EGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain

K Venkateswarlu, Francis James Gunn-Moore, JM Tavaré, PJ Cullen

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56 Citations (Scopus)

Abstract

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4,5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane, This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIE production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 mu M) and by coexpression with a dominant negative p85, This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

Original languageEnglish
Pages (from-to)1957-1965
Number of pages9
JournalJournal of Cell Science
Volume112
Publication statusPublished - Jun 1999

Keywords

  • cytohesin-1
  • PC12 cell
  • PI 3-kinase
  • ARF
  • neurite outgrowth
  • GUANINE-NUCLEOTIDE-EXCHANGE
  • ADP-RIBOSYLATION-FACTOR
  • PROTEIN-KINASE-B
  • PLECKSTRIN-HOMOLOGY DOMAINS
  • BRUTONS TYROSINE KINASE
  • EPIDERMAL GROWTH-FACTOR
  • PHOSPHATIDYLINOSITOL 3-KINASE
  • PHOSPHOLIPASE-D
  • FACTOR ARNO
  • INOSITOL 1,3,4,5-TETRAKISPHOSPHATE

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