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Myosin motor Myo1c and its receptor NEMO/IKK-gamma promote TNF-alpha-induced serine307 phosphorylation of IRS-1 - PubMed

  • ️Sun Jan 01 2006

Myosin motor Myo1c and its receptor NEMO/IKK-gamma promote TNF-alpha-induced serine307 phosphorylation of IRS-1

Yoshitaka Nakamori et al. J Cell Biol. 2006.

Abstract

Tumor necrosis factor-alpha (TNF-alpha) signaling through the IkappaB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor kappaB essential modulator (NEMO)/IKK-gamma subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-alpha- induced phosphorylation of Ser307-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-alpha-induced Ser307-IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-beta-binding domain or silencing NEMO blocked the TNF-alpha signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-alpha-induced insulin resistance.

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Figures

Figure 1.
Figure 1.

Intracellular localization of NEMO in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes (A) or adipocytes expressing eGFP-NEMO (B) were serum starved and either left untreated (A) or were pretreated with 5 μM latrunculin B (LatB) or 30 μM nocodazole (Noc) for 60 min (B). They were then incubated with 20 ng/ml TNF-α or 100 nM insulin for 15 min at 37°C. The cells were fixed, and F-actin was visualized by AlexaFluor596-phalloidin.

Figure 2.
Figure 2.

Insulin promotes the interaction between NEMO and Myo1c. (A) 3T3-L1 adipocytes expressing myc-tagged WT NEMO were serum starved and stimulated with 100 nM insulin or 20 ng/ml TNF-α for 20 min at 37°C. NEMO-binding proteins were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by silver staining. Bands were proteolytically digested and analyzed by mass spectrometry. Myo1c and actin (arrows) were identified. (B) 3T3-L1 adipocytes were serum starved for 2 h and treated with 100 nM insulin or 20 ng/ml TNF-α for 20 min at 37°C. The NEMO–Myo1c interaction was determined by immunoprecipitation using anti-NEMO or anti-Myo1c antibodies.

Figure 3.
Figure 3.

Effect of Myo1c expression on the intracellular localization of NEMO. 3T3-L1 adipocytes were coexpressed with eGFP-NEMO and WT Myo1c or the Myo1c cargo domain (A) or were infected with adenovirus encoding shRNA (Myo1c) or vector alone. After 2 h of serum starvation, the cells were stimulated with or without 100 nM insulin for 20 min at 37°C, stained with anti-Flag antibody followed by Cy3-labeled secondary antibody (A) or anti-NEMO antibody and anti-Myo1c antibody followed by FITC and Cy3-labeled secondary antibodies (B), and were observed by confocal microscopy.

Figure 4.
Figure 4.

Myo1c modulates IRS-1–NEMO interaction. (A) 3T3-L1 adipocytes were serum starved for 2 h and stimulated with or without 100 nM insulin for 15 min at 37°C. Cells were fixed and stained with anti–IRS-1 and anti-NEMO antibody followed by FITC and Cy3-labeled secondary antibodies. (B) eGFP-tagged IRS-1 and Xpress-fused NEMO were coexpressed in 3T3-L1 adipocytes. After 2 h of serum starvation, the cells were stimulated with or without 100 nM insulin for 15 min and were then fixed. Expressed NEMO was visualized by anti-Xpress antibody and a Cy3-labeled second antibody. (C) High resolution view of the boxed area outlined in B. (D) 3T3-L1 adipocytes were infected with adenovirus encoding WT Myo1c and dominant inhibitory Myo1c. The cells were stimulated with or without 100 nM insulin for 15 min. The cell lysates were immunoprecipitated with anti-NEMO antibody, and precipitates were immunoblotted with anti–IRS-1 antibody. (E) 3T3-L1 adipocytes were left untreated or treated with 20 ng/ml TNF-α for 10 min and were incubated with various concentrations (1–100 nM) of insulin for 10 min at 37°C. The cells were lysed and immunoprecipitated by anti–IRS-1 antibody, and the precipitates were blotted with antiphospho-Ser307 IRS-1 and anti–IRS-1 antibody.

Figure 5.
Figure 5.

NEMO and Myo1c for insulin signaling and glucose uptake in 3T3-L1 adipocytes. (A–E) 3T3-L1 adipocytes were infected with recombinant adenovirus encoding wild type (WT) NEMO, ΔN-NEMO, shRNA (NEMO), Myo1c WT, Myo1c cargo domain, and vector alone (for control) at an MOI of 50. (A and C) The cells were serum starved for 2 h, treated with or without 20 ng/ml TNF-α for 15 min, and stimulated with or without 100 nM insulin for 10 min at 37°C. The cell lysates were immunoprecipitated with anti–insulin receptor β (IR-β) or anti–IRS-1 antibody, and the precipitates were immunoblotted with antiphosphotyrosine, anti–IR-β, anti–IRS-1, antiphospho-Ser307 IRS-1, antiphospho-Ser473 Akt, and anti-Akt antibodies. (B, D, and E) The cells were serum starved for 2 h in Krebs-Ringer phosphate buffer and treated with 20 ng/ml TNF-α for 4 h. Glucose uptake was measured. Each bar represents the mean ± SD (error bars) value of at least three independent experiments. (F) Schematic model of Myo1c-mediated IRS-1–IKK complex formation.

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