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首页> 外文学位 >The distinct roles of Ras and Rac in PI3-kinase dependent protrusion during EGF-stimulated cell migration.
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The distinct roles of Ras and Rac in PI3-kinase dependent protrusion during EGF-stimulated cell migration.

机译：在EGF刺激的细胞迁移过程中，Ras和Rac在PI3激酶依赖性突出中的独特作用。

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摘要

Phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol, generating secondary messengers that direct a variety of cellular responses. In EGF-stimulated carcinoma cells, PI3K activity is required for the formation of actin-rich protrusions, such as lamellipodia. There are two actin polymerization transients in EGF-stimulated MTLn3 cells, with peaks of barbed end formation at the leading edge at 1 and 3 min. PI3K is only required for the late peak of barbed end formation at 3 min.; Given the differential requirement of PI3K activity in the two-actin polymerization transients, it is important to define the spatial and the temporal regulation of PI3K activation and PIP3 production during EGF-stimulated protrusion. We measured the kinetics of EGF-stimulated PIP3 production at the leading edge of the lamellipod using a saponin-permeabilization/fixation method and a monoclonal anti-PIP3 antibody. Anti-PIP3 immunostaining exhibited a rapid and persistent response, as the activity reached maximal level at 1 min and remained elevated at 3 min. However, membrane PIP3 turnover is extremely fast, as Ly294002 treatment abolished leading edge PIP3 within 10sec. This means that the sustained elevation of leading edge PIP3 in EGF-stimulated cells requires persistent PI3K activation.; Both Rac and Ras have been implicated in growth factor-induced membrane ruffling and have been shown to activate Class IA PI3K. However, the coordination of Rac and Ras activation with PI3K dependent lamellipod extension has not been characterized during EGF-stimulated protrusion. Inhibition of Rac1 activity by siRNA-mediated knockdown or pharmacological inhibition had no effect on EGF-stimulated lamellipod extension or PIP3 production at the leading edge of carcinoma cells. In contrast, microinjection of inhibitory anti-Ras antibodies or pharmacological inhibition of Ras abolished EGF-stimulated protrusion and PIP3 production at the leading edge. Moreover, the activation kinetics of Ras, but not Rac, was coincident with those of leading edge PIP 3 production. Despite the fact that lamellipod extension was independent of Rac, Rac activity was required for cell motility, as Rac1 siRNA treatment inhibited cell migration. We conclude that Ras, but not Rac, plays a dominant role in regulating PI3K activation and PIP3-dependent lamellipod extension in EGF-stimulated cells.
机译：磷酸肌醇3激酶（PI3K）使膜脂质磷脂酰肌醇磷酸化，产生指导各种细胞反应的次级信使。在EGF刺激的癌细胞中，PI3K活性对于形成富含肌动蛋白的突起（例如片状脂膜）是必需的。在EGF刺激的MTLn3细胞中有两个肌动蛋白聚合瞬变，在第1分钟和第3分钟的前缘有带刺的末端形成峰。 PI3K仅在3分钟的带刺末端形成的后期达到峰值。考虑到在两个肌动蛋白聚合过程中对PI3K活性的不同要求，在EGF刺激的突出过程中定义PI3K激活和PIP3产生的时空调节非常重要。我们使用皂苷通透/固定方法和单克隆抗PIP3抗体测量了在lamellipod前沿的EGF刺激的PIP3产生的动力学。抗-PIP3免疫染色表现出快速而持久的反应，因为活性在1分钟时达到最大水平，并在3分钟时保持升高。然而，由于Ly294002处理在10秒内取消了前沿PIP3，膜PIP3的转换非常快。这意味着在EGF刺激的细胞中，前沿PIP3的持续升高需要持续的PI3K激活。 Rac和Ras均与生长因子诱导的膜起皱有关，并已显示出能激活IA PI3K类。但是，Rac和Ras激活与PI3K依赖的lamellipod延伸的协调尚未在EGF刺激的突出过程中表征。 siRNA介导的敲低或药理学抑制作用抑制Rac1活性对癌细胞前缘EGF刺激的lamellipod延伸或PIP3产生没有影响。相反，抑制性抗Ras抗体的显微注射或Ras的药理抑制作用消除了EGF刺激的前突和前缘PIP3的产生。此外，Ras而不是Rac的活化动力学与前沿PIP 3生产的动力学一致。尽管lamellipod的延伸独立于Rac，但Rac活性对于细胞运动是必需的，因为Rac1 siRNA处理抑制了细胞迁移。我们得出结论，在EGF刺激的细胞中，Ras（而非Rac）在调节PI3K激活和PIP3依赖的lamellipod延伸中起着主导作用。

著录项

  • 作者

    Yip, Shu-chin Jenny.;

  • 作者单位

    Yeshiva University.;

  • 授予单位 Yeshiva University.;
  • 学科 Biology Cell.
  • 学位 Ph.D.
  • 年度 2006
  • 页码 142 p.
  • 总页数 142
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;
  • 关键词

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