Abstract
Dynamic regulation of the actin cytoskeleton is an essential feature of cell motility. Action of Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP), a family of conserved actin-elongating proteins, is an important aspect of regulation of the actin cytoskeletal architecture at the leading edge that controls membrane protrusion and cell motility. In this study, we performed mutagenesis experiments in overexpression and knockdown-rescue settings to provide, for the first time, direct evidence of the role of the actin-binding protein profilin1 (Pfn1) in VASP-mediated regulation of cell motility. We found that VASP's interaction with Pfn1 is promoted by cell-substrate adhesion and requires down-regulation of PKA activity. Our experimental data further suggest that PKA-mediated Ser137 phosphorylation of Pfn1 potentially negatively regulates the Pfn1-VASP interaction. Finally, Pfn1's ability to be phosphorylated on Ser137 was partly responsible for the anti-migratory action elicited by exposing cells to a cAMP/PKA agonist. On the basis of these findings, we propose a mechanism of adhesion-protrusion coupling in cell motility that involves dynamic regulation of Pfn1 by PKA activity.
Highlights
Dynamic regulation of the actin cytoskeleton is an essential feature of cell motility
Because the Ena/VASP homology 1 (EVH1) domain is responsible for VASP’s targeting to membrane and focal adhesions, and because the EVH2 domain of VASP contains binding sites for G-actin, F-actin, and coiled-coiled regions, we predicted that the P119E/L209E mutations in the PLP regions should not interfere with VASP’s ability to localize at the leading edge, focal adhesions, and actin stress fibers
Based on evidence that Pfn1 profoundly enhances the F-actin elongation capability of Ena/VASP [29] and that both Pfn1 and the PLP region of VASP are required for efficient actin-driven intracellular motility of bacterial pathogens [19, 29], it has been widely postulated that Ena/VASP’s interaction with Pfn1 plays a role in membrane protrusion and cell motility
Summary
Ena, Enabled; VASP, vasodilator-stimulated phosphoprotein; PLP, polyproline; SH, Src homology; MEF, mouse embryonic fibroblast; aa, amino acids; FSK, forskolin; EGFP, enhanced GFP. The random 2D motility of mouse embryonic fibroblast (MEFs) was found to be enhanced in the absence of Ena/ VASP activity [28]. The rate of actin assembly by VASP is dramatically enhanced by its PLP interaction with Pfn (the major isoform of Pfn and a key promoter of membrane protrusion) in vitro [29, 31]. These findings are consistent with enriched Pfn1-VASP interaction at the leading edge of motile cells [32]. We directly demonstrate, for the first time, that VASP regulates cell motility through its interaction with Pfn and that this interaction is regulated by cell adhesion in a PKA-dependent manner that likely involves phosphorylation of Pfn on its Ser137 residue
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
