Role of VASP phosphorylation for the regulation of microglia chemotaxis via the regulation of focal adhesion formation/maturation.
Microglia activation and migration are known to play crucial roles for the response to brain injuries. Extracellular ADP was reported to induce microglia chemotaxis and membrane ruffles through P2Y12 receptor. In this study, we examined the role of VASP phosphorylation in ADP-induced microglia chemotaxis and membrane ruffle formation. ADP stimulation transiently increased intracellular cAMP level, VASP phosphorylation at Ser153, membrane ruffle formation, and chemotaxis. PKA inhibitor effectively inhibited VASP phosphorylation and chemotaxis, indicating that P2Y12-mediated activation of PKA and subsequent VASP phosphorylation are involved in the regulation of microglia chemotaxis. Forskolin and okadaic acid induced sustained VASP phosphorylation at a high level, causing a significant reduction of the retraction of membrane ruffles and chemotaxis. In forskolin- or okadaic acid-treated cells, phosphorylated VASP remained at the membrane cortex, and size and number of mature focal adhesions were not increased, indicating that prolonged phosphorylation of VASP could inhibit transformation of focal complexes into focal adhesions. VASP knockdown cells showed markedly reduced frequency and distance of membrane ruffling upon ADP stimulation, reinforcing the idea that VASP is required for the ruffle formation. Cells expressing GFP-VASP(S153A) also showed a significant reduction of protrusion distance during ruffle formation, but the frequency and the distance of retraction were not affected by FSK at all. This result suggests that dephosphorylation of VASP might be required for the growth of adhesion strength during membrane retraction. Our results suggest that VASP phosphorylation by PKA plays an important role in membrane ruffle formation and chemotaxis via the regulation of focal adhesion formation/maturation.
Lee
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Department of Pharmacology, Vanderbilt University Medical Center, 468 Robinson Research Building (MRB I), 1215 21st Ave., South@Pierce, Nashville, TN 37232-6600, USA.
Chung
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D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D022081:Cell Surface Extensions / Q000378:metabolism / Q000648:ultrastructure; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D022001:Focal Adhesions / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D017628:Microglia / Q000166:cytology / Q000502:physiology*; D051419:Paxillin / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D000244:Adenosine Diphosphate; D000262:Adenylyl Cyclases; D015815:Cell Adhesion Molecules; D017868:Cyclic AMP-Dependent Protein Kinases; D008840:Microfilament Proteins; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D051057:Proto-Oncogene Proteins c-akt; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D000098662:Vasodilator-Stimulated Phosphoprotein; C548577:P2ry12 protein, mouse