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Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE - J. Cell Biol. (2001)

Bottom Line: Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10.We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains.These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

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The endomembrane trafficking and plasma membrane subdomain compartmentalization of TC10 is defined by the COOH-terminal domain. (A) Amino acid sequence comparison of the COOH-terminal 22 amino acids of TC10, H-Ras, K-Ras, TC10/H-Ras chimera, and TC10/K-Ras chimera. (B) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the HA epitope–tagged TC10 (a), H-Ras (b), K-Ras (c), TC10/H-Ras chimera (d), and TC10/K-Ras chimera (e) cDNAs as described in Materials and methods. 18 h later, the cells were fixed and the subcellular localization was determined by confocal fluorescent microscopy. (C) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), TC10 (c and d), H-Ras (e and f), K-Ras (g and h), TC10/H-Ras chimera (i and j), and TC10/K-Ras chimera (k and l) cDNAs. 18 h later, the cells were then incubated for 30 min in the absence (a, c, e, g, i, and k) or presence (b, d, f, h, j, and l) of 100 nM insulin. The cells were then fixed and the subcellular localization was determined by confocal fluorescent microscopy. These are a representative field of cells from five or six independent determinations. (D) Quantitation of the number of cells displaying GLUT4-EGFP plasma membrane fluorescent was determined from the counting of 175–200 cells that were coexpressing both the TC10 constructs and GLUT4-EGFP in five or six independent experiments. Bar, 10 μM.
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fig2: The endomembrane trafficking and plasma membrane subdomain compartmentalization of TC10 is defined by the COOH-terminal domain. (A) Amino acid sequence comparison of the COOH-terminal 22 amino acids of TC10, H-Ras, K-Ras, TC10/H-Ras chimera, and TC10/K-Ras chimera. (B) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the HA epitope–tagged TC10 (a), H-Ras (b), K-Ras (c), TC10/H-Ras chimera (d), and TC10/K-Ras chimera (e) cDNAs as described in Materials and methods. 18 h later, the cells were fixed and the subcellular localization was determined by confocal fluorescent microscopy. (C) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), TC10 (c and d), H-Ras (e and f), K-Ras (g and h), TC10/H-Ras chimera (i and j), and TC10/K-Ras chimera (k and l) cDNAs. 18 h later, the cells were then incubated for 30 min in the absence (a, c, e, g, i, and k) or presence (b, d, f, h, j, and l) of 100 nM insulin. The cells were then fixed and the subcellular localization was determined by confocal fluorescent microscopy. These are a representative field of cells from five or six independent determinations. (D) Quantitation of the number of cells displaying GLUT4-EGFP plasma membrane fluorescent was determined from the counting of 175–200 cells that were coexpressing both the TC10 constructs and GLUT4-EGFP in five or six independent experiments. Bar, 10 μM.

Mentions: Since TC10 is predicted to be both farnesylated and palmitoylated at the COOH-terminus (Fig. 2 A), TC10 most likely transits through the secretory membrane system en route to the plasma membrane, in an identical manner to that established for H-Ras and several other proteins that undergo posttranslational prenylation and palmitoylation (Choy et al., 1999; Resh, 1999; Apolloni et al., 2000; Michaelson et al., 2001). Indeed, the TC10 expression pattern was essentially indistinguishable from the subcellular distribution of H-Ras, both in control cells and cells treated with BFA or nocodazole (Fig. 1 D, a–i). Furthermore, incubation of cells with cycloheximide subsequent to transfection resulted in the rapid and identical chase (<4 h) of the entire endomembrane pool of both H-Ras and TC10 to the plasma membrane (data not shown). Thus, TC10 appears to be processed through the secretory membrane system and perinuclear–recycling endosomes during its transit to the plasma membrane in a manner similar to that described recently for H-Ras (Prior and Hancock, 2001).


Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE - J. Cell Biol. (2001)

The endomembrane trafficking and plasma membrane subdomain compartmentalization of TC10 is defined by the COOH-terminal domain. (A) Amino acid sequence comparison of the COOH-terminal 22 amino acids of TC10, H-Ras, K-Ras, TC10/H-Ras chimera, and TC10/K-Ras chimera. (B) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the HA epitope–tagged TC10 (a), H-Ras (b), K-Ras (c), TC10/H-Ras chimera (d), and TC10/K-Ras chimera (e) cDNAs as described in Materials and methods. 18 h later, the cells were fixed and the subcellular localization was determined by confocal fluorescent microscopy. (C) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), TC10 (c and d), H-Ras (e and f), K-Ras (g and h), TC10/H-Ras chimera (i and j), and TC10/K-Ras chimera (k and l) cDNAs. 18 h later, the cells were then incubated for 30 min in the absence (a, c, e, g, i, and k) or presence (b, d, f, h, j, and l) of 100 nM insulin. The cells were then fixed and the subcellular localization was determined by confocal fluorescent microscopy. These are a representative field of cells from five or six independent determinations. (D) Quantitation of the number of cells displaying GLUT4-EGFP plasma membrane fluorescent was determined from the counting of 175–200 cells that were coexpressing both the TC10 constructs and GLUT4-EGFP in five or six independent experiments. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196453&req=5

fig2: The endomembrane trafficking and plasma membrane subdomain compartmentalization of TC10 is defined by the COOH-terminal domain. (A) Amino acid sequence comparison of the COOH-terminal 22 amino acids of TC10, H-Ras, K-Ras, TC10/H-Ras chimera, and TC10/K-Ras chimera. (B) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the HA epitope–tagged TC10 (a), H-Ras (b), K-Ras (c), TC10/H-Ras chimera (d), and TC10/K-Ras chimera (e) cDNAs as described in Materials and methods. 18 h later, the cells were fixed and the subcellular localization was determined by confocal fluorescent microscopy. (C) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), TC10 (c and d), H-Ras (e and f), K-Ras (g and h), TC10/H-Ras chimera (i and j), and TC10/K-Ras chimera (k and l) cDNAs. 18 h later, the cells were then incubated for 30 min in the absence (a, c, e, g, i, and k) or presence (b, d, f, h, j, and l) of 100 nM insulin. The cells were then fixed and the subcellular localization was determined by confocal fluorescent microscopy. These are a representative field of cells from five or six independent determinations. (D) Quantitation of the number of cells displaying GLUT4-EGFP plasma membrane fluorescent was determined from the counting of 175–200 cells that were coexpressing both the TC10 constructs and GLUT4-EGFP in five or six independent experiments. Bar, 10 μM.
Mentions: Since TC10 is predicted to be both farnesylated and palmitoylated at the COOH-terminus (Fig. 2 A), TC10 most likely transits through the secretory membrane system en route to the plasma membrane, in an identical manner to that established for H-Ras and several other proteins that undergo posttranslational prenylation and palmitoylation (Choy et al., 1999; Resh, 1999; Apolloni et al., 2000; Michaelson et al., 2001). Indeed, the TC10 expression pattern was essentially indistinguishable from the subcellular distribution of H-Ras, both in control cells and cells treated with BFA or nocodazole (Fig. 1 D, a–i). Furthermore, incubation of cells with cycloheximide subsequent to transfection resulted in the rapid and identical chase (<4 h) of the entire endomembrane pool of both H-Ras and TC10 to the plasma membrane (data not shown). Thus, TC10 appears to be processed through the secretory membrane system and perinuclear–recycling endosomes during its transit to the plasma membrane in a manner similar to that described recently for H-Ras (Prior and Hancock, 2001).

Bottom Line: Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10.We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains.These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

Show MeSH
Related in: MedlinePlus