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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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Expression of a dominant-interfering caveolin 3 mutant inhibits insulin-stimulated TC10 activation. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA-TC10/WT cDNA plus 200 μg of Cav3/WT (A) or Cav3/DGV (B) cDNAs. 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 three independent determinations.
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fig10: Expression of a dominant-interfering caveolin 3 mutant inhibits insulin-stimulated TC10 activation. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA-TC10/WT cDNA plus 200 μg of Cav3/WT (A) or Cav3/DGV (B) cDNAs. 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 three independent determinations.

Mentions: Since the insulin activation of TC10 requires the appropriate COOH-terminal domain targeting sequences (Fig. 3), we next determined the effect of Cav3/DGV on insulin-stimulated TC10 activation (Fig. 10) . Coexpression of HA-TC10/WT with Cav3/WT had no significant effect on the insulin stimulation of TC10 activation (Fig. 10 A, lanes 1–5). In contrast, expression of Cav3/DGV resulted in marked inhibition in the time and extent of insulin-stimulated TC10 activation (Fig. 10 B, lanes 1–5). Quantitation of these data demonstrated that the maximum insulin-stimulated activation of TC10/WT in the Cav3/WT expressing cells was 1.6 ± 0.3-fold. However, in the presence of Cav3/DGV insulin was only able to activate TC10 1.0 ± 0.1-fold. The effect of Cav3/DGV was specific for TC10 activation, as there was no detectable effect on insulin-stimulated insulin receptor autophosphorylation, IRS tyrosine phosphorylation, PI(3,4,5)P3 production, or PKB/Akt activation (data not shown).


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)

Expression of a dominant-interfering caveolin 3 mutant inhibits insulin-stimulated TC10 activation. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA-TC10/WT cDNA plus 200 μg of Cav3/WT (A) or Cav3/DGV (B) cDNAs. 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 three independent determinations.
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Related In: Results  -  Collection

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fig10: Expression of a dominant-interfering caveolin 3 mutant inhibits insulin-stimulated TC10 activation. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA-TC10/WT cDNA plus 200 μg of Cav3/WT (A) or Cav3/DGV (B) cDNAs. 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 three independent determinations.
Mentions: Since the insulin activation of TC10 requires the appropriate COOH-terminal domain targeting sequences (Fig. 3), we next determined the effect of Cav3/DGV on insulin-stimulated TC10 activation (Fig. 10) . Coexpression of HA-TC10/WT with Cav3/WT had no significant effect on the insulin stimulation of TC10 activation (Fig. 10 A, lanes 1–5). In contrast, expression of Cav3/DGV resulted in marked inhibition in the time and extent of insulin-stimulated TC10 activation (Fig. 10 B, lanes 1–5). Quantitation of these data demonstrated that the maximum insulin-stimulated activation of TC10/WT in the Cav3/WT expressing cells was 1.6 ± 0.3-fold. However, in the presence of Cav3/DGV insulin was only able to activate TC10 1.0 ± 0.1-fold. The effect of Cav3/DGV was specific for TC10 activation, as there was no detectable effect on insulin-stimulated insulin receptor autophosphorylation, IRS tyrosine phosphorylation, PI(3,4,5)P3 production, or PKB/Akt activation (data not shown).

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