Critical roles of phosphorylation and actin binding motifs, but not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migration.
The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Joseph J
JJ
Loureiro
Douglas A
DA
Rubinson
James E
JE
Bear
Gretchen A
GA
Baltus
Adam V
AV
Kwiatkowski
Frank B
FB
Gertler
D000199:Actins / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010641:Phenotype; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C102176:Enah protein, mouse; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C495772:Wasl protein, mouse; C495770:Wasl protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D000098662:Vasodilator-Stimulated Phosphoprotein; D016596:Vinculin; D049452:Green Fluorescent Proteins; D013912:Threonine; D012694:Serine; D011392:Proline; D017868:Cyclic AMP-Dependent Protein Kinases