Localized α4 integrin phosphorylation directs shear stress-induced endothelial cell alignment

Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized alpha 4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial alpha 4 integrins became phosphorylated on Ser(988). In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced alpha 4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable alpha 4(S(988)A) mutation failed to align in response to shear stress, thus establishing alpha 4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent alpha 4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress.