The Effect Of VASP Phosphorylation On Cell Migration And Its Regulatory Mechanism

Cell Migration is an important process involved in embryonic development, wound healing, angiogenesis, and tumor metastasis. Cell migration is a dynamic procedure based on the remodeling of cell cytoskeleton. During migration actin polymerization and depolymerization are required for extending a leading edge in the direction of movement.An actin binding protein Vasodilator-stimulated phosphoprotein (VASP) is a key substrate for PKA, which promote nucleation and assembly of actin filaments. VASP is a critical role in many actin associated processes. However, the role of VASP phosphorylation in cell migration is not entirely clear. In addition, how extracellular stimulus direct the signals to VASP that regulate cell migration are still obscure.Recent findings have shown that a growing family of cellular organizing proteins, A-kinase anchoring proteins (AKAPs) that anchor inactive PKA holoenzymes to defined subcellular compartments, therby efficiently activating catalytic subunits of PKA and selecting appropriate substrate in those regions.WAVE-1, a member of the Wiskott-Aldrich syndrome family (WASP) of adaptor proteins involved in Rac induced the formation of the lamellipodial protrusions, has recently been identified as an AKAP that targets to actin cytoskeleton. Thus, We wonder the effect of WAVE-1 binding to PKA on cell migration and the regulation of WAVE-1 on PKA-VASP signal transduction.In present study, we investigated the role of VASP phosphorylation in cell migration and the effect of WAVE-1 on expression of PKA and VASP through cell experiments in vitro and animal model. The aim is to investigate the effect of VASP phosphorylation on cell migration and its regulatory mechanism. The study was divided into following four parts. Part 1 The effect of phosphorylation and the site-directed mutagenesis of phosphorylation sites of VASP on cell migrationAIMS:Phosphorylation of VASP is critical for the regulation of its function during cell migration. Human VASP has three phosphorylation sites at Ser-157, Ser-239 and Thr-278, which were phosphorylated by PKA/PKG kinases. Our study investigated the expression and distribution of VASP in migrating VSMC and the effect of the site-directed mutagenesis of phosphorylation sites of VASP on cell migration.METHODS:(1)The cell migration velocity of ECV304 cell line was detected by cell scratch wound assays. Phosphorylation level of VASP at Ser 157 site and non-phosphorylation level of VASP were detected respectively by western blot assay. In addition, redistribution of actin cytoskeleton and VASP was examined through immunofluorescence microscopy.(2) Followly, the effect of the site-directed mutagenesis of phosphorylation sites of VASP on cell migration was observed assessed using transwell migration chambers. The mutant VASP-S157A based on the expression vector pcDNA3.1+ were constructed to encode human VASP in which the phosphorylation site on Ser157 was site-directed mutated to a non-phosphorylatable alanine residue. Similarly, VASP-S239A was site-directed mutated to an alanine residue on Ser239.RESULTS:(1) PDGF promote obviously cell migration of ECV304. Meanwhile, PDGF stimulation led to high phosphorylation expression of VASP and reduced non-phosphorylation expression of VASP compared with that of control cells. Immunofluorescence result show a few actin filaments randomly oriented in different directions and VASP show sporadic dot distribution Originally. After PDGF stimulation, cells exhibited thick actin fibers, which aligned mainly in numerous elongated pseudopods along the direction of migration. VASP translocated at the two extremities of actin fibers and partly accumulated at leading edge of pseudopods.(2) VASP-S157A, VASP-S239A and corresponding control vector were transiently transfected into ECV304 cells for 24 hours, and then the cells were used in the experiments. VASP-S157A blocked VASP phosphorylation on Serl57 when expressed in ECV304 cells, which led to diminution of migration under PDGF treatment. VASP-S239A blocked VASP phosphorylation on Ser239 when expressed in cells, which also arrest migration of ECV304 cells. However, no significant difference in the inhibitory effect on cell migration betweenVASP-S157A and ASP-S239A.CONCLUSION:Phosphorylation of VASP has a positive role in the regulation of cell migration. Ser-157 and Ser-239 are both important sites for VASP phosphorylation on promoting migration of cell migration. Part 2 The regulation of PKA and AKAP on PDGF induced cell migrationAIMS:To investigate the effect of AKAP and PKA on PKA-VASP pathway and cell migration, PKA activity was regulated by blocking the interaction of AKAP and PKA or inhibiting PKA activation.METHODS:The cell migration velocity of ECV304 cell line was detected by cell scratch wound assays. Activity of PKA within the cells was detected by PepTag(?) assay for non-radioactive detection. Also, phosphorylation level of VASP at Ser 157 site and non-phosphorylation level of VASP were detected respectively by western blot assay. In addition,we examined redistribution of actin cytoskeleton and VASP through immunofluorescence microscopy.RESULTS:PDGF promoted obviously cell migration of ECV304. Meanwhile, PDGF stimulation