Modulation of Cellular Signaling by Sprouty Proteins

Sprouty 蛋白对细胞信号传导的调节

• 批准号: 7109291
• 负责人: TARUN B. PATEL
• 金额: $ 36.17万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7109291
• 关键词: HeLa cells; biological signal transduction; cell migration; cell proliferation; enzyme activity; epidermal growth factor; fibroblasts; gene expression; genetic regulation; genetically modified animals; guanine nucleotide exchange factors; laboratory mouse; membrane proteins; protein protein interaction; protein structure function; protein tyrosine phosphatase

DESCRIPTION (provided by applicant): The Sprouty (Spry) proteins antagonize the actions of growth factors and thereby inhibit tracheal branching, angiogenesis and development of limbs and cerebellum. Among the four mammalian Sprouty (Spry) isoforms, other and we have shown that Spry1, Spry2, and Spry4 inhibit growth factor and serum induced cellular migration and proliferation. However, the precise mechanisms by which Spry proteins inhibit these processes remains to be determined. Therefore, the overall goal of this project is to identify the signaling molecules and pathways in EOF stimulated migration and proliferation of cells that are modulated by human Sprouty 2 (hSpry2). Toward this end, we have shown that hSpry2 expression increases the amount of soluble protein tyrosine phosphatase 1B (PTP1B) and decreases the particulate form of this protein. Indeed, in the presence of a dominant negative (DN) form of PTP1B or in PTP1B-/- cells the ability of hSpry2 to inhibit cell migration is attenuated; DN-PTP1B does not modify the ability of hSpry2 to inhibit cell proliferation. Interestingly, activation of Rac1 by EOF is decreased by hSpry2 and constitutively active Rac1 obliterates the ability of hSpry2 to inhibit cell migration without any effects on the ability of hSpry2 to inhibit cell proliferation. Therefore, we propose that the anti-migratory actions of hSpry2 are mediated, at least in part, by elevation in soluble PTP1B amount and activity. Since EGF-stimulated phosphorylation of p130Cas, a PTP1B substrate is decreased by hSpry2, we propose that the guanine nucleotide exchange factor activity of the p130Cas/CrkII/DOCK180/ELMO complex for Rac1 may be attenuated by hSpry2. Thus specific aims 1 and 2 of the application will investigate the role of PTP1B, Rac1, and p130Cas in modulation of EGF-stimulated cell migration by hSpry2. Under aim 3, we will investigate the role of PTEN in modulation of EGF-induced cell proliferation by hSpry2. These latter studies are based upon the findings that in hSpry2 expressing cells the amount of PTEN is increased and the activation of Akt by EGF is decreased. Our approaches will involve the use of cells in which one of the critical signaling elements (e.g., PTP1B or PTEN) is knocked out or in which Spry2 is knocked down by the use of siRNA and shRNA technology or conditionally knocked out. Elucidation of the mechanisms by which hSpry2 increases the amount of soluble PTP1B as well as total PTEN levels together with the identification of the signaling pathways that are modulated to antagonize growth factor-mediated activation of cell proliferation and migration will provide critical information that can be used to develop novel methods to inhibit angiogenesis, and growth of tumors as well as decrease the incidence of restenosis following vascular injury.

描述（由申请人提供）： Sprouty（Spry）蛋白拮抗生长因子的作用，从而抑制气管分支、血管生成以及四肢和小脑的发育。在四种哺乳动物Sprouty（Spry）同种型中，其他的和我们已经表明Spry 1、Spry 2和Spry 4抑制生长因子和血清诱导的细胞迁移和增殖。然而，Spry蛋白抑制这些过程的确切机制仍有待确定。因此，本项目的总体目标是鉴定由人Sprouty 2（hSpry 2）调节的细胞的细胞刺激的迁移和增殖中的信号分子和途径。为此，我们已经表明，hSpry 2表达增加可溶性蛋白酪氨酸磷酸酶1B（PTP 1B）的量，并减少这种蛋白质的颗粒形式。 事实上，在PTP 1B的显性阴性（DN）形式存在下或在PTP 1B-/-细胞中，hSpry 2抑制细胞迁移的能力减弱; DN-PTP 1B不改变hSpry 2抑制细胞增殖的能力。有趣的是，Rac 1被hSpry 2降低，并且组成型活性Rac 1消除了hSpry 2抑制细胞迁移的能力，而对hSpry 2抑制细胞增殖的能力没有任何影响。因此，我们提出hSpry 2的抗迁移作用至少部分是通过可溶性PTP 1B量和活性的升高介导的。由于EGF刺激的p130 Cas（PTP 1B底物）的磷酸化被hSpry 2降低，我们认为p130 Cas/CrkII/DOCK 180/埃尔莫复合物对Rac 1的鸟嘌呤核苷酸交换因子活性可能被hSpry 2减弱。因此，本申请的具体目标1和2将研究PTP 1B、Rac 1和p130 Cas在hSpry 2调节EGF刺激的细胞迁移中的作用。在目标3下，我们将研究PTEN在hSpry 2调节EGF诱导的细胞增殖中的作用。这些后面的研究是基于这样的发现，即在表达hSpry 2的细胞中，PTEN的量增加，并且EGF对Akt的激活减少。我们的方法将涉及使用细胞，其中一个关键的信号传导元件（例如，PTP 1B或PTEN）被敲除或其中Spry 2通过使用siRNA和shRNA技术被敲除或条件性敲除。阐明hSpry 2增加可溶性PTP 1B的量以及总PTEN水平的机制，以及鉴定被调节以拮抗生长因子介导的细胞增殖和迁移活化的信号传导途径，将提供可用于开发抑制血管生成的新方法的关键信息，和肿瘤生长以及降低血管损伤后再狭窄的发生率。