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首页> 外文学位 >A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: Recruitment of C-terminal Src kinase.
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A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: Recruitment of C-terminal Src kinase.

机译：磷酸酪氨酸依赖性蛋白相互作用筛选揭示了酪氨酸14上小窝蛋白1磷酸化的作用：招募C端Src激酶。

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摘要

Caveolae are plasma membrane invaginations that have been implicated in the physical organization of the components of multiple signaling pathways. The major structural protein of caveolae, caveolin-1, is phosphorylated on tyrosine 14 by Src family kinases (SFKs) in response to several stimuli including insulin, cellular stress, endothelin-1, and angiotensin II. To investigate the functions of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A 3T3-L1 adipocyte cDNA library was screened using the amino terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified c-terminal Src kinase (Csk) as a protein that binds specifically to phosphocaveolin. We proposed a model in which phosphorylation of caveolin by Src family kinases would create a feedback inhibition loop through the recruitment of Csk. This feedback mechanism was supported by experimental results that were consistent with a number of predictions that can be made based on this model. Multiple stimuli that activate caveolin phosphorylation increase caveolin-Csk binding. Csk activity increases concomitantly with the increase of caveolin phosphorylation, consistent with the recruitment and activation of Csk by phosphocaveolin. As predicted in a feedback inhibition loop, caveolin phosphorylation is self-limiting by a mechanism requiring Csk. In response to cellular stress, caveolin, phosphocaveolin, and Csk translocate to a pellet fraction enriched in paxillin, indicating that this feedback loop acts at focal adhesions. The localization of phosphocaveolin to focal adhesions was confirmed by immunofluorescence microscopy. Significantly, in cells attaching and spreading on fibronectin, caveolin was only transiently phosphorylated in newly formed focal adhesions. Many lines of evidence link caveolin to the actin cytoskeleton. We propose that this feedback loop is involved in the transient activation of Src kinases at points of contact of the actin cytoskeleton with the plasma membrane. This transient activation may be necessary for controlled actin cytoskeleton remodeling.
机译：小窝是质膜的内陷，已牵涉多个信号传导途径的成分的物理组织。响应于包括胰岛素，细胞应激，内皮素-1和血管紧张素II在内的多种刺激，Src家族激酶（SFK）将小窝蛋白的主要结构蛋白小窝蛋白1磷酸化在酪氨酸14上。要研究小窝蛋白1磷酸化的功能，我们修改了基于Gal4的酵母双杂交系统，以筛选磷酸化依赖性蛋白相互作用。使用caveolin-1的氨基末端作为诱饵在表达Abl催化结构域的酵母菌株中筛选了3T3-L1脂肪细胞cDNA文库。我们确定c端Src激酶（Csk）为一种特异性结合磷酸小肠磷脂的蛋白质。我们提出了一个模型，其中Src家族激酶使小窝蛋白磷酸化，将通过募集Csk产生反馈抑制环。这种反馈机制得到了实验结果的支持，这些实验结果与基于此模型可以做出的许多预测一致。激活caveolin磷酸化的多个刺激增加caveolin-Csk的结合。 Csk活性随小窝蛋白磷酸化的增加而增加，这与磷酸小泡蛋白对Csk的募集和活化相一致。如反馈抑制回路中所预测的，小窝蛋白的磷酸化是通过需要Csk的机制自我限制的。响应于细胞应激，小窝蛋白，磷酸小窝蛋白和Csk易位至富含Paxillin的沉淀级分，表明该反馈环作用于粘着斑。免疫荧光显微镜检查证实了磷酸小泡素对粘着斑的定位。重要的是，在附着和扩散于纤连蛋白上的细胞中，小窝蛋白仅在新形成的粘着斑中被瞬时磷酸化。许多证据表明小窝蛋白与肌动蛋白细胞骨架有关。我们建议该反馈回路参与肌动蛋白细胞骨架与质膜接触点的Src激酶的瞬时激活。此短暂激活可能是受控的肌动蛋白细胞骨架重塑所必需的。

著录项

  • 作者

    Cao, Haiming.;

  • 作者单位

    University of Nevada, Reno.;

  • 授予单位 University of Nevada, Reno.;
  • 学科 Biology Molecular.; Biology Cell.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;细胞生物学;
  • 关键词

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