Differential phosphorylation regulates the shear stress-induced polar activity of Rho-specific guanine nucleotide dissociation inhibitor α

The activity of Rho-specific guanine nucleotide dissociation inhibitor alpha (RhoGDI alpha) is regulated by its own phosphorylation at different amino acid sites. These phosphorylation sites may have a crucial role in local Rho GTPases activation during cell migration. This paper is designed to explore the influence of phosphorylation on shear stress-induced spatial RhoGDI alpha activation. Based on the fluorescence resonance energy transfer biosensor sl-RhoGDI alpha, which was constructed to test the RhoGDI alpha activity in living cells, new RhoGDI alpha phosphomimetic mutation (sl-S101E/S174E, sl-Y156E, sl-S101E, sl-S174E) and phosphorylation-deficient mutation (sl-S101A/S174A, sl-Y156A, sl-S101A, sl-S174A) biosensors were designed to test their effects on RhoGDI alpha activation upon shear stress application in human umbilical vein endothelial cells (HUVECs). The results showed lower RhoGDI alpha activity at the downstream of HUVECs (the region from the edge of the nucleus to the edge of the cell along with the flow). The overall decrease in RhoGDI alpha activity was inhibited by Y156A-mutant, whereas the polarized RhoGDI alpha and Rac1 activity were blocked by S101A/S174A mutant. It is concluded that the Tyr156 phosphorylation mainly mediates shear stress-induced overall RhoGDI alpha activity, while Ser101/Ser174 phosphorylation mediates its polarization. This study demonstrates that differential phosphorylation of RhoGDI alpha regulates shear stress-induced spatial RhoGDI alpha activation, which could be a potential target to control cell migration.