Modulation of Cellular Signaling by Sprouty Proteins

Sprouty（Spry）蛋白拮抗生长因子的作用，从而阻断气管 分支、血管生成和四肢和小脑的发育。在四种哺乳动物中， 我们已经表明，Spry 1、Spry 2和Spry 4抑制生长因子和血清 诱导细胞迁移和增殖。然而，Spry蛋白质的精确机制 抑制这些过程仍有待确定。因此，本项目的总体目标是确定 受调节的细胞的迁移和增殖中的信号分子和途径 人类Sprouty 2（hSpry 2）。为此，我们已经表明，hSpry2表达增加了细胞内的细胞数量。 可溶性蛋白酪氨酸磷酸酶IB（PTP1B）的，并减少这种蛋白质的颗粒形式。 事实上，在PTP1B的显性阴性（DN）形式的存在下或在PTP1B β-细胞中， DN-PTP1B不改变hSpry2抑制细胞迁移的能力， 增殖有趣的是，hSpry 2和组成型活性的Racl降低了Racl的活化。 消除了hSpry 2抑制细胞迁移的能力，而对hSpry 2抑制细胞迁移的能力没有任何影响。 细胞增殖因此，我们认为hSpry 2的抗迁移作用是介导的，至少在 部分，通过可溶性PTP1B量和活性的升高。由于EGF刺激的p! 30Cas， PTP1B底物被hSpry 2降低，我们提出， Rac1的p130 Cas/Crk II/DOCK 180/埃尔莫复合物可被hSpry 2减弱。具体目标1 和2的应用将调查PTP1B，Racl，和p! 30Cas调节EGF- 通过hSpry 2刺激细胞迁移。在目标3中，我们将研究PTEN在调节细胞凋亡中的作用。 EGF诱导的hSpry 2细胞增殖。后面这些研究是基于hSpry 2中的发现， 在表达细胞中，PTEN的量增加，EGF对Akt的激活减少。我们 方法将涉及使用细胞，其中一个关键的信号元件（例如PTP1B或PTEN） 或其中Spry2通过使用siRNA和shRNA技术被敲除，或 有条件地被击倒阐明hSpry 2增加可溶性蛋白质的量的机制。 PTP1B以及总的PTEN水平，以及识别信号通路， 被调节以拮抗生长因子介导的细胞增殖和迁移的活化， 可用于开发抑制血管生成和肿瘤生长的新方法的关键信息 以及降低血管损伤后再狭窄的发生率。

The Sprouty (Spry) proteins antagonize the actions of growth factors and thereby inhibittracheal branching, angiogenesis and development of limbs and cerebellum. Among the four mammalian Sprouty (Spry) isoforms, other and we have shown that Spryl, 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 IB (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 Racl by EOF is decreased by hSpry2 and constitutivelyactive Racl 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 p!30Cas, a PTP1B substrate is decreased by hSpry2, we propose that the guanine nucleotide exchange factor activity of the pl30Cas/CrkII/DOCK180/ELMO complex for Racl may be attenuated by hSpry2. Thus specific aims 1 and 2 of the application will investigate the role of PTP1B, Racl, and p!30Cas 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 signalingelements (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 followingvascular injury.