Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts
Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts
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DOI：
10.1016/j.bbamcr.2008.10.001
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发表时间：
2009-02-01
影响因子：
5.1
通讯作者：
Donella-Deana, Arianna
中科院分区：
文献类型：
--
作者：
Frasson, Martina;Vitadello, Maurizio;Donella-Deana, Arianna
内质网伴侣Grp 94是参与天然免疫应答、中胚层诱导和肌肉发育的分子的细胞表面输出所必需的，但负责Grp 94募集的信号仍然不清楚。在这里，我们首次表明，Grp 94经历酪氨酸磷酸化分化肌源性C2 C12细胞。通过磷酸化蛋白质组学和免疫沉淀分析，并使用Src特异性抑制剂，我们证明，Src-酪氨酸激酶Fyn变得活跃后早期诱导C2 C12细胞分化，在平行的招聘和酪氨酸磷酸化的Grp 94，在6小时分化的峰值。Grp 94在内质网内被腔内Fyn酪氨酸磷酸化，如荧光和电子显微镜免疫定位、化学交联后的免疫共沉淀以及用蛋白酶K处理完整的内质网囊泡所示。此外，分馏的细胞膜室和双免疫荧光研究表明，酪氨酸磷酸化的Grp 94是必要的蛋白质从内质网到高尔基体的易位。这些结果表明，Fyn催化的酪氨酸磷酸化的Grp 94是一个事件，需要促进伴侣出口从内质网发生在成肌细胞分化的早期阶段。(c)2008 Elsevier B. V.保留所有权利。
The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation. (c) 2008 Elsevier B.V. All rights reserved.