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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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Expressed TC10 localizes to the plasma membrane and a subset of endomembrane compartments in 3T3L1 adipocytes. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the cDNA encoding for the full length TC10 protein containing an NH2- terminal HA-epitope tag as described in Materials and methods. 18 h later, the cells were either left untreated (a–c) or incubated with 10 μM BFA (d–f) for 60 min or 33 μM nocodazole (g–i) for 3 h at 37°C. The cells were then fixed and colabeled with a polyclonal HA antibody (b, e, and h) and either (A) monoclonal antibody for p115 (a, d, and g), (B) monoclonal antibody for syntaxin 6 (a, d, and g), or (C) monoclonal antibody for TfR (a, d, and g). (D) The cells were cotransfected with HA-HRas (50 μg) and Myc-TC10 (50 μg) and treated as described above. The cells were then labeled with a polyclonal HA antibody (a, d, and g) and a monoclonal Myc antibody (b, e, and h). The merged images for each individual condition are presented in c, f, and i. These are representative cells from three independent determinations. Bar, 10 μM.
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fig1: Expressed TC10 localizes to the plasma membrane and a subset of endomembrane compartments in 3T3L1 adipocytes. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the cDNA encoding for the full length TC10 protein containing an NH2- terminal HA-epitope tag as described in Materials and methods. 18 h later, the cells were either left untreated (a–c) or incubated with 10 μM BFA (d–f) for 60 min or 33 μM nocodazole (g–i) for 3 h at 37°C. The cells were then fixed and colabeled with a polyclonal HA antibody (b, e, and h) and either (A) monoclonal antibody for p115 (a, d, and g), (B) monoclonal antibody for syntaxin 6 (a, d, and g), or (C) monoclonal antibody for TfR (a, d, and g). (D) The cells were cotransfected with HA-HRas (50 μg) and Myc-TC10 (50 μg) and treated as described above. The cells were then labeled with a polyclonal HA antibody (a, d, and g) and a monoclonal Myc antibody (b, e, and h). The merged images for each individual condition are presented in c, f, and i. These are representative cells from three independent determinations. Bar, 10 μM.

Mentions: To examine the mechanism by which TC10 modulates insulin signaling and GLUT4 translocation, we first colabeled expressed hemagglutinin (HA)-tagged TC10 with various intracellular markers in 3T3L1 adipocytes (Fig. 1) . In addition, we capitalized on the differential effects of brefeldin A (BFA) and nocodazole on endomembrane compartments to further define the TC10 localization pattern. BFA causes the Golgi stacks to collapse into the endoplasmic reticulum and the TGN to coalesce with endosomal membranes in the microtubule-organizing center (Banting and Ponnambalam, 1997; Chardin and McCormick, 1999). In contrast, the microtubule inhibitor nocodazole disrupts the Golgi apparatus into ministacks and blocks retrograde traffic between the Golgi membranes and the endoplasmic reticulum, without inhibiting anterograde traffic to the plasma membrane (Lippincott-Schwartz et al., 1990; Kok et al., 1992).


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)

Expressed TC10 localizes to the plasma membrane and a subset of endomembrane compartments in 3T3L1 adipocytes. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the cDNA encoding for the full length TC10 protein containing an NH2- terminal HA-epitope tag as described in Materials and methods. 18 h later, the cells were either left untreated (a–c) or incubated with 10 μM BFA (d–f) for 60 min or 33 μM nocodazole (g–i) for 3 h at 37°C. The cells were then fixed and colabeled with a polyclonal HA antibody (b, e, and h) and either (A) monoclonal antibody for p115 (a, d, and g), (B) monoclonal antibody for syntaxin 6 (a, d, and g), or (C) monoclonal antibody for TfR (a, d, and g). (D) The cells were cotransfected with HA-HRas (50 μg) and Myc-TC10 (50 μg) and treated as described above. The cells were then labeled with a polyclonal HA antibody (a, d, and g) and a monoclonal Myc antibody (b, e, and h). The merged images for each individual condition are presented in c, f, and i. These are representative cells from three independent determinations. Bar, 10 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196453&req=5

fig1: Expressed TC10 localizes to the plasma membrane and a subset of endomembrane compartments in 3T3L1 adipocytes. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of the cDNA encoding for the full length TC10 protein containing an NH2- terminal HA-epitope tag as described in Materials and methods. 18 h later, the cells were either left untreated (a–c) or incubated with 10 μM BFA (d–f) for 60 min or 33 μM nocodazole (g–i) for 3 h at 37°C. The cells were then fixed and colabeled with a polyclonal HA antibody (b, e, and h) and either (A) monoclonal antibody for p115 (a, d, and g), (B) monoclonal antibody for syntaxin 6 (a, d, and g), or (C) monoclonal antibody for TfR (a, d, and g). (D) The cells were cotransfected with HA-HRas (50 μg) and Myc-TC10 (50 μg) and treated as described above. The cells were then labeled with a polyclonal HA antibody (a, d, and g) and a monoclonal Myc antibody (b, e, and h). The merged images for each individual condition are presented in c, f, and i. These are representative cells from three independent determinations. Bar, 10 μM.
Mentions: To examine the mechanism by which TC10 modulates insulin signaling and GLUT4 translocation, we first colabeled expressed hemagglutinin (HA)-tagged TC10 with various intracellular markers in 3T3L1 adipocytes (Fig. 1) . In addition, we capitalized on the differential effects of brefeldin A (BFA) and nocodazole on endomembrane compartments to further define the TC10 localization pattern. BFA causes the Golgi stacks to collapse into the endoplasmic reticulum and the TGN to coalesce with endosomal membranes in the microtubule-organizing center (Banting and Ponnambalam, 1997; Chardin and McCormick, 1999). In contrast, the microtubule inhibitor nocodazole disrupts the Golgi apparatus into ministacks and blocks retrograde traffic between the Golgi membranes and the endoplasmic reticulum, without inhibiting anterograde traffic to the plasma membrane (Lippincott-Schwartz et al., 1990; Kok et al., 1992).

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