Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239.
The initial step in directed cell movement is lamellipodial protrusion, an action driven by actin polymerization. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family proteins are key regulators of this actin polymerization and can control lamellipodial protrusion rate. Ena/VASP proteins are substrates for modification by cyclic-nucleotide-dependent protein kinases at a number of sites. Phosphorylation of Ser239 of VASP in vitro inhibits its anti-capping and filament-bundling activity but the effects of this modification on lamellipodial structure and function are unknown. To examine the functional effects of this modification in living cells, we studied VASP phosphorylation at Ser239 by nitric oxide (NO) stimulation of cGMP-dependent protein kinase. Using live cell imaging of primary cells transfected with GFP-VASP constructs, we found that NO produced rapid retraction of lamellipodia together with cell rounding that was dependent on guanylate cyclase and type II cGMP-dependent protein kinase. In cells expressing a mutant VASP (Ser239Ala) lacking the site preferentially phosphorylated by this kinase, NO had no effect. Phosphorylation of Ser239 of VASP results in loss of lamellipodial protrusions and cell rounding, and is a powerful means of controlling directed actin polymerization within lamellipodia.
Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow Biomedical Research Centre, 120, University Place, Glasgow, G12 8TA, UK.
Susan L
SL
Lindsay
Sara
S
Ramsey
Michael
M
Aitchison
Thomas
T
Renné
Thomas J
TJ
Evans
D000199:Actins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D048430:Cell Shape; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011554:Pseudopodia / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D012694:Serine; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase