PKA and PKGDependent Phosphorylation of EnaVASP Proteins

Ena/VASP proteins show tightly regulated and reversible phosphorylation. VASP was initially isolated from human platelets as a 46-kDa membrane-associated protein that is phosphorylated by PKA and PKG (Halbrugge and Walter 1989; Halbrugge et al. 1990). Three phosphorylation sites were identified on VASP: Ser157, Ser239 and Thr274, all of which can be phosphorylated by either PKA or PKG (Butt et al. 1994). The phosphorylation of VASP at Ser157 results in the apparent mobility shift from 46 to 50 kDa observed on SDS-PAGE. Ser157 is the preferred site of phosphorylation for PKA, which phosphorylates VASP at this site with faster kinetics than at Ser239 either in vitro or in vivo. Ser239 is the preferred phosphorylation site for PKG in vitro. Experiments in intact human platelets showed that PKG

phosphorylates either Ser157 or Ser239 with similar kinetics, but the level of Ser157 phosphorylation is only around 50% of that induced by PKA activation (Smolenski et al. 1998). This and further evidence suggested a predominant role of PKG in the Ser239 phosphorylation of VASP in platelets and other cells (Ibarra-Alvarado et al. 2002; Cook and Haynes 2007). On the other hand, opposing evidence has appeared in the literature (Li et al. 2003), which reports that in human platelets cGMP-dependent phosphorylation of VASP at Ser239 is predominantly PKA-dependent. Thr274 is phosphorylated by both PKA and PKG, but only following phosphorylation of VASP on Ser157 and Ser239.

PKA is responsible also for the phosphorylation of other members of the Ena/ VASP family. EVL is phosphorylated on Ser156 (corresponding to VASP Ser157) in a PKA-dependent manner both in vitro and in vivo (Lambrechts et al. 2000). As for VASP Ser157, the phosphorylation of EVL on Ser156 induces a mobility shift in SDS-PAGE. The other two phosphorylation sites of VASP (Ser239 and Thr278) are not conserved in EVL, where the corresponding residues are instead mutated to glutamine and alanine, respectively. Mena, the other mammalian member of the Ena/VASP family, is also phosphorylated in a PKA-dependent manner (Gertler et al. 1996). Mena contains two serine-phosphorylation sites corresponding to VASP Ser157 and Ser239, but not the Thr-phosphorylation site, and their phosphorylation leads to a mobility shift similar to the one observed for VASP (Gertler et al. 1996; Lebrand et al. 2004).

0 0

Post a comment