Cooke, F. T.

Domin, J.

Hammond, G.

Meunier, F. A.

Osborne, S. L.

Parker, P. J.

Schiavo, G.

University of Queensland eSpace

58th Annual Meeting ⋅ Society of General Physiologists 19a
pholipase C 1 pleckstrin homology (PH) domain in-
hibit Ca2-triggered catecholamines release from per-
meabilized PC12 cells. Furthermore, functional studies
using mutant versions of syt indicate that PIP2 steering
of the cytoplasmic domain of syt toward the plasma
membrane plays a role in exocytosis, perhaps by driving
the close apposition of the vesicle and target mem-
brane in response to Ca2. (Supported by NIH grants
GM 56827 and MH61876, American Heart Association
grant 9750326N. Jihong Bai is supported by an AHA
postdoctoral Fellowship.)
44. Regulation of Phosphoinositide 3-kinase Signal-
ing by PI-5-P 4-kinases VALERIE CARRICABURU,
DEBORAH SARKES, VANESSA JUNG, and LUCIA E.
RAMEH, Boston Biomedical Research Institute, Water-
town, MA
Phosphatidylinositol-5-phosphate (PI-5-P) is a newly
identified phosphoinositide with characteristics of a sig-
naling lipid but with no known cellular function. PI-5-P
levels are controlled by the type II PI-5-P 4-kinases
(PIP4k II), a family of kinases that converts PI-5-P into
PI-4,5-P2. The PI-5-P pathway is an alternative route for
PI-4,5-P2 synthesis as the bulk of this lipid is generated
by the canonical pathway in which phosphatidylinosi-
tol-4-phosphate (PI-4-P) is the intermediate. PI-5-P lev-
els in cells dramatically increase following Shigella flex-
neri infection, due to the activity of the virulence factor
IpgD, a bacterial phosphatase that dephosphorylates
PI-4,5-P2 to generate PI-5-P (Niebuhr et al. 2002. EMBO
J. 21:5069–5078). Therefore, IpgD reverts the reaction
catalyzed by PIP4k II. We have recently shown that over-
expression of PIP4k II reduced PI-3,4,5-P3 levels in cells
stimulated with insulin or cells expressing activated
phosphoinositide 3-kinase (PI3k). This reduction in PI-
3,4,5-P3 levels resulted in decreased activation of the
downstream protein kinase, Akt/PKB. Conversely, ex-
pression of IpgD resulted in Akt activation and this ef-
fect was partially reversed by PIP4k II. Since PIP4k II
expression did not impair insulin-dependent associa-
tion of PI3k with IRS1 but abbreviated Akt activation,
our data support a model in which the PI-5-P pathway
controls insulin signaling that leads to Akt activation by
regulating a PI-3,4,5-P3 phosphatase (Carricaburu et al.
2003. Proc. Natl. Acad. Sci. USA.100:9867–9872). To fur-
ther understand the role of PI-5-P and PIP4k II in
growth-factor signaling and in PI-3,4,5-P3 regulation,
we suppressed the expression of PIP4k II, II, and/or
II in various mammalian cell lines using RNA interfer-
ence. Consistent with our previous results, we find that
suppression of PIP4k II can up-regulate insulin and
EGF-induced PI3k signaling that leads to Akt phos-
phorylation and activation. (Supported by NIH grant
DK063219 and US Army, DOD, DAMD 017–01-1-0155.)
45. Class II PI3-kinase C2 Is Essential for ATP-depen-
dent Priming of Neurosecretory Granule Exocytosis
FRÉDÉRIC A. MEUNIER,1 FRANK T. COOKE,2
SHONA L. OSBORNE,4 GERALD HAMMOND,1 JAN
DOMIN,3 PETER J. PARKER,1 and GIAMPIETRO
SCHIAVO,1 1Cancer Research UK, London Research Insti-
tute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn
Fields, London WC2A 3PX, UK; 2Biochemistry and Molecu-
lar Biology, University of College, London, Darwin Building,
Gower Street, W1E 6BT, London, UK; 3Renal Section, Faculty
of Medicine, Imperial College, London W12 0NN, UK;
4School of Biomedical Sciences, University of Queensland, St.
Lucia, Queensland 4072, Australia
Phosphoinositide-3 kinase (PI3K) activity is impor-
tant for several aspects of neuronal differentiation and
plasticity. However, its direct involvement in regulated
exocytosis is unclear, despite clear evidence for a re-
quirement for phosphoinositides in this process. Neu-
rotransmitter release from synaptosomes and hor-
monal secretion from chromaffin cells is only sensitive
to high concentrations of the PI3K inhibitors wortman-
nin and LY294002 pointing to a possible role for the
less sensitive PI3K-C2. In neurosecretory cells PI3K-
C2 was detected on a subpopulation of mature secre-
tory granules, abutting the plasma membrane. Both
PI3K-C2 antibodies and PI3K inhibitors selectively
prevented ATP-dependent priming in permeabilized
chromaffin cells. Transient expression of PI3K-C2 po-
tentiated secretion, whereas its catalytically inactive mu-
tant abolished exocytosis, suggesting a possible role of
the main catalytic product of this enzyme, PtdIns-3-
phosphate (PtdIns3P), in this process. Treatment of
PC12 cells expressing the PtdIns3P-binding FYVE do-
main with a low concentration of wortmannin selec-
tively abolished early endosomal staining and revealed
a full colocalisation with PI3K-C2 on PC12 granules.
Finally, sequestration of PtdIns3P by the overexpression
of FYVE domain abolished secretion from PC12 cells.
Together these data demonstrate that production of
PtdIns3P along with PtdIns4,5P2 is required in the ac-
quisition of fusion competence that secretory vesicles
undergo during or following docking to the plasma
membrane.
46. Dolichol Phosphate Mannose Synthase Dpm1p
Recruits the Lipid Phosphatase Sac1p to the Endoplas-
mic Reticulum FRANK FAULHAMMER, GERLINDE
KONRAD, BEN BRANKATSCHK, ANDREAS KNOEDLER
 o
n
 O
ctober 19, 2015
jgp.rupress.org
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Published June 28, 2004


Class II PI3-kinase C2 alpha is essential for ATP-dependent printing of neurosecretory granule exocytosis

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Class II PI3-kinase C2 alpha is essential for ATP-dependent printing of neurosecretory granule exocytosis

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