The Identification And Functional Study Of ACAP4 Associated Protein

Cytoskeleton contains microfilament,microtubule,intermediate filaments.Cytoskeleton is involved in many cellular processes,specially in endocytosis,vesicular transport,mitosis.In this processes,ligands bind to the specific receptor to activate the intracellular signaling,then adaptor proteins localize to the plasmalemma and recruit the binding moleculars to the specific locations,which regulates cytoskeleton rearrangement and membrane dynamics,affecting the dynamic change of adhension and the traffick of the intracellular organic matter.ADP-ribosylation factor belongs to the superfamily of Ras GTPase,Arfs regulate its acivity by binding GTP or GDP,GEFs and GAPs can activate and inactivate Arfs,respectively.Arf6 is the lowest conserved protein of Arf family.Arf6 GTPase localizes to plasma membrane and endosome,regulating membrane traffic and acting remodeling of endocytosis.Arf6 is associated with many cellular processes,including integrinsdependent cell migration,regulating the cytoskeletal rearrangement in processes of cell migraton and invasion.Our previous studies had identified a novel Arf6 GAP regulatory protein—ACAP4.It contains many domains,including BAR domain,PH domain,GAP domain and ANK repeat sequence.Recent research indicated ACAP4 had a GAP activity to Arf6,ACAP4 was associated with the foamation of focal adhensions and memberan ruffles,but it did not localize to the invapodias and podosomes.Our present research showed ACAP4 interacted with Crk?,an adaptor protein.The amino acid sequences of ACAP4 and Crk? were 560-660 and 130-304 amino acid,respectively.Through bioinformatics analysis,we found the Tyr632 was most likely to the site of phosphorylation,we supposed that Tyr632 may the site of the interaction between ACAP4 and Crk?.Therefore we established the mutant of Tyr632,a series of experiments proved our pevious suppose: ACAP4 interacts with Crk? through the phosphorylation of ACAP4Tyr632.Epidermal growth factor stimulation of HeLa cells in culture can induce the phosphorylation of ACAP4-Tyr632,which promotes its recruitment to the plasma membrane and regulates the activity of Arf6.This study will help us to understand the role and activation mechanism of Arf6 regulating the cell migration and the dynamics of actin cytoskeleton.