|
|
|
|
|
|
|
||
J. Cell Biol.,
Volume 140, Number 5, March 9, 1998 1211-1225
* The Center for Molecular and Cellular Biology, and the Department of Physiology and Pharmacology, University of
Queensland, Brisbane, Queensland, 4072, Australia
Phosphatidylinositide (PI) 3-kinase binds to
tyrosyl-phosphorylated insulin receptor substrate-1
(IRS-1) in insulin-treated adipocytes, and this step
plays a central role in the regulated movement of the
glucose transporter, GLUT4, from intracellular vesicles
to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may
be mediated by differential localization of PI 3-kinase
in response to insulin versus PDGF activation. Using
subcellular fractionation in 3T3-L1 adipocytes, we show
that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet
(HSP) and in the plasma membrane (PM), respectively.
The HSP is also enriched in IRS-1, insulin-stimulated
tyrosyl-phosphorylated IRS-1 and intracellular
GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate
the HSP into two separate subfractions: one enriched in
IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as
well as cytoskeletal elements, and another enriched in
membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI
3-kinase remain insoluble. Conversely, at high ionic
strength, membranes remain intact, whereas IRS-1 and
PI 3-kinase become freely soluble. We further show
that this IRS-1-PI 3-kinase complex exists in CHO cells
overexpressing IRS-1 and, in these cells, the cytosolic
pool of IRS-1 and PI 3-kinase is released subsequent to
permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These
data suggest that IRS-1, PI 3-kinase, as well as other
signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the
cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow
confer the absolute specificity of insulin signaling in
these cells.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
