Modulation of Cellular Signaling by Sprouty Proteins

Sprouty 蛋白对细胞信号传导的调节

• 批准号: 7487315
• 负责人: TARUN B. PATEL
• 金额: $ 35.34万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487315
• 关键词: Address; Attenuated; Blood Vessels; Cell Migration Inhibition function; Cell Migration Inhibition measurement; Cell Proliferation; Cells; Cerebellum; Chromosomes, Human, Pair 10; Color; Complex; Dominant-Negative Mutation; Drosophila sprouty protein; EGF gene; Elements; Elevation; Embryo; Epidermal Growth Factor; Fibroblasts; Figs - dietary; Goals; Growth Factor; Guanine Nucleotide Exchange Factors; Homologous Gene; Human; Human Activities; Incidence; Injury; Knock-out; Limb Development; Mediating; Methods; Mus; Numbers; Other Finding; Particulate; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Process; Protein Isoforms; Protein Tyrosine Phosphatase; Proteins; Proto-Oncogene Proteins c-akt; Role; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Study Section; Technology; Tyrosine Phosphorylation; angiogenesis; base; cell motility; human BCAR1 protein; knock-down; migration; novel; protein tyrosine phosphatase 1B; restenosis; small hairpin RNA; tensin; tumor growth

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)亚型中，我们已经发现Spry1、SPRY2和SPRY4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确定在EOF刺激的细胞迁移和增殖过程中受人类Sprouty 2(HSpry2)调控的信号分子和信号通路。为此，我们已经证明，hSpry2的表达增加了可溶性蛋白酪氨酸磷酸酶1B(PTP1B)的数量，并减少了该蛋白的颗粒形式。事实上，在PTP1B或PTP1B-/-细胞中存在显性负性(DN)形式的PTP1B时，hSpry2抑制细胞迁移的能力减弱；dN-PTP1B不改变hSpry2抑制细胞增殖的能力。有趣的是，hSpry2降低了EOF对rac1的激活，并且hSpry2的结构性活性rac1消除了hSpry2抑制细胞迁移的能力，而不影响hSpry2抑制细胞增殖的能力。因此，我们认为hSpry2的抗迁移作用至少部分是通过增加可溶性PTP1B的数量和活性来实现的。由于表皮生长因子刺激的p130Cas的磷酸化，PTP1B底物被hSpry2降低，我们认为p130Cas/CrkII/DOCK180/Elmo复合体对rac1的鸟核苷酸交换因子活性可能被hSpry2减弱。因此，本申请的特定目标1和2将研究PTP1B、rac1和p130Cas在hSpry2调节EGF刺激的细胞迁移中的作用。在目标3中，我们将研究PTEN在hSpry2调控EGF诱导的细胞增殖中的作用。后者的研究是基于在表达hSpry2的细胞中，PTEN的数量增加，而EGF对Akt的激活减少。我们的方法将涉及使用细胞的使用，其中一个关键的信号元件(例如，PTP1B或PTEN)被敲除，或者SPRY2通过使用siRNA和shRNA技术被敲除，或有条件地被敲除。阐明hSpry2增加可溶性PTP1B和总PTEN水平的机制，以及识别被调控以拮抗生长因子介导的细胞增殖和迁移激活的信号通路，将为开发新的方法来抑制血管生成和肿瘤生长以及降低血管损伤后再狭窄的发生率提供关键信息。