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. ? ?
