Role of the p70S6K pathway in regulating the actin cytoskeleton and cell migration
Role of the p70S6K pathway in regulating the actin cytoskeleton and cell migration
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DOI：
10.1016/j.yexer.2003.12.032
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发表时间：
2004-06-10
影响因子：
3.7
通讯作者：
Crouch, MF
中科院分区：
文献类型：
--
作者：
Berven, LA;Willard, FS;Crouch, MF
我们研究了内源性70 kDa S6激酶（p70（S6K））在瑞士3T3成纤维细胞肌动蛋白细胞骨架组织和细胞迁移中的作用。通过p70（S6K）与F-肌动蛋白的共沉淀和亚细胞分级分离（其中在F-肌动蛋白细胞骨架组分中测量p70（S6K）活性）证明了p70（S6K）与肌动蛋白细胞骨架的关联。免疫细胞化学研究表明，p70（S6K），Akt1，PDK1，和p85磷酸肌醇3-激酶（PI 3-激酶）定位于肌动蛋白，小窝蛋白丰富的细胞骨架结构位于迁移细胞的前缘。使用针对哺乳动物雷帕霉素靶蛋白（mTOR）的磷酸化特异性抗体，我们发现激活的mTOR在肌动蛋白弧处富集，表明p70（S6K）信号通路的激活对细胞迁移是重要的。使用肌动蛋白弧评估迁移，发现表皮生长因子（EGF）刺激诱导肌动蛋白弧形成，这种作用被雷帕霉素治疗阻断。我们进一步表明，肌动蛋白应力纤维可能起到下调p70（S6K）。纤连蛋白刺激应力纤维的形成在生长因子的情况下，并导致p70（S6K）的失活。相反，细胞松弛素D和Rho激酶抑制剂Y-27632，这两者都导致应力纤维破坏，增加p70（S6K）的活性。这些研究提供的证据表明，p70（S6K）途径是重要的信号在两个F-肌动蛋白微结构域的细胞和调节细胞迁移。(C)2004爱思唯尔公司All rights reserved.
We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin are, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration. (C) 2004 Elsevier Inc. All rights reserved.