Role Of Phosphorylation In Shear Stress-induced RhoGDI? Activation

Introduction: Rho guanine nucleotide dissociation inhibitor ?(RhoGDI?)is a vital negative regulator of Rho GTPases.The interaction of RhoGDI? and Rho GTPases affects Rho GTPases localized activation in cell migration directly.However,little is known about the role of RhoGDI? phosphorylation in regulating the affinity of RhoGDI? for Rho GTPaeses in response to shear stress.Methods: A classical parallel plate flow system is used for applying shear stress on human umbilical vein endothelial cells(HUVEC),and the affinity of RhoGDI? and Rho GTPases is measured with a fluorescence resonance energy transfer(FRET)biosensor(sl-RhoGDI?).RhoGDI? phosphomimetic mutations(sl-S101E/S174 E,sl-Y156 E,sl-S101 E,sl-S174E)and phosphorylation-deficient mutations(sl-S101A/S174 A,sl-Y156 A,sl-S101 A,sl-S174A)biosensors are designed to explore the role of phosphorylation in RhoGDI? activation upon shear stress application.PBD-GFP biosenseor is used for detecting active Rac1 distrubution.Fluorescence images are acquired by a FRET microscope and analyzed by Matlab software.Results: 1.Sequencing results show that the phosphorylation sites of RhoGDI? domain in sl-RhoGDI? are mutated to alanine or glutamic acid as expected,and these biosensors can express normally in HUVEC and do not affect cell function.2.The FRET ratio of sl-RhoGDI?(control group),sl-Y156 A and sl-Y156 E all decrease obviously upon shear stress application within 30 min,with lower FRET ratio at the downstream of cells.The decreased FRET ratio is found higher in sl-Y156 A than control group,while lower in sl-Y156 E.3.The shear stress-induced decrease of sl-RhoGDI? FRET ratio,including the difference between the upstream and the downstream,are depressed significantly through PAK1 inhibition.Similarly,the decrease of overall FRET ratio in sl-S101A/S174 A is found lower than control group,while no differenc can be observed between the upstream and downstream.4.The FRET ratio is found higher in sl-S101 A and sl-S174 E than control group,while no difference between upstream and downstream in sl-S101 A and sl-S174 A.5.Shear stress promotes PBD-GFP fluorescence accumulating at the downstream side of HUVEC,and this polarity is inhibited by S101A/S174 A,S174A or S101 A overexpression.Conclusions: This study successfully constructs the FRET biosensors that report the affinity of RhoGDI? mutants for Rho GTPases.Shear stress-induced down-regulated affinity of RhoGDI? for Rho GTPases is mainly affected by the phosphorylation of Tyr156 in RhoGDI?.On the other hand,shear stress-induced polar affinity of Rho GDI? for Rho GTPases and active Rac1 accumulating at downstream side are regulated by the PAK1-mediated phosphorylation of Ser101 and Ser174 sites,but not phosphorylation of Tyr156 in RhoGDI?.This study demonstrates that differential phosphorylation of RhoGDI? plays different roles in regulating the overall and polar affinity of RhoGDI? for Rho GTPases,which provides deep understanding for mechanotransduction in endothelial cells.