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

Biochem J. 1999 Jun 1;340 ( Pt 2)(Pt 2):359-63.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factors
  • Adaptor Proteins, Signal Transducing
  • Animals
  • Base Sequence
  • Blood Proteins / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cloning, Molecular
  • DNA, Complementary
  • DNA-Binding Proteins / metabolism*
  • Enzyme Activation
  • Fungal Proteins / metabolism*
  • GTP-Binding Proteins / metabolism*
  • GTPase-Activating Proteins*
  • Genetic Complementation Test
  • Humans
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • PC12 Cells
  • Phosphatidylinositol Phosphates / metabolism*
  • Phosphoproteins*
  • Protein Binding
  • Rats
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*

Substances

  • ADAP1 protein, human
  • Adap1 protein, rat
  • Adaptor Proteins, Signal Transducing
  • Blood Proteins
  • Carrier Proteins
  • DNA, Complementary
  • DNA-Binding Proteins
  • Fungal Proteins
  • GCS1 protein, S cerevisiae
  • GTPase-Activating Proteins
  • Nerve Tissue Proteins
  • Phosphatidylinositol Phosphates
  • Phosphoproteins
  • Saccharomyces cerevisiae Proteins
  • phosphatidylinositol 3,4,5-triphosphate
  • platelet protein P47
  • GTP-Binding Proteins
  • ADP-Ribosylation Factors

Associated data

  • GENBANK/AJ006422