Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577 - Role of Src family kinases and Rho family GTPases
Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577 - Role of Src family kinases and Rho family GTPases
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DOI：
10.1074/jbc.m314132200
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发表时间：
2004-10-22
影响因子：
4.8
通讯作者：
Rozengurt, E
中科院分区：
文献类型：
--
作者：
Lunn, JA;Rozengurt, E
用蔗糖或山梨醇处理瑞士3T3细胞引起的高渗应激，迅速而有力地刺激内源性粘着斑激酶(FAK)在主要的自磷酸化位点Tyr-397和激酶激活环内的Tyr-577的磷酸化。高渗应激刺激的FAK在Tyr-397处的磷酸化是通过不依赖于Src的途径发生的，而暴露于Src家族的激酶抑制剂PP-2则完全阻断了Tyr-577的磷酸化。抑制p38MAPK或磷脂酰肌醇3-激酶不能阻止高渗应激刺激的FAK磷酸化。N17RhoA的过表达不能减少高渗应激介导的磷酸化粘着斑激酶定位于局灶性接触者，Rho相关激酶抑制剂Y-27632的治疗也不能阻止高渗透应激反应中的粘着斑激酶转位和酪氨酸磷酸化。N17RAC的过度表达仅轻微改变了高渗应激介导的磷酸化FAK对局灶性接触的定位。相反，CDC42的N17突变体的过表达破坏了高渗应激刺激的FAK Tyr-397定位到焦点接触。此外，艰难梭菌毒素B处理细胞可有效抑制高渗应激诱导的FAK酪氨酸磷酸化。此外，与含有FAK的对照组相比，FAK缺失的成纤维细胞对持续高渗应激的敏感性显著增加，表现为随后的细胞凋亡。我们的结果表明，FAK在保护细胞免受高渗应激中起着基础性的作用，并且介导FAK在Tyr-397上自动磷酸化的途径(S)可以与许多其他刺激所利用的途径不同，这些刺激包括神经肽和生物活性脂(Rho和Rho相关的激酶依赖)、酪氨酸激酶受体激动剂(磷脂酰肌醇3-激酶依赖)和整合素(Src依赖)。
Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop. Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2. Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress. Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress. Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts. In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts. Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation. Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress. Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).