PKA-Mediated Regulation of Profilin-1 - Implication in Sprouting Angiogenesis

Angiogenesis is a process of vessel outgrowth from a pre-existing capillary that is implicated in many physiological and pathological conditions. Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, has been previously shown to be up-regulated in vascular endothelial cells during capillary morphogenesis and required for endothelial cell migration, morphogenesis and invasion in vitro. In the first part of this study, we demonstrated that depletion of Pfn1 interferes with sprouting angiogenesis in vitro and ex vivo. In the second part, we further explored how Pfn1 might be biochemically regulated. Our studies suggested that a significant fraction of Pfn1 could exist in a number of phosphorylated states in cells. We showed that Pfn1 can be phosphorylated by Protein Kinase A (PKA) in vitro and in a PKA-dependent manner in vivo. By mass-spectrometry, we identified several potential PKA phosphorylation sites of Pfn1, one of which, T89, at least also appeared to be a bona fide modification site of Pfn1 in vivo. We performed biochemical and in silico analyses to determine the potential consequence of this phosphorylation on the properties of Pfn1. Finally, we showed that activating the PKA pathway affects ligand binding of Pfn1 in cells and negatively impacts both endothelial cell migration and sprouting angiogenesis. Collectively, these observations implicate a possible role for Pfn1 post-translational modification in the PKA-mediated regulation of sprouting angiogenesis