Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling

Abstract

The 3 Akt protein kinase isoforms have critical and distinct functions in the regulation of metabolism, cell growth, and apoptosis, yet the mechanisms by which their signaling specificity is achieved remain largely unclear. Here, we investigated potential mechanisms underlying Akt isoform functional specificity by using Akt2-specific regulation of glucose transport in insulin-stimulated adipocytes as a model system. We found that insulin activates both Akt1 and Akt2 in adipocytes, but differentially regulates the subcellular distribution of these Akt isoforms. The greater accumulation of Akt2 at the plasma membrane (PM) of insulin-stimulated adipocytes correlates with Akt2-specific regulation of the trafficking of the GLUT4 glucose transporter. Consistent with this pattern, Akt constructs that do not accumulate at the PM to the same degree as Akt2 fail to regulate GLUT4 translocation to the PM, whereas enhancement of Akt1 PM association through mutation in Akt1 PH domain is sufficient to overcome Akt-isoform specificity in GLUT4 regulation. Indeed, we found that this distinct insulin-induced PM accumulation of Akt kinases is translated into a differential regulation by the Akt isoforms of AS160, a RabGAP that regulates GLUT4 trafficking. Our data show that Akt2 specifically regulates AS160 phosphorylation and membrane association providing molecular basis for Akt2 specificity in the modulation of GLUT4 trafficking. Together, our findings reveal the stimulus-induced subcellular compartmentalization of Akt kinases as a mechanism contributing to specify Akt isoform functions.

Fig. 1.

Insulin differentially regulates the plasma membrane accumulation of Akt isoforms in adipocytes. (A) Immunoblot analyses of cell extracts from adipocytes stably expressing Flag-Akt1 or Flag-Akt2. (B) Immunoblot analyses of the phosphorylation pattern and in vitro activity of immunoprecipitated Flag-Akt1 or Flag-Akt2 after 10 nM insulin stimulation. In vitro activity kinase assay was performed using as substrate a GST-GSK3β fusion peptide. (C) Densitometric analyses of insulin-induced Flag-Akt1 and Flag-Akt2 phosphorylation at Thr^308/9 and Ser^473/4. Each data point represents the mean ± SE, n = 3–4. (D) Densitometric analyses of in vitro Akt activity measurements. Each data point represents the mean ± SE, n = 3–4. (E) Quantification of insulin-induced Flag-Akt1 and Flag-Akt2 redistribution to the PM using TIRF microscopy. Indirect immunofluorescence of the Flag epitope in basal or insulin-stimulated cells was measured in the epifluorescence mode and in the TIRF mode. The TIRF is normalized to the anti-Flag fluorescence in the epifluorescence mode. For each experiment the data are normalized to the basal state of Flag-Akt1 expressing cells. Each data point represents the mean ± SE, n ≥ 80 cells. *, P < 0.001 (t test). (F) Time-lapse TIRF microscopy of Akt1-GFP and Akt2-GFP. Images were acquired every min for 35 min, 10 nM insulin was added after 4 min of recording. The average GFP intensity for each cell in the first frame has been subtracted and the pseudo colored images have been equally scaled to allow direct comparison of insulin-induced changes in TIRF for the different constructs. Pseudo color is from purple (low signal) to red (highest signal). (G) Quantification Akt1-GFP and Akt2-GFP TIRF versus epifluorescence in live adipocytes in basal conditions. The data are normalized to the Akt2-GFP TIRF/Epifluorescence ratio. Each data point represents the mean ± SE, n ≥ 120 cells. (H) Quantification of time-lapse TIRF microscopy of GFP, Akt1-GFP, and Akt2-GFP. TIR GFP fluorescence for each cell was measured in every frame, background fluorescence was subtracted and fluorescence was normalized to the first frame TIRF value. The data shown are the mean ± SE, n ≥ 12 cells.

Fig. 2.

