Identification of centaurin-alpha1 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)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.
Venkateswarlu
K
K
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, U.K.
Oatey
P B
PB
Tavaré
J M
JM
Jackson
T R
TR
Cullen
P J
PJ
D020727:ADP-Ribosylation Factors; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D005816:Genetic Complementation Test; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010750:Phosphoproteins; D011485:Protein Binding; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C100956:ADAP1 protein, human; C491062:Adap1 protein, rat; D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C089147:GCS1 protein, S cerevisiae; D020690:GTPase-Activating Proteins; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C039077:platelet protein P47; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors