Regulation of Mitogenesis and Apoptosis by G Proteins

描述(申请人提供)：G12亚家族的异源三聚体G蛋白，已被认为是细胞过程中的一个信号成分，从细胞骨架的变化到有丝分裂和凋亡。我们先前提议的三个主要目标是研究G12和G13蛋白的GA亚基诱导有丝分裂或凋亡的特定信号通路，以及鉴定和表征由GA12和Ga13形成的新的生化和功能蛋白质复合体。我们将扩大我们对G12亚家族调控有丝分裂和凋亡反应的细胞和分子机制的研究。我们的初步数据表明，GA12和Ga13与重要的信号蛋白丝氨酸/苏氨酸蛋白磷酸酶PP5相互作用并诱导激活。值得注意的是，PP5通过抑制抑制生长的信号通路和激活促进生长的信号通路，被证明在有丝分裂途径的调节中起着关键作用。其他重要数据表明，GA12和Ga13与丝氨酸/苏氨酸蛋白激酶LIM K1、LIMK1相互作用并诱导其激活。重要的是，LIMK1在有丝分裂过程中被激活，并参与肌动蛋白细胞骨架的重组。因此，这项提议将分析新的分子机制，允许GA12和Ga13与PP5和LIMK1形成特定的信号复合体，从而产生在细胞中既能产生有丝分裂反应又能产生凋亡反应的信号。具体的具体目标是：目的1.研究GA12和GA13与PP5的相互作用在信号转导、有丝分裂和细胞凋亡中的作用。(I)我们将研究带有PP5的GA12/Ga13在信号通路中在调节有丝分裂或凋亡反应中的生理作用。(Ii)由于PP5与热休克蛋白90相互作用，Hsp90和Hsp90是GA12介导的增殖反应所必需的，我们将研究GA12在PPF/Hsp90功能中的作用。(3)由于PP5可以相互作用并失活凋亡信号调节激酶ASK1，我们的研究结果表明GA12/Ga13激活了ASK1，从而促进了细胞凋亡，因此我们将研究GA12/Ga13在PP5/ASK1功能中的作用。利用从ASK1基因敲除小鼠获得的成纤维细胞和内皮细胞，我们将研究GA12/Ga13-PP5/ASK1通路的生理相关性。目的2.探讨GA12和Ga13与LIMK1的相互作用在信号转导、有丝分裂和细胞凋亡中的作用。(I)我们将描述LIMK1与GA12和Ga13的生化相互作用的机制。利用体外重组实验，我们将确定LIMK1(II)在GA12和Ga13依赖的激活LIMK1中的作用，因为LIMK1调节肌动蛋白细胞骨架，我们已经证明LIMK1与细胞内的微管细胞骨架相关，我们将研究GA12/Ga13诱导的LIMK1在调节肌动蛋白和微管细胞骨架中的作用。(Iii)利用从LIMK1基因敲除小鼠获得的成纤维细胞和内皮细胞，我们将研究GA12/Ga13-LIMK1途径的生理相关性。这些研究对于了解组织增殖和凋亡的控制，以及确定潜在的治疗干预部位至关重要。

DESCRIPTION (provided by applicant): The G12 subfamily of the heterotrimeric G proteins, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to mitogenesis and apoptosis. The three primary goals of our previous proposal were to investigate the specific signaling pathways leading to mitogenesis or apoptosis induced by the Ga subunits of G12 and G13 proteins, and to identify and characterize biochemically and functionally novel protein complexes formed by Ga12 and Ga13. We will extend our studies of the cellular and molecular mechanisms that are responsible for the mitogenic and apoptotic responses regulated by G12 subfamily. Our preliminary data indicated that Ga12 and Ga13 interact with and induce activation of the important signaling protein, serine/threonine protein phosphatase PP5. Notably, PP5 was shown to be critically involved in the regulation of mitogenic pathways by inhibiting the growth-suppressing signaling cascades and by activating growth-promoting signaling pathways. Additional important data showed that Ga12 and Ga13 interact with and induce activation of the serine/threonine protein kinase, LIM kinase 1, LIMK1. Importantly, LIMK1 is activated during mitosis and is involved in actin cytoskeleton reorganization. Therefore, this proposal will analyze novel molecular mechanisms that allow Ga12 and Ga13 to form specific signaling complexes with PP5 and LIMK1, thereby generating signals that both create either mitogenic or apoptotic responses in the cells. The detailed Specific Aims are: Aim 1. To address the role of the interaction of Ga12 and Ga13 with PP5 in signaling, mitogenesis, and apoptosis. (i) We will study the physiological role of Ga12/Ga13 with PP5 in signaling pathway in the regulation of the mitogenic or apoptotic responses. (ii) As PP5 interacts with heat shock protein 90, Hsp90 and Hsp90 is required for the Ga12-mediated proliferation response, we will study the role of Ga12 in PPf/Hsp90 function. (iii) As PP5 was shown to interact and inactivate apoptosis-signal-regulating kinase, ASK 1, and our data suggest that Ga12/Ga13 activate ASK1, thereby promoting apoptosis, we will study the role of Ga12/Ga13 in PP5/ASK1 function. Using fibroblasts and endothelial cells obtained from ASK 1 knock-out mice, we will address the physiological relevance of Ga12/Ga13-PP5/ASK1 pathway. Aim 2. To address the role of interaction of Ga12 and Ga13 with LIMK1 in signaling, mitogenesis and apoptosis. (i) We will characterize the mechanisms of the biochemical interactions of LIMK1 with Ga12 and Ga13. Using in vitro reconstitution assay, we will determine the role of protein interaction in Ga12 and Ga13-dependent activation of LIMK1 (ii) As LIMK1 regulates actin cytoskeleton and we have shown that LIMK1 is associated with the microtubule cytoskeleton in the cells, we will address the role of Ga12 /Ga13-induced LIMK1 in regulation of the actin and microtubule cytoskeleton. (iii) Using fibroblasts and endothelial cells obtained from LIMK1 knock-out mice, we will address the physiological relevance of the Ga12/Ga13-LIMK1 pathway. These studies are critical for understanding of the control of tissue proliferation and apoptosis, and to identify potential sites for therapeutic intervention.