Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin.
The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.
Harbeck
B
B
Department of Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.
Hüttelmaier
S
S
Schluter
K
K
Jockusch
B M
BM
Illenberger
S
S
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003285:Contractile Proteins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006023:Glycoproteins; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008667:Metalloproteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D051304:Profilins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D016596:Vinculin / Q000378:metabolism; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D015815:Cell Adhesion Molecules; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D006023:Glycoproteins; D008024:Ligands; D008667:Metalloproteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; C495739:Pfn1 protein, mouse; D010750:Phosphoproteins; D051304:Profilins; D011994:Recombinant Proteins; C092842:ZYX protein, human; C498285:Zyx protein, mouse; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein; D016596:Vinculin; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases