Identification of centaurin-α1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1

Abstract

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica

“…These PH domains mediate translocation of centaurin-␣1 from the cytoskeleton to the plasma membrane upon stimulation of cells with growth factors (64,86). Our finding that centaurin-␣1 is needed for accumulation of F-actin during invasin-or InlB-mediated uptake (see Table S5 in the supplemental material) is in general agreement with results indicating a role for this human protein's GAP activity in growth factor- induced remodeling of the actin cytoskeleton (64).…”

p42IP4/Centaurin α1, a Brain-specific PtdIns(3,4,5)P3/Ins(1,3,4,5)P4-binding Protein: Membrane Trafficking Induced by Epidermal Growth Factor is Inhibited by Stimulation of Phospholipase C-coupled Thrombin Receptor

“…Studies from our laboratory and others have shown that p42 IP4 /centaurin a1 binds in vitro with high affinity and specificity to two second messengers, Ins(1,3,4,5)P 4 and PtdIns(3,4,5)P 3 (8,14,15). Moreover, p42 IP4 is recruited to the plasma membrane upon stimulation of PI-3 kinase (8,10). In vitro studies on p42 IP4 from pig cerebellum have shown that p42 IP4 dissociates from cerebellar membranes by incubation with Ins(1,3,4,5)P 4 .…”

“…PH domains, which contain approximately 120 amino acids, are found in many proteins, such as protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinase-1 (PDK1) and phospholipase C (PLC)-c1 [reviewed in (3,4)]. PH domains occur in proteins of the cytohesin family [cytohesin-1, Arf nucleotide-binding-site opener (ARNO; cytohesin-2), general receptor for phosphoinositides-1: GRP1 (cytohesin-3)] [reviewed in (5)], in Ras GTPase activating proteins (GAP1 m and GAP1 IP4BP ) (6) and in members of the centaurin family (p42 IP4 /centaurin a1; PIP3BP, and centaurin a2) (7)(8)(9)(10). For these proteins in vitro binding to phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P 3 ) and inositol (1,3,4,5) tetrakisphosphate (Ins(1,3,4,5)P 4 ) has been reported [reviewed in (5,11)].…”

“…There is also a N-terminal zinc finger (ZF) motif, which is highly homologous to the catalytic region of rat ADP-ribosylation factor 1 (ARF1) GTPase activating protein (GAP) and yeast ARF-GAP, Gcs1 (8,9,12,13). Studies from our laboratory and others have shown that p42 IP4 /centaurin a1 binds in vitro with high affinity and specificity to two second messengers, Ins(1,3,4,5)P 4 and PtdIns(3,4,5)P 3 (8,14,15). Moreover, p42 IP4 is recruited to the plasma membrane upon stimulation of PI-3 kinase (8,10).…”

“…The C-terminal PH domain (PHC) of the centaurin family, which shows a high degree of homology with the PH domain of the cytohesins, was proposed to be responsible for PtdIns(3,4,5)P 3 binding (8). In previous studies two highly conserved arginine residues within the PH domains of p42 IP4 /centaurin a1 were mutated.…”