Characterization of EVL-I as a protein kinase D substrate.
EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPbeta and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell-cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell-cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.
Janssens
Katrien
K
Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Belgium.
De Kimpe
Line
L
Balsamo
Michele
M
Vandoninck
Sandy
S
Vandenheede
Jackie R
JR
Gertler
Frank
F
Van Lint
Johan
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003599:Cytoskeleton; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism / Q000648:ultrastructure
D015815:Cell Adhesion Molecules; C102177:Evl protein, mouse; D010703:Phorbol Esters; C089212:protein kinase D; D011493:Protein Kinase C