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
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.