PI-3-kinase-dependent membrane recruitment of centaurin-α2 is essential for its effect on ARF6-mediated actin cytoskeleton reorganisation

Abstract

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-␣ 2 , which is structurally related to the centaurin-␣ 1 ARF6 GAP. Centaurin-␣ 2 contains an N-terminal GAP domain followed by two pleckstrin homology (PH) domains (N-PH and C-PH). In vitro, GFP-centaurin-␣ 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, GFPcentaurin-␣ 2 was recruited to the plasma membrane from the cytosol in EGF-stimulated cells in a PI-3-kinasedependent manner. Moreover, the C-PH domain is sufficient and necessary for membrane recruitment of centaurin-␣ 2 . Centaurin-␣ 2 shows sustained kinetics of PI-3-kinase-mediated membrane recruitment in EGFstimulated cells, owing to its binding to PI 3,4-P 2 . Centaurin-␣ 2 prevents ARF6 translocation to, and cortical actin formation at, the plasma membrane, which are phenotypic indications for ARF6 activation in EGFstimulated cells. Moreover, the constitutively active mutant of ARF6 reverses the effect of centaurin-␣ 2 on cortical actin formation. The membrane targeted centaurin-␣ 2 is constitutively active. Together, these studies indicate that centaurin-␣ 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.

ADAP2in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome

“…ADAP2 is known to regulate microtubule stability10 and the activity of ARF6, a GTPase involved in cellular motility, adhesion and polarity by regulating cytoskeleton remodelling and cortical actin formation 9. The alteration of these functions might impair adhesion and migration properties of AV valve cells, explaining their disorganisation and the irregular valve architecture observed in adap2 morphants.…”

“…Centaurin-α-2 is recruited to the plasma membrane where it specifically regulates actin cytoskeleton remodelling via ARF6, indicating an important role in exocytosis and cell motility 9. Moreover, it was recently shown to interact with microtubules and to increase their stability 10…”

Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6)

“…The EFA6R E134K mutant was generated using a QuikChange II site-directed mutagenesis kit (Stratagene). The GFP-EFA6R and its deletion mutants were targeted to membranes by attaching a C-terminal C AAX motif using PCR with a 3′-primer containing the coding sequence for the C AAX motif from K-Ras (KDGKKKKKKSKTKCVIM) as described previously (23). The ARF6-HA/pXS, ARF1-HA/pXS, ARF6 T27N -HA/pXS, and ARF1 T31N -HA/pXS constructs were used to express ARF proteins with a C-terminal hemagglutinin (HA) epitope tag in mammalian cells (24).…”

“…The in vitro PI binding assay was performed as described previously (23). Briefly, COS7 cells transfected for 2 days with GFP, GFP-tagged EFA6R or its mutants, or GFP-PLCδPH were treated with lysis buffer (10 m m HEPES, pH 7.4, 150 m m NaCl, 0.5% Nonidet P-40, and 5 m m dithiothreitol) containing 1% mammalian protease inhibitor mixture.…”

Cloning of a centaurin-α1 like gene MjCent involved in WSSV infection from shrimp Marsupeneaus japonicus

“…Several studies have confirmed that this protein family is involved in the cell cytoskeleton [9][10][11] and regulates actin cytoskeleton organization in vivo as a GTPaseactivating protein (GAP) of ARF6 [12], a member of the ARF family of GTPases [13]. In addition, identifications of associations of centaurin-a1 with casein kinase Ia (CKIa) [14,15], Ran binding protein in microtubule-organizing center (RanBPM) [16], kinesin motor protein (KIF13B) [17,18] and protein kinase C (PKC) [19] further indicates the significant potential of centaurin-a1 in cellular cytoskeletal remodeling, vesicle trafficking and thus cellular innate immunity.…”