Regulation of EnaVASP Protein Functions by Phosphorylation

The phosphorylation of Ena/VASP proteins has different functional consequences. First of all, the regulation of actin filament structure by Ena/VASP proteins is regulated by phosphorylation. In vitro phosphorylation of VASP resulted in the down-regulation of its filament bundling and anti-capping activities (Harbeck et al. 2000; Barzik et al. 2005). Profilin enhances VASP anti-capping activity in a dose-dependent manner. Phosphorylation also inhibits VASP anti-capping activity in the presence of profilin (Barzik et al. 2005). Similarly to VASP, PKA-dependent phosphorylation of EVL was shown to impair the ability of this protein to enhance actin polymerisation (Lambrechts et al. 2000). Finally, contrasting evidence has been reported regarding the regulation of VASP's ability to bind actin. PKA-dependent phosphorylation of VASP on Ser157 was reported to potentiate F-actin binding by Laurent et al. (1999), whilst Harbeck et al. (2000) described a reduction of F-actin binding as a consequence of the same post-translational modification. A possible explanation for this contradiction lies in the different salt concentrations used for the co-sedimentation assays in these two reports. Nonetheless, the acceptance of a negative regulatory role for the PKA-dependent phosphorylation of Ena/VASP proteins on their anti-capping activity suggests that this post-translational modification should also negatively regulate F-actin binding, therefore in agreement with Harbeck et al. (2000).

The PKA-dependent phosphorylation of Ena/VASP family members regulates their ability to interact with other proteins. Lambrechts et al. (2000) showed that the interaction of EVL with Abl and n-Src SH3 domains is selectively abolished by PKA-dependent phosphorylation of this Ena/VASP protein. Other protein-protein interactions (e.g. with Lyn SH3 domain, Fe65 WW domain or profilin) were not affected by the PKA-dependent phosphorylation of EVL. Moreover, the PKA-dependent phosphorylation of VASP was shown to inhibit its interaction with Abl tyrosine kinase (Howe et al. 2002). On the other hand, the interaction of VASP with profilin, vinculin and zyxin is not affected by PKA-dependent phosphorylation (Harbeck et al. 2000). Finally, the Tyr-phosphorylation of Drosophila Ena was shown to inhibit its interaction with Abelson kinase (Comer et al. 1998).

Importantly, the phosphorylation state of Ena/VASP appears to play an important role in the regulation of cell morphology and motility. In neurons, PKA-dependent phosphorylation of Ena/VASP proteins, and in particular the phosphorylation of Mena Ser236, leads to filopodia production and neurite growth (Lebrand et al. 2004). Hyperphosphorylation of VASP Ser157 in human platelets is associated with hyperpro-duction of filopodia and increased platelet aggregation in response to collagen

(Pula et al. 2006). In neutrophils, activation of PKG induced both VASP phosphorylation and cell spreading (Lawrence and Pryzwansky 2001). On the contrary, in cultured fibroblastic cells, there is evidence that the PKA/PKG-dependent phosphorylation of Mena negatively regulates fibroblast migration (Loureiro et al. 2002). Finally, in human umbilical vein endothelial cells (HUVECs), there is evidence that PKG activation induces VASP phosphorylation and reduces cell migration (Smolenski et al. 2000).

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