cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domains.
Proteins of the Ena/VASP family are implicated in processes that require dynamic actin remodeling such as axon guidance and platelet activation. In this work, we explored some of the pathways that likely regulate actin dynamics in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, were highly expressed in hematopoietic cells of thymus and spleen. In CD3-activated T-cells, EVL was found in F-actin-rich patches and at the distal tips of the microspikes that formed on the activated side of the T-cells. Like the other family members, EVL localized to focal adhesions and the leading edge of lamellipodia when expressed in fibroblasts. EVL was a substrate for the cAMP-dependent protein kinase, and this phosphorylation regulated several of the interactions between EVL and its ligands. Unlike VASP, EVL nucleated actin polymerization under physiological conditions, whereas phosphorylation of both EVL and VASP decreased their nucleating activity. EVL bound directly to the Abl, Lyn, and nSrc SH3 domains; the FE65 WW domain; and profilin, likely via its proline-rich core. Binding of Abl and nSrc SH3 domains, but not profilin or other SH3 domains, was abolished by cAMP-dependent protein kinase phosphorylation of EVL. We show strong cooperative binding of two profilin dimers on the polyproline sequence of EVL. Additionally, profilin competed with the SH3 domains for binding to partially overlapping binding sites. These data suggest that the function of EVL could be modulated in a complex manner by its interactions with multiple ligands and through phosphorylation by cyclic nucleotide dependent kinases.
Lambrechts
A
A
Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Chemistry, Faculty of Medicine, Ghent University, Ledeganckstraat 35, 9000 Gent, Belgium.
Kwiatkowski
A V
AV
Lanier
L M
LM
Bear
J E
JE
Vandekerckhove
J
J
Ampe
C
C
Gertler
F B
FB
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001704:Biopolymers / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry*; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002478:Cells, Cultured; D003285:Contractile Proteins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008213:Lymphocyte Activation; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D051304:Profilins; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D018909:src Homology Domains; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D001704:Biopolymers; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; C484449:EVL protein, human; C102176:Enah protein, mouse; C102177:Evl protein, mouse; D008840:Microfilament Proteins; C495739:Pfn1 protein, mouse; C495740:Pfn1 protein, rat; D010750:Phosphoproteins; D051304:Profilins; D011506:Proteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D011392:Proline; D017868:Cyclic AMP-Dependent Protein Kinases