Molecular Mechanisms Of ARF6-ACAP4-Acapin Signaling Axis In Cell Migration

Cell migration plays a central role in a wide variety of biological phenomena,such as embryonic development,wound healing,bacterial infections,immune,cancer metastasis.Therefore,the study of cell migration has always been one of the priorities for the present scientific research.For well study of cell migration protein can not only explain the molecular mechanisms of cell migration,but also provide a theoretical basis for the treatment of malignant tumors of the migration.As a key member in Ras protein superfamily,ARF6,plays an important role in the regulation of vesicle transport and actin cytoskeleton rearrangement.More and more data indicates that ARF6 activity is critical for cell migration.Membrane trafficking and remodeling of the actin cytoskeleton are critical activities contributing to cellular events,such as cell growth,migration and tumor invasion.ADP-ribosylation factor 6(Arf6)GTPase activating proteins,ACAP4,have crucial roles in these processes.ACAP4 can reduce ARF6 activity by regulating hydrolysis of GTP bound to Arf6 proteins.In addition,ACAP4 has multiple functional domains,also affects the actin cytoskeleton and membranes by specific interactions with lipids and proteins.Recently results revealed that ACAP4 regulated integrin ?1 recycling in response to activated EGF signal transducted by the adaptor protein Grb2.In addition,EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration.A description of these functions provides insights into the molecular mechanisms by which ACAP4 regulate physiological and pathological cellular events.As an ARF6 GTPase-activation protein,ACAP4 is critical to EGF-elicited cell migration.However,how ACAP4 regulates the proper level of active ARF6 is not well understood.Here we identify a new protein,Acapin,that inhibits ACAP4 mediated inactivation of ARF6 in a dose-dependent manner and show that it can compete with ARF6 for binding ACAP4.Knock-down of Acapin could decrease the cellular ARF6-GTP level,whereas Acapin overexpression would increase the level of cellular ARF6-GTP.Both Acapin and ACAP4 exhibit peripheral membrane localization,the localization of Acapin is essential for EGF-elicited membrane remodeling.Significantly,our biochemical characterization demonstrated that EGF stimulation can induce the active Akt phosphorylated Acapin at serine 247,which strengthens the association Acapin with ACAP4.In addition,the overexpression of the phospho-mimicking mutant of Acapin promotes ARF6 activation and cell migration.Our data uncover Acapin as a modifier of cellular ARF6-GTP through regulation of ACAP4.Acapin phosphorylation facilitates cell migration by sequestering ACAP4 at the cell peripheral membrane to stabilize active ARF6 in response to EGF stimulation.Collectively,our data disclose a prevously uncharcaterized molecular mechanism involved in cell motility and underline how appropriate and timely activation ARF6 is essential for cell migration.