Small GTPase Polybasic Regions: Function and Regulation

小 GTP 酶多碱基区域：功能和调节

• 批准号: 7030289
• 负责人: Carol Lucille Williams
• 金额: $ 27.81万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030289
• 关键词: cell migration; cell proliferation; genetic regulation; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanosinetriphosphatases; immunoprecipitation; phosphorylation; protein degradation; protein localization; protein protein interaction; protein sequence; protein structure function; tissue /cell culture; ubiquitin; vascular smooth muscle

DESCRIPTION (provided by applicant): Members of the Ras and Rho families of small GTPases participate in atherosclerosis by regulating the proliferation and migration of vascular smooth muscle cells (VSMC). Many of these small GTPases are activated by the guanine nucleotide exchange factor SmgGDS. The ability of SmgGDS to activate multiple small GTPases belonging to different families is very unique, and makes SmgGDS a strong candidate to regulate overlapping signaling pathways utilized by different members of the Ras and Rho families. SmgGDS preferentially activates small GTPases that have a C-terminal polybasic region (PBR). We recently reported that the PBRs of some Ras and Rho family members contain the canonical nuclear localization signal (NLS) sequence K-K/R-x-K/R. According to our model, the PBR allows small GTPases to associate with SmgGDS. If a small GTPase has a PBR that contains an NLS, the small GTPase and SmgGDS will accumulate in the nucleus. Conversely, if a small GTPase has a PBR that lacks an NLS, the small GTPase and SmgGDS will remain in the cytoplasm. We propose that this nucleocytoplasmic shuttling directs the small GTPases to different signaling and ubiquitination/degradation pathways in the nucleus and cytoplasm, affecting their ability to regulate VSMC proliferation and migration. This model will be tested by completing the following specific aims. Aim 1: Test the hypothesis that the PBR determines the ability of a small GTPase to regulate VSMC proliferation and migration. Aim 2: Test the hypothesis that the intracellular localization of small GTPases in quiescent and serum- or PDGF-BB-stimulated cells depends on the presence of NLS sequences and phosphorylation in the PBR. Aim 3: Test the hypothesis that the PBR sequence determines whether a small GTPase is able to A) associate with SmgGDS, B) undergo guanine nucleotide exchange, and C) promote the nuclear accumulation of SmgGDS in response to serum or PDGF-BB stimulation. Aim 4: Test the hypothesis that the ubiquitination and degradation of small GTPases is affected by NLS sequences in the PBR and by their interactions with SmgGDS. These hypotheses will be tested by examining the functional and physical interactions of wildtype or mutant small GTPases (including Rac1, RhoA, Rap1 A, and K-Ras4B) with wildtype or mutant SmgGDS when the proteins are transiently expressed in quiescent and serum- or PDGF-BB-stimulated rat aortic smooth muscle cells.

描述（由申请人提供）：小GTPases的RAS和RHO家族的成员通过调节血管平滑肌细胞的增殖和迁移（VSMC），参与动脉粥样硬化。这些小的GTPases中的许多是通过鸟嘌呤核苷酸交换因子SMGGD激活的。 SMGGD激活属于不同家族的多个小型GTP酶的能力非常独特，并使SMGGD成为强大的候选者，以调节RAS和RHO家族的不同成员使用的重叠信号通路。 SMGGD优先激活具有C末端多边区（PBR）的小GTP酶。我们最近报道说，一些RA和RHO家族成员的PBR包含规范核定位信号（NLS）序列K-K/R-X-K/R。根据我们的模型，PBR允许小的GTPase与SMGGDS关联。如果小的GTPase具有包含NLS的PBR，则小的GTPase和SMGGD会在细胞核中积聚。相反，如果一个小的GTPase具有缺乏NLS的PBR，则小的GTPase和SMGGD将保留在细胞质中。我们提出，这种核细胞质穿梭将小的GTPase引导到核和细胞质中的不同信号传导和泛素化/降解途径，影响了它们调节VSMC增殖和迁移的能力。该模型将通过完成以下特定目标来测试。目标1：检验PBR确定小GTPase调节VSMC增殖和迁移的能力的假设。目标2：测试假说，即在静态和血清或PDGF-BB刺激的细胞中，小GTP酶的细胞内定位取决于NLS序列的存在和PBR中的磷酸化。 AIM 3：检验PBR序列确定小GTPase是否能够a）与SMGGDS相关的假设，b）进行鸟嘌呤核苷酸交换，c）促进SMGGD的核积累，以响应血清或PDGF-BB刺激。 AIM 4：检验以下假设：小GTPases的泛素化和降解受PBR中NLS序列以及它们与SMGGD的相互作用的影响。这些假设将通过检查WildType或突变体小GTPases（包括Rac1，RhoA，RAP1 A和K-RAS4B）的功能和物理相互作用，当蛋白质在Quiescent和quiescent和pdgf-bb-bb-bb-bb-bb-stimpunpunpational stampunpunpational tamplatience smallization parmime sploomple syort syort mussce s s s s smggs中时，蛋白质或突变体SMGGDs的功能和物理相互作用。