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suggesting that the Gbg plays a central role in activating the response
downstream from mammalian GPCR chemoattractant receptors (Stephens
et al., 1997; Krugmann et al., 1999). Genetic disruption of p110g and
treatment of macrophages or neutrophils with PI3K inhibitors LY294002/
Wortmannin have been used to demonstrate PH domain translocation is also
PI3K-dependent in mammalian cells. Macrophages and neutrophils isolated
from p110g-null mice show no accumulation of PI(3,4,5)P
3
after chemo-
attractant stimulation (Hirsch et al., 2000; Hannigan et al., 2002; Li et al.,
2000; Rickert et al., 2000; Sasaki et al., 2000; Stephens et al., 2002;
Vanhaesebroeck et al., 1999).
PH domain localization and chemotaxis eciency were linked through the
examination of chemotaxis of neutrophils, macrophages and Dictyostelium
cells in which PI3K activity was abrogated by creation of PI3K gene
knockouts, through the injection of PI3K isoform-specific antibodies and
treatment of cells with PI3K inhibitors. Dictyostelium pi3k1/2 null cells or cells
treated with LY294002 exhibit a loss of cell polarity and defective cell
migration (Chung et al., 2001a,b; Meili et al., 1999; Funamoto et al., 2001).
Activation of Akt/PKB is abolished, translocations of PH domains are
abrogated in response to global stimulation, and PH domains do not localize
to the leading edge. Mammalian neutrophils and macrophages experience a
similar loss of chemotaxis e
ciency, Akt activation, and PH domain
localization (Hirsch et al., 2000; Hannigan et al., 2002; Li et al., 2000; Rickert
et al., 2000; Sasaki et al., 2000; Stephens et al., 2002; Vanhaesebroeck et al.,
1999). Those observations indicate that localized activation of PI3K recruits
the PH domain to the plasma membrane, leading to actin assembly, cell
polarization and directional movement.
PI3K translocates upon stimulation with a chemoattractant
In order to investigate the basis of PH domain localization at the leading edge,
we used GFP fusions of PI3K1 and PI3K2 in Dictyostelium. We showed that
PI3K1-GFP or PI3K2-GFP rapidly and transiently translocates to the plasma
membrane upon stimulation by a chemoattractant (Funamoto et al., 2002).
We discovered rapid accumulation of PI3K1-GFP and PI3K2-GFP to a new
leading edge in response to movement of the chemoattractant source
(Figure 17.1B). PI3K1 and PI3K2, like mammalian Class I PI3Ks, possess a
Ras binding domain, a C2 domain, and a lipid kinase and associated domains.
Surprisingly none of these domains are important for membrane transloca-
tion. Expression of truncated mutants of PI3K1 revealed that the N-terminal
region (1-492) is necessary and su
cient for PI3K translocation to the
membrane. This N-terminal region is unique in the protein database,
