Modulation of G proteins by Growth Factors

描述(申请人提供)：异源三聚体G蛋白是控制信号转导的分子开关，其失调可预示肿瘤的发生。生长因子受体酪氨酸激酶(RTK)和它们启动的无数信号/通路是另一条作用毋庸置疑的、也被广泛参与推动肿瘤生长和侵袭的途径。此外，这两条看似不同的通路之间的串扰可以相互激活，而由于通过激活招募多个受体而产生的不受限制的信号在介导癌症进展方面是至关重要的。尽管G蛋白中间体对RTK的间接反式激活已被证实，但对直接刺激RTK后G蛋白如何反式激活缺乏深入的了解。这项拟议的研究旨在揭示生长因子受体调节G蛋白信号的机制。这项赠款中展示的初步工作报告了在G蛋白和生长因子信号通路的十字路口发现了一些独特的信号复合体，并揭示了这些复合体在细胞迁移、有丝分裂和自噬过程中的含义，这些过程对肿瘤的发展至关重要。我们对GIV、三聚体G蛋白G？I以及生长因子RTK家族的原型成员--表皮生长因子受体对GIV的研究，使我们能够定义一个新的信号放大层次。这项提案的总体目标是获得对这一层的工作原理及其与癌症进展的相关性的机械性见解。我们的具体目标是：1)使用体外和体内磷酸化试验、线性离子陷阱技术用于磷酸质谱分析、磷蛋白的生物物理分析、与GEF的蛋白质-蛋白质相互作用分析、GTP酶激活蛋白(GAP)、胍解离抑制物(GDIs)和GPCRs的相互作用、计算机模拟以及划痕和有丝分裂试验来评估RTK在细胞迁移和增殖过程中三聚体G蛋白G？I的磷酸化的结果(S)；2)结合上述方法、免疫荧光、G蛋白酶学和蛋白质-蛋白质相互作用分析来鉴定和表征生长因子在肿瘤细胞迁移和增殖过程中对GIV的磷酸化作用；3)研究RTK-GIV-G？I三元复合体在细胞内组装的方式、时间和地点，以及它们对PI3K信号转导、上述方法结合的细胞迁移/有丝分裂的影响、活细胞成像的FRET分析和核磁共振波谱。这些研究有望为G蛋白和生长因子之间在调节信号转导中的串扰提供新的信息和机制方面的见解。这项拟议的研究有望揭开在两条通路的十字路口组装的信号转导的独特界面，这不仅可能成为有吸引力和有效的治疗靶点，而且所获得的见解也将有助于破译、获取和操纵癌细胞的复杂信号密码。

DESCRIPTION (provided by applicant): Heterotrimeric G proteins are molecular switches that control signal transduction and their dysregulation can herald oncogenesis. Another pathway with undisputed roles and also widely implicated in driving cancer growth and invasion comprises of growth factor receptor tyrosine kinases (RTKs) and the myriad of signals/pathways they initiate. Furthermore, cross-talk between these two seemingly distinct pathways can transactivate each other, and unrestricted signaling due to recruitment of multiple receptors via transactivation is critical in mediating cancer progression. Although indirect transactivation of RTKs by G protein intermediates is well established, insights into how G proteins are transactivated after direct stimulation of RTKs is lacking. The proposed research aims to expose the mechanisms by which growth factor receptors regulate G protein signaling. The preliminary work showcased in this grant reports the discovery of a number of unique signaling complexes at the cross-roads of G protein- and growth factor signaling pathways and unravels the implications of these complexes during cell migration, mitosis and autophagy; processes that are vital for tumor progression. Our studies with GIV, a non-receptor guanidine exchange factor (GEF), trimeric G protein, G?i, and their regulation by the epidermal growth factor receptor, the prototype member of the growth factor RTK family have allowed us to define a novel stratum for signal amplification. The overall goal in this proposal is to gain mechanistic insights into the workings of this stratum and its relevance in cancer progression. Our specific aims are: 1) to assess the consequence(s) of phosphorylation of trimeric G protein, G?i by RTKs during cell migration using in vitro and in vivo phosphorylation assays, linear ion-trap technology for phospho-Mass Spectrometry, biophysical analysis of phosphoprotein, protein-protein interaction assays with GEFs, GTPase activating proteins (GAPs), guanidine dissociation inhibitors (GDIs) and GPCRs, computational modeling, and scratch-wound and mitosis assays; 2) to identify and characterize the phosphomodifications on GIV triggered by growth factors during tumor cell migration and proliferation using a combination of aforementioned approaches, immunofluorescence, G protein enzymology, and protein-protein interaction assays; and 3) to study how, when and where the RTK-GIV- G?i ternary complexes are assembled in cells, and their consequences on PI3K signaling, cell migration/mitosis by a combination of above approaches, FRET assays by live cell imaging, and NMR spectroscopy. These studies are expected to yield novel information and mechanistic insights into the cross-talk between G proteins and growth factors in regulating signaling transduction. The proposed study is expected to unravel unique interfaces for signal transduction assembled at the cross-roads of the two pathways which may not only serve as attractive and effective therapeutic targets, but insights gained will also help decipher, access, and manipulate the cancer cell's complex signaling code.