ELMO1复合体作为Rac的双体鸟嘌呤核苷酸交换因子，在细胞迁移、吞噬、癌细胞侵袭等多种细胞生理病理过程中发挥重要作用。在src家族酪氨酸激酶（SFKs）中，有报道称Hck直接磷酸化ELMO1，通过促进Rac1的激活来调节吞噬；然而，其他sfk在ELMO1磷酸化中的作用尚不清楚。在这里，我们发现了被SFKs磷酸化的ELMO1的新的酪氨酸(Y)残基，并研究了对Rac1活性、细胞粘附、扩散和细胞外基质（ECM）细胞运动的影响。在本研究中，我们在体内和体外磷酸化实验中发现Src和Fyn可以诱导ELMO1的酪氨酸磷酸化。突变分析发现ELMO1的Y720和Y724残基都是src介导的磷酸化位点，优先在Y724上。Y724单次替换为Phe取消了Src触发的Rac1激活。为了阐明pY724的生物学功能，我们建立了NIH3T3细胞，与Dock180一起稳定表达野生型ELMO1或其Y724F突变体。其中，y724缺陷细胞即使在ecm刺激下也表现出Rac1活性的消耗和ELMO1磷酸化的降低。值得注意的是，带有ELMO1 Y724F的NIH3T3细胞在纤维连接蛋白包被的培养皿上明显缺乏促进细胞扩散的能力，同时表现出肌动蛋白应激纤维的不成熟组装和局灶粘连。最终，ELMO1 Y724F显著损害细胞迁移。这些结果表明，src介导的Y724磷酸化在ELMO1中通过激活Rac1对细胞扩散起关键作用，从而促进细胞迁移。由于Src的过表达和/或过度激活已在多种人类癌症中得到证实，Src介导的ELMO1中Y724的磷酸化可能调节癌细胞对ECM的粘附、向周围组织的侵袭以及随后的远处转移。本文的在线版本（doi:10.1186/s12964-015-0113-y）包含补充材料，仅供授权用户使用。
The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM). In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration. These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis. The online version of this article (doi:10.1186/s12964-015-0113-y) contains supplementary material, which is available to authorized users.