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

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P-a. In this report we have addressed whether centaurin-alpha(1) a human homologue of centaurin-alpha(1) binds PtdIns(3,4,5)P-3, in vivo and furthermore, identified a potential physiological function for centaurin-alpha(1). Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha(1) (GFP-centaurin-alpha(1)), we demonstrated the rapid plasma membrane recruitment of cytosolic GFPcentaurin-alpha(1), following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha(1) pleckstrin homology domains and was blocked by the PtdIns(4,5)P-2, 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 mu M), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha(1) 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-alpha(1). Taken together, our data demonstrated that centaurin-alpha(1) 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)P-3,.