Abstract
Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) proteins are key regulators that promote or inhibit actin-based motility, cell adhesion, and various aspects of axon guidance. However, a conclusive concept of Ena/VASP functions remains elusive. Here, we report that VASP-deficient fibroblasts, despite normal mammalian Enabled (Mena) and Ena-VASP-like (Evl) expression levels, are highly spread. VASP(-/-) cells cover about twice the substrate surface area as wild type cells, while cell volumes are unchanged. In accordance with these observations, activation of the Rac/p21-activated kinase (PAK) pathway, a crucial element in the regulation of cell spreading, is markedly enhanced in VASP(-/-) cells. Thus, in the absence of VASP Rac activation is dramatically prolonged, and PAK activity is elevated after stimulation with platelet-derived growth factor or serum, respectively. Moreover, VASP-deficient cells show compromised migration and reorientation in a wound healing assay. Collectively, our results reveal a VASP-dependent modulation of the Rac/PAK pathway and Rac/PAK-regulated processes, like cell motility and polarization.
Highlights
The Ena1/VASP family of proteins comprises Drosophila Enabled (Ena), its mammalian counterparts mammalian Enabled (Mena), VASP, and Evl (Ena-VASP-like)
Analysis using an antibody directed against this phosphorylated autophosphorylation site showed that wild type cells displayed a transient p21-activated kinase (PAK) phosphorylation ceasing within about 5 min of fetal calf serum stimulation, whereas a profound and more sustained elevation of phospho-PAK was observed with VASP (Ϫ/Ϫ) mouse cardiac fibroblast cell lines (MCFB) (Fig. 6A)
VASP-dependent Inhibition of the Rac/PAK Pathway—The absence of VASP expression leads to increased cell spreading, a characteristic feature of Rac/PAK pathway activation (19, 38 – 40)
Summary
Ena, Enabled; VASP, vasodilator-stimulated phosphoprotein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CRIB, Cdc42/Rac-interactive binding; DIG, digoxigenin; DMEM, Dulbecco’s modified Eagle’s medium; Evl, EnaVASP-like; GST, glutathione S-transferase; GTP␥S, guanosine-5Ј-O-(3thiotriphosphate); IRSp53, insulin receptor substrate p53; MCFB, mouse cardiac fibroblasts; PAK(s), p21-activated kinase(s); PBD, p21binding domain; PDGF, platelet-derived growth factor; PIX, PAK-interacting exchange factor; PKL, paxillin kinase linker; Scar, suppressor of cAR; WASP, Wiskott-Aldrich-Syndrome protein; Mena, mammalian Enabled; SH, Src homology; LIMK, LIM kinase These proteins are involved in actin filament formation at the plasma membrane [4], plasma membrane protrusion [5, 6], the acceleration of actin-based listerial motility [7, 8], and in the establishment of cell-cell adhesion [9]. Our results demonstrate an unexpected functional link between VASP and small GTP-binding proteins and help to resolve the apparently contrary effects of Ena/VASP proteins on actin-based motility
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