Regulation of Mitogenesis and Apoptosis by G Proteins

描述（由申请人提供）：异源三聚体G蛋白的G12亚家族，在细胞骨架变化、有丝分裂和凋亡等细胞过程中被认为是一个信号元件。我们之前提出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形成的生物化学和功能上的新蛋白复合物。我们将进一步研究G12亚家族调控的有丝分裂和凋亡反应的细胞和分子机制。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相互作用并诱导其活化。值得注意的是，PP5被证明通过抑制生长抑制信号级联和激活生长促进信号通路，在有丝分裂途径的调节中起关键作用。其他重要数据显示，Ga12和Ga13与丝氨酸/苏氨酸蛋白激酶LIM激酶1 （LIMK1）相互作用并诱导其活化。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重组。因此，本提案将分析允许Ga12和Ga13与PP5和LIMK1形成特定信号复合物的新分子机制，从而产生在细胞中产生有丝分裂或凋亡反应的信号。具体目标如下：目标1。探讨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Ga12/Ga13与PP5在信号通路中调控有丝分裂或凋亡反应的生理作用。（ii）由于PP5与热休克蛋白90相互作用，Ga12介导的增殖反应需要Hsp90和Hsp90，我们将研究Ga12在PPf/Hsp90功能中的作用。（iii）由于PP5被证明与凋亡信号调节激酶ASK1相互作用并失活，我们的数据表明Ga12/Ga13激活ASK1，从而促进细胞凋亡，我们将研究Ga12/Ga13在PP5/ASK1功能中的作用。利用ASK1基因敲除小鼠的成纤维细胞和内皮细胞，我们将探讨Ga12/Ga13-PP5/ASK1通路的生理相关性。目标2。探讨Ga12和Ga13与LIMK1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的生化相互作用机制。使用体外重构实验，我们将确定蛋白质相互作用在Ga12和ga13依赖性LIMK1激活中的作用（ii）由于LIMK1调节肌动蛋白细胞骨架，并且我们已经证明LIMK1与细胞中的微管细胞骨架相关，我们将解决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敲除小鼠中获得的成纤维细胞和内皮细胞，我们将研究Ga12/Ga13-LIMK1通路的生理相关性。这些研究对于理解组织增殖和凋亡的控制以及确定治疗干预的潜在位点至关重要。

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.