PI-3-kinase-dependent membrane recruitment of centaurin-alpha2 is essential for its effect on ARF6-mediated actin cytoskeleton reorganisation.
GTPase activating proteins (GAPs) of the centaurin family regulate the actin cytoskeleton and vesicle trafficking through inactivation of the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. We report the functional characterisation of centaurin-alpha(2), which is structurally related to the centaurin-alpha(1) ARF6 GAP. centaurin-alpha(2) contains an N-terminal GAP domain followed by two pleckstrin homology (PH) domains (N-PH and C-PH). In vitro, GFP-centaurin-alpha(2) specifically binds the phosphatidylinositol (PI) 3-kinase lipid products, PI 3,4-P(2) and PI 3,4,5-P(3) (PIP(3)), through its C-terminal PH domain. In agreement with this observation, GFP-centaurin-alpha(2) was recruited to the plasma membrane from the cytosol in EGF-stimulated cells in a PI-3-kinase-dependent manner. Moreover, the C-PH domain is sufficient and necessary for membrane recruitment of centaurin-alpha(2). centaurin-alpha(2) shows sustained kinetics of PI-3-kinase-mediated membrane recruitment in EGF-stimulated cells, owing to its binding to PI 3,4-P(2). centaurin-alpha(2) prevents ARF6 translocation to, and cortical actin formation at, the plasma membrane, which are phenotypic indications for ARF6 activation in EGF-stimulated cells. Moreover, the constitutively active mutant of ARF6 reverses the effect of centaurin-alpha(2) on cortical actin formation. The membrane targeted centaurin-alpha(2) is constitutively active. Together, these studies indicate that centaurin-alpha(2) is recruited in a sustained manner to the plasma membrane through binding to PI 3,4-P(2) and thereby regulates actin reorganisation via ARF6.
Venkateswarlu
Kanamarlapudi
K
Department of Pharmacology, The University of Bristol, Bristol, BS8 1TD, UK. k.venkateswalru@bristol.ac.uk
Brandom
Kevin G
KG
Yun
Hongruo
H
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013997:Time Factors; D014162:Transfection
D020727:ADP-Ribosylation Factors; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D004815:Epidermal Growth Factor; D020690:GTPase-Activating Proteins; D049452:Green Fluorescent Proteins; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C100956:ADAP1 protein, human