led to enhanced PKA activity, high phosphorylation expression of VASP compared with control cells. However, cell migration upon the treatment of PDGF was significantly inhibit in the presence of St-Ht31. In comparison with PDGF group, decreased PKA activity, reduced phosphorylated VASP were also observed by adding PDGF into cells pretreated with St-Ht31. H-89 treatment also caused the similar results. Compared with obvious actin remodeling and VASP localization in leading edge of pseudopods induced by PDGF, actin stress fiber formation is reduced and VASP show dispersive dots localized at focal adhension of cytomembrance in response to St-Ht31. Furthermore, H89 blocked actin stress fiber formation. Occasional week dots of VASP were diffusely localized in the cytoplasm.CONCLUSION:PDGF induced actin cytoskeleton remodeling and cell migration can be blocked by inhibiting the activity of PKA. Also, the interaction of AKAP with PKA is involved in the regulation of actin cytoskeleton and cell migration. Part 3 The exploration of the regulatory mechanism of WAVE-1 on cell migrationAIMS:WAVE-1, a recently identified member of A-kinase-anchoring proteins (AKAPs) family, was reported to anchor cyclic AMP-dependent protein kinase (PKA) with various components to dynamic sites of the actin cytoskeleton. However, the function of WAVE-1 binding to PKA in cell migration has not been evaluated yet. Our study is to investigate the effect of WAVE-1-PKA interaction on cell migration and the role of putative effector VASP in this process via WAVE-1-PKA signaling pathway.METHODS:We stably transfected a plasmid encoding WAVE-1 into ECV304 endothelial cells, and examined the effects on cell migration using transwell migration chambers, PKA activity and VASP phosphorylation. We examined redistribution of PKA in WAVE-1 stable cells through immunofluorescence microscopy.RESULTS:We found that the cell migration was arrested and the PKA activity was significantly elevated in response to PDGF stimulation in the cells. Disruption of the WAVE-1-PKA interaction reversed the effect on cell migration and decreased PKA activity. In addition, PKA accumulated at actin ring structures at the periphery of the nuclear within WAVE-1 overexpressed cells, in contrast to sporadic distribution within control cells, PDGF stimulation resulted in redistribution of PKA to cytoplasm. Another, alteration of VASP phosphorylation level was consistent with the alteration of PKA levels in WAVE-1 stable cells, and overexpression of a constitutive active mutant form of VASP (VASP-S157A) led to further diminution of cell migration.CONCLUSION:Our study suggests that overexpression of WAVE-1 facilitates PKA activation and VASP phosphorylation, which is regulated by the interaction of WAVE-land PKA. Also, hyper-activation of PKA maybe accounted for down regulation of haptotactic mobility and Chemotaxis in WAVE-1 stable cells. Meanwhile, overexpression of WAVE-1 lead to abnormal PKA subcellular localization.In addition, VASP phosphorylation on Ser157 has a positive role in maintaining cell migratory ability during this process. Part 4 The expression of PKA and VASP within migrating VSMC in type 2 diabetic rats macroangiopathy modelAIMS:To investigate the relationship between VSMC migration and VASP expression in type 2 diabetic rats macroangiopathy model.METHODS:Eight-week-old male SD rats were divided into two groups:control(n=10) and type 2 diabetes (n=12). The control group was fed with a normal chow diet; the latter was fed with a high-fat diet and intraperitoneally injected with STZ (25mg/kg) for one time to induce type 2 diabetes. Feeding was administered daily for 8 weeks. The plasma glucose, HbAlc,blood cholesterin and Insulin were detected by biochemical methods. The aortas of type 2 diabetic rats were used in experiments, morphological changes of aorta and cell migration of VSMC were observed by HE staining,transmission electron microscope and immunohistochemistry. The expression and redistribution of VSMC,PKA and phosphorylated VASP were observed by immunohistochemistry.RESULTS:The plasma glucose, HbAlc and blood cholesterin were significantly increased in type 2 diabetes rats model group. Insulin sensitivity was reduced, identified with an glucose test using HOMA-IR index. Morphological changes of aorta were observed in type 2 diabetes model group. Compared with smooth intima and orderly arranged medial VSMC of aorta in the control rats, the thickness of media was increased, the elastic lamella within the intima was destroyed and the VSMCs in the media proliferated, some VSMCs in the media migrated to the intima in type 2 diabetic rats. Immunohistochemistry with anti-PKA antibody showed that expression of PKA was distributed in the cytoplasm of VSMC. VSMCs expressed PKA were diffused in media in control group. However, the number of VSMCs expressed PKA was increased and mainly distributed in the region near the intima in type 2 diabetic rats. Expression of VASP was distributed in the cytoplasm of VSMC. Similarly, the distribution of VSMCs expressed VASP was consistent with that of PKA.CONCLUSION:The animal model of type 2 diabetic macroangiopathy was successfully established. Our results indicate expression of PKA and phosphorylated VASP were correlated with cell migration of VSMCs.