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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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Insulin activates TC10 only when specifically targeted by the TC10 and H-Ras COOH-terminal domains. Differentiated 3T3L1 adipocytes were electroporated with (A) 50 μg of HA-TC10/WT, (B) HA-TC10/H-Ras chimera, and (C) HA-TC10/K-Ras chimera cDNAs as described in Materials and methods. 48 h later, the cells were incubated in the absence (lane 1) or the presence of 100 nM insulin for 1 (lane 2), 2 (lane 3), 5 (lane 4), and 10 (lane 5) min. Cell lysates were then prepared and either directly immunoblotted for TC10 expression (Lysate) or precipitated with 6 μg of the GST-Pak1 Crib domain fusion. The precipitates were then solubilized and immunoblotted for TC10 (GST-Pak1). This is a representative immunoblot from two to four independent determinations.
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fig3: Insulin activates TC10 only when specifically targeted by the TC10 and H-Ras COOH-terminal domains. Differentiated 3T3L1 adipocytes were electroporated with (A) 50 μg of HA-TC10/WT, (B) HA-TC10/H-Ras chimera, and (C) HA-TC10/K-Ras chimera cDNAs as described in Materials and methods. 48 h later, the cells were incubated in the absence (lane 1) or the presence of 100 nM insulin for 1 (lane 2), 2 (lane 3), 5 (lane 4), and 10 (lane 5) min. Cell lysates were then prepared and either directly immunoblotted for TC10 expression (Lysate) or precipitated with 6 μg of the GST-Pak1 Crib domain fusion. The precipitates were then solubilized and immunoblotted for TC10 (GST-Pak1). This is a representative immunoblot from two to four independent determinations.

Mentions: We next determined whether TC10 subdomain compartmentalization was important for its activation by insulin (Fig. 3) . Using a GST-Pak1 pull down assay to precipitate the GTP-bound TC10 protein, we observed that insulin produced a time-dependent activation of the expressed wild-type TC10 protein (Fig. 3 A, lanes 1–5). Similarly, insulin activated the TC10/H-Ras chimera over the same time frame (Fig. 3 B, lanes 1–5). However, insulin was unable to activate the TC10/K-Ras chimera under the identical conditions (Fig. 3 C, lanes 1–5). In parallel, immunoblots of cell lysates demonstrated equal amounts of TC10 expression under all these conditions. Quantitation of these data demonstrated that insulin activated TC10/WT and TC10/H-Ras 1.7 ± 0.4- and 1.7 ± 0.3-fold, respectively. In contrast, TC10/K-Ras was not significantly activated by insulin (0.9 ± 0.1). Thus, these data demonstrate that the upstream pathway required for insulin-dependent activation of TC10 is also confined to the same specific lipid raft microdomain defined by the COOH-terminal H-Ras and TC10 targeting sequences.


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)

Insulin activates TC10 only when specifically targeted by the TC10 and H-Ras COOH-terminal domains. Differentiated 3T3L1 adipocytes were electroporated with (A) 50 μg of HA-TC10/WT, (B) HA-TC10/H-Ras chimera, and (C) HA-TC10/K-Ras chimera cDNAs as described in Materials and methods. 48 h later, the cells were incubated in the absence (lane 1) or the presence of 100 nM insulin for 1 (lane 2), 2 (lane 3), 5 (lane 4), and 10 (lane 5) min. Cell lysates were then prepared and either directly immunoblotted for TC10 expression (Lysate) or precipitated with 6 μg of the GST-Pak1 Crib domain fusion. The precipitates were then solubilized and immunoblotted for TC10 (GST-Pak1). This is a representative immunoblot from two to four independent determinations.
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Related In: Results  -  Collection

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

fig3: Insulin activates TC10 only when specifically targeted by the TC10 and H-Ras COOH-terminal domains. Differentiated 3T3L1 adipocytes were electroporated with (A) 50 μg of HA-TC10/WT, (B) HA-TC10/H-Ras chimera, and (C) HA-TC10/K-Ras chimera cDNAs as described in Materials and methods. 48 h later, the cells were incubated in the absence (lane 1) or the presence of 100 nM insulin for 1 (lane 2), 2 (lane 3), 5 (lane 4), and 10 (lane 5) min. Cell lysates were then prepared and either directly immunoblotted for TC10 expression (Lysate) or precipitated with 6 μg of the GST-Pak1 Crib domain fusion. The precipitates were then solubilized and immunoblotted for TC10 (GST-Pak1). This is a representative immunoblot from two to four independent determinations.
Mentions: We next determined whether TC10 subdomain compartmentalization was important for its activation by insulin (Fig. 3) . Using a GST-Pak1 pull down assay to precipitate the GTP-bound TC10 protein, we observed that insulin produced a time-dependent activation of the expressed wild-type TC10 protein (Fig. 3 A, lanes 1–5). Similarly, insulin activated the TC10/H-Ras chimera over the same time frame (Fig. 3 B, lanes 1–5). However, insulin was unable to activate the TC10/K-Ras chimera under the identical conditions (Fig. 3 C, lanes 1–5). In parallel, immunoblots of cell lysates demonstrated equal amounts of TC10 expression under all these conditions. Quantitation of these data demonstrated that insulin activated TC10/WT and TC10/H-Ras 1.7 ± 0.4- and 1.7 ± 0.3-fold, respectively. In contrast, TC10/K-Ras was not significantly activated by insulin (0.9 ± 0.1). Thus, these data demonstrate that the upstream pathway required for insulin-dependent activation of TC10 is also confined to the same specific lipid raft microdomain defined by the COOH-terminal H-Ras and TC10 targeting sequences.

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