Centaurin-alpha1 is an in vivo phosphatidylinositol 3,4,5-trisphosphate-dependent GTPase-activating protein for ARF6 that is involved in actin cytoskeleton organization.
The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6GDP. Centaurin-alpha1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we show that centaurin-alpha1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-alpha1 also inhibited cortical actin formation in a PIP3-dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-alpha1. An artificially plasma membrane-targeted centaurin-alpha1 bypasses the requirement of PIP3 for its involvement in ARF6 inactivation, suggesting that PIP3 is required for recruitment of centaurin-alpha1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-alpha1 negatively regulates ARF6 activity by functioning as an in vivo PIP3-dependent ARF6 GAP.
Venkateswarlu
Kanamarlapudi
K
Department of Pharmacology, School of Medical Sciences, The University of Bristol, Bristol BS8 1TD, UK. k.venkateswarlu@bristol.ac.uk
Brandom
Kevin G
KG
Lawrence
Joanna L
JL
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014162:Transfection
C100956:ADAP1 protein, human; D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human