Association of Impaired Phosphatidylinositol 3-Kinase Activity in GLUT1-Containing Vesicles with Malinsertion of Glucose Transporters into the Plasma Membrane of Fibroblasts from a Patient with Severe Insulin Resistance and Clinical Features of Werner Syndrome

2000 ◽  
Vol 85 (2) ◽  
pp. 905-918 ◽  
Author(s):  
C. Kausch
Keyword(s):  
Insulin Resistance ◽  
Plasma Membrane ◽  
Clinical Features ◽  
Kinase Activity ◽  
Werner Syndrome ◽  
Glucose Transporters ◽  
Phosphatidylinositol 3 Kinase ◽  
Severe Insulin Resistance ◽  
Phosphatidylinositol 3

scholarly journals Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

2000 ◽  
Vol 279 (2) ◽  
pp. E266-E274 ◽  
Author(s):  
Paul G. Drake ◽  
Alejandro Balbis ◽  
Jiong Wu ◽  
John J. M. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Metabolic Effects ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

scholarly journals Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells

1996 ◽  
Vol 313 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Jing YANG ◽  
James F. CLARKE ◽  
Catriona J. ESTER ◽  
Paul W. YOUNG ◽  
Masato KASUGA ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Time Course ◽  
Glucose Transporter ◽  
Kinase Activity ◽  
Insulin Receptor Substrate ◽  
Low Density ◽  
Intracellular Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (≈ 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti-[insulin receptor substrate 1 (IRS1)] and anti-α-p85 antibodies show the same level of insulin-stimulated activity, ≈ 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of α-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

scholarly journals S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3

2001 ◽  
Vol 21 (7) ◽  
pp. 2423-2434 ◽  
Author(s):  
Antonio Chiloeches ◽  
Clive S. Mason ◽  
Richard Marais
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Ras Activation ◽  
High Concentrations ◽  
Dependent Pathway ◽  
K Activity ◽  
Phosphatidylinositol 3

ABSTRACT The Raf-1 serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-1 to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-1 activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (Pl3-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of Pl3-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when Pl3-K activity is completely blocked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-1 kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-1 to the plasma membrane. Taken together, our data show that growth factor-stimulated Raf-1 activity is independent of Pl3-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.

scholarly journals Human Placental Growth Hormone Increases Expression of the P85 Regulatory Unit of Phosphatidylinositol 3-Kinase and Triggers Severe Insulin Resistance in Skeletal Muscle

Endocrinology ◽  
2004 ◽  
Vol 145 (3) ◽  
pp. 1144-1150 ◽  
Author(s):  
Linda A. Barbour ◽  
Jianhua Shao ◽  
Liping Qiao ◽  
Wayne Leitner ◽  
Marianne Anderson ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Normal Pregnancy ◽  
Dominant Negative ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Cellular Mechanisms ◽  
Severe Insulin Resistance ◽  
Placental Growth Hormone ◽  
Glut 4 Translocation ◽  
Phosphatidylinositol 3

Abstract The insulin resistance of normal pregnancy is necessary to divert fuels to the fetus to meet fetal growth demands and is mediated by placental hormones. We recently demonstrated that human placental GH (hPGH) can trigger severe insulin resistance in transgenic (TG) mice. In this study we sought to elucidate the cellular mechanisms by which hPGH interferes with insulin signaling in muscle in TG mice. Insulin-stimulated GLUT-4 translocation to the plasma membrane (PM) was reduced in the TG compared with wild-type (WT) mice (P = 0.05). Insulin receptor (IR) levels were modestly reduced by 19% (P < 0.01) in TG mice, but there were no changes in phosphorylation of IR or IR substrate-1 (IRS-1) between WT and TG mice. A singular finding was a highly significant increase in the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase; P < 0.001), yet a reduced ability of insulin to stimulate IRS-1-associated PI 3-kinase activity (P < 0.05). Although the levels of the p110 catalytic subunit protein of PI 3-kinase and IRS-1 were unchanged in the TG mice, insulin’s ability to stimulate p110 association with IRS-1 was markedly reduced (P < 0.0001). We demonstrate a unique mechanism of insulin resistance and suggest that hPGH may contribute to the insulin resistance of normal pregnancy by increasing the expression of the p85α monomer, which competes in a dominant negative fashion with the p85-p110 heterodimer for binding to IRS-1 protein.

scholarly journals Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

1995 ◽  
Vol 92 (22) ◽  
pp. 10142-10146 ◽  
Author(s):  
N. Bonnefoy-Berard ◽  
Y. C. Liu ◽  
M. von Willebrand ◽  
A. Sung ◽  
C. Elly ◽  
...  
Keyword(s):  
T Cells ◽  
Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3
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