Small GTPase Polybasic Regions: Function and Regulation

描述（由申请人提供）：小gtpase的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。许多这些小gtpase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个小gtpase的能力是非常独特的，这使得SmgGDS成为调节Ras和Rho家族不同成员使用的重叠信号通路的强有力候选。SmgGDS优先激活具有c端多基区（PBR）的小gtpase。我们最近报道了一些Ras和Rho家族成员的pbr含有典型的核定位信号（NLS）序列K-K/R-x- k /R。根据我们的模型，PBR允许小gtpase与SmgGDS关联。如果小GTPase的PBR中含有NLS，则小GTPase和SmgGDS将在细胞核中积累。相反，如果小GTPase的PBR缺乏NLS，则小GTPase和SmgGDS将留在细胞质中。我们认为这种核细胞质穿梭将小gtpase引导到核和细胞质中不同的信号通路和泛素化/降解途径，从而影响它们调节VSMC增殖和迁移的能力。该模型将通过完成以下具体目标进行测试。目的1：验证PBR决定小GTPase调节VSMC增殖和迁移能力的假设。目的2：验证静止和血清或pdgf - bb刺激的细胞中小gtpase的细胞内定位取决于PBR中NLS序列的存在和磷酸化的假设。目的3：验证PBR序列决定小GTPase是否能够a)与SmgGDS关联，B)进行鸟嘌呤核苷酸交换，以及C)在血清或PDGF-BB刺激下促进SmgGDS的核积累的假设。目的4：验证小gtpase的泛素化和降解受PBR中NLS序列及其与SmgGDS的相互作用影响的假设。这些假设将通过检测野生型或突变型小gtpase（包括Rac1、RhoA、rap1a和K-Ras4B）与野生型或突变型SmgGDS在静止和血清或pdgf - bb刺激的大鼠主动脉平滑肌细胞中短暂表达时的功能和物理相互作用来验证。

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.