Insulin-induced accumulation of Akt2 at the PM depends on both the PH-linker and catalytic-regulatory domains and necessary for GLUT4 translocation. (A) Schematic representation of Akt constructs tagged with eGFP at the carboxy-terminus. Akt1: full length Akt1; Akt2: full length Akt2; PHL-Akt1: Akt1 residues 1–149; PHL-Akt2 residues 1–151 Akt2; Akt12: chimera Akt1 (1–149)-Akt2 (152–481); Akt21: chimera Akt2 (1–151)-Akt1 (150–480). (B) Quantification PHL-Akt1-GFP and PHL-Akt2-GFP TIRF versus epifluorescence in live adipocytes in basal conditions. The data are normalized to the Akt2-GFP TIRF/Epifluorescence ratio. Each data point represents the mean ± SE, n ≥ 76 cells. *, P < 0.01 t test. (C) Quantification of time-lapse TIRF microscopy of PHL-Akt1-GFP and PHL-Akt2-GFP. Data are process as in Fig. 1H. The data shown are the mean ± SE from n ≥ 13 cells per condition. (D) Quantification of Akt12-GFP and Akt21-GFP TIRF versus epifluorescence in living adipocytes in basal conditions. The data are normalized to Akt2-GFP TIRF/Epifluorescence ratio. Each data point represents mean ± SE, n ≥ 77 cells. (E) Quantification of time-lapse TIRF microscopy of Akt12-GFP and Akt21-GFP. Data quantified as described in Fig. 1H. Quantification of time-lapse TIRF of Akt2-GFP from Fig. 1H has been included for direct comparison. The data shown are the mean ± SE, n = 12 cells. (F) Immunoblot analyses prepared from 3T3-L1 adipocytes stably expressing Flag-Akt2, Flag-Akt12 or Flag-Akt21. Adipocytes were treated with 10 nM insulin and Akt constructs were immunoprecipitated with an anti-Flag epitope antibody. (G) Box-and-whisker diagram showing the increase in TIR fluorescence of GFP tagged Akt constructs in adipocytes after 30 min of insulin stimulation. Data are derived from graphs in Figs. 1H and 2E. *, P < 0.001 (ANOVA). (H) Immunoblot analyses of cell extracts prepared from adipocytes coelectroporated with Akt siRNAs and cDNAs as noted. (I) Surface-to-total distribution of HA-GLUT4-GFP in adipocytes electroporated with control or Akt2 siRNA and cDNA encoding Akt constructs: A1, Akt1; A2, Akt2; A12, Akt12 chimera; A21, Akt21 chimera. Each bar represents the mean ± SE, n = 2–6. In each experiment the surface-to-total GLUT4 distribution was normalized to that of control cells stimulated with 1 nM insulin.

Fig. 3.

E17K mutation in Akt1 leads to PM membrane accumulation, activation and GLUT4 translocation. (A) The E17K mutation leads to Akt1 and Akt2 activation. Flag-tagged wild type Akt2 and E17K Akt1 and Akt2 mutants were pulled down from unstimulated and 10 nM insulin treated adipocytes and Akt activity was assessed in vitro using GSK3β peptide fused to GST. (B) Quantification Akt2-GFP, Akt1^E17K-GFP and Akt2^E17K-GFP TIRF versus epifluorescence in serum starved and 10 nM insulin treated adipocytes. The data are normalized to the Akt2-GFP basal TIRF/Epifluorescence ratio. Each data point represents the mean ± SE, n = 40 cells. (C) Images of HA-GLUT4-GFP cellular distribution in control and Akt E17K mutants expressing adipocytes. (D) Surface-to-total distribution of HA-GLUT4-GFP in 3T3-L1 adipocytes electroporated with Akt E17K mutants. Each bar represents the mean ± SE, n = 4. In each experiment data were normalized to that of control cells stimulated with 1 nM insulin.

Fig. 4.

Akt2 interacts with AS160 at the PM environment and regulates AS160 phosphorylation and membrane release in response to insulin. (A) Left, immunoblot of AS160 phosphorylation in adipocytes electroporated with noted siRNAs. B, Basal, and Ins, insulin, 10 nM for 15 min. Right, densitometric analyses of AS160 phosphorylation. Each data point represents the mean ± SE, n = 3. *, P < 0.05 (ANOVA). (B) Adipocytes electroporated with Flag-AS160, HA-GLUT4-GFP and siRNAs as noted. Serum-starved or insulin stimulated cells were permeabilized for 1 min to leak out cytosolic contents and the amount of membrane bound AS160 was determined by indirect immunofluorescence as described in Methods. (C) Confocal images of permeabilized adipocytes overexpressing Akt1-GFP or Akt2-GFP and Flag-AS160 in the absence or presence of 10 nM insulin. The Akt-GFP constructs display some nuclear localization, consistent with previous reports; however, it is of note that this nuclear fraction can be dynamically mobilized upon stimulation. (D) Adipocytes were electroporated with Flag-AS160 or Flag-AS160–4P and HA-GLUT4-GFP and membrane bound AS160 was determined as in B. Each bar represents the mean ± SE, n = 3. (E) Confocal images of permeabilized adipocytes overexpressing Akt1-GFP or Akt2-GFP and Flag-AS160–4P in the absence or presence of 10 nM insulin.