Identification of the Ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate protein in vivo

Citation
Pj. Lockyer et al., Identification of the Ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate protein in vivo, CURR BIOL, 9(5), 1999, pp. 265-268
Citations number
16
Categorie Soggetti
Experimental Biology
Journal title
CURRENT BIOLOGY
ISSN journal
09609822 → ACNP
Volume
9
Issue
5
Year of publication
1999
Pages
265 - 268
Database
ISI
SICI code
0960-9822(19990311)9:5<265:IOTRGP>2.0.ZU;2-L
Abstract
GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (G APs) [1]. In vitro, it has been shown to bind inositol 1,3,4,5-tetrakisphos phate (IP4), the water-soluble inositol head group of the lipid second mess enger phosphatidylinositol 3,4,5-trisphosphate (PIP3) [2,3]. This has led t o the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4] . Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m ) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane, This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH d omain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GA P1(m) translocation, Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significa ntly, the PIP3-induced recruitment of GAP1(m) did not appear to result in a ny detectable enhancement in its basal Pas GAP activity. From these results , we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the p lasma membrane, but does not appear to be activated, following agonist stim ulation of PI 3-kinase. (C) Elsevier Science Ltd ISSN 0960-9822.