Small GTPase Polybasic Regions: Function and Regulation

小 GTP 酶多碱基区域：功能和调节

• 批准号: 7215668
• 负责人: Carol Lucille Williams
• 金额: $ 27.01万
• 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/7215668
• 关键词: 26S proteasome; Affect; Atherosclerosis; Binding; Blood Vessels; Boxing; C-terminal; Cell Nucleus; Cell Proliferation; Cells; Charge; Complementary DNA; Complex; Cytoplasm; Degradation Pathway; Dominant-Negative Mutation; Epitopes; Family; Family member; Goals; Green Fluorescent Proteins; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Triphosphate Phosphohydrolases; Mass Spectrum Analysis; Modeling; Monomeric GTP-Binding Proteins; Mutate; Mutation; Nuclear; Nuclear Localization Signal; Peptide Signal Sequences; Phosphorylation; Platelet-Derived Growth Factor; Precipitation; Protein Isoforms; Proteins; Rattus; Regulation; Reporting; Research; Research Personnel; Role; Serine; Serum; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Testing; Ubiquitination; inhibitor/antagonist; member; migration; mutant; nucleocytoplasmic transport; platelet-derived growth factor BB; protein B; response; rho; vascular smooth muscle cell migration; vascular smooth muscle cell proliferation

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（由申请方提供）：小GTP酶Ras和Rho家族成员通过调节血管平滑肌细胞（VSMC）的增殖和迁移参与动脉粥样硬化。这些小GTP酶中的许多被鸟嘌呤核苷酸交换因子SmgGDS激活。SmgGDS激活属于不同家族的多种小GTP酶的能力是非常独特的，并且使得SmgGDS成为调节Ras和Rho家族的不同成员所利用的重叠信号传导途径的强有力的候选者。SmgGDS优先激活具有C末端多碱基区（PBR）的小GTP酶。我们最近报道了一些Ras和Rho家族成员的PBRs含有经典的核定位信号（NLS）序列K-K/R-x-K/R。根据我们的模型，PBR允许小GTP酶与SmgGDS相关联。如果一个小的GTdR有一个包含NLS的PBR，那么小的GTdR和SmgGDS将在细胞核中积累。相反，如果小GTdR具有缺乏NLS的PBR，则小GTdR和SmgGDS将保留在细胞质中。我们认为，这种核质穿梭指导小GTP酶在细胞核和细胞质中的不同信号传导和泛素化/降解途径，影响其调节VSMC增殖和迁移的能力。将通过完成以下具体目标来检验这一模式。目的1：检验PBR决定小GT3调节VSMC增殖和迁移的能力的假设。目标二：检验以下假设：在静止和血清或PDGF-BB刺激的细胞中，小GTP酶的细胞内定位取决于PBR中NLS序列和磷酸化的存在。目标3：检验以下假设：PBR序列决定小GTdR是否能够A）与SmgGDS结合，B）进行鸟嘌呤核苷酸交换，以及C）促进SmgGDS在血清或PDGF-BB刺激后的核蓄积。目标4：检验小GTP酶的泛素化和降解受PBR中NLS序列及其与SmgGDS相互作用的影响的假设。当蛋白质在静止和血清或PDGF-BB刺激的大鼠主动脉平滑肌细胞中瞬时表达时，将通过检查野生型或突变型小GTP酶（包括Rac 1、RhoA、Rap 1 A和K-Ras 4 B）与野生型或突变型SmgGDS的功能和物理相互作用来检验这些假设。