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Functional consequence of targeting protein kinase B/Akt to GLUT4 vesicles

Pierre-Henri Ducluzeau^*, Laura M. Fletcher^*, Gavin I. Welsh and Jeremy M. Tavaré

Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK
^* These authors contributed equally to this work

View larger version (57K): [in a new window]   Fig. 1. Subcellular localisation of PKB mutants in 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with plasmids encoding wild-type PKB (A; PKB[WT]), kinase-inactive PKB (B; PKB[KD]), constitutively active PKB (C; PKB[DD]), constitutively active myristoylated PKB (D; Myr-PKB), constitutively active PKB fused to the N-terminus of GLUT4 (E,F; PKB[DD]G4) and kinase-inactive PKB fused to the N-terminus of GLUT4 (G,H; PKB[KD]G4). 16 hours later the cells were serum starved for 2 hours and then incubated in the absence (A-E,G) or presence (F,H) of transferrin-Alexa568 for 30 minutes (as indicated by `Tf-Alexa') and then fixed and stained with anti-HA antibodies to visualise the PKB construct (as indicated in the panel) and anti-IRAP antibodies (as indicated by `IRAP'), as required. A merged image is also provided of the regions indicated by the yellow box. Bars, 10 µm. The schematic of the PKB-GLUT4 fusion proteins illustrate the position of the N-terminal HA-tag (red), PKB (blue), GLUT4 (cyan) and the activatory (T308D/S473D) and inhibitory (K179A) mutations.

View larger version (38K): [in a new window]   Fig. 2. Insulin induces the translocation of PKB[DD]-GLUT4 but not PKB[KD]-GLUT4 to the plasma membrane of 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with plasmids encoding constitutively active PKB fused to the N-terminus of GLUT4 (A; PKB[DD]-GLUT4) and kinase-inactive PKB fused to the N-terminus of GLUT4 (B; PKB[KD]-GLUT4). 16 hours later the cells were serum starved for 2 hours and then incubated, as required, in the presence or absence of insulin (30 minutes at 100 nM), as indicated. The cells were fixed and stained with anti-HA antibodies and imaged by confocal microscopy.

View larger version (48K): [in a new window]   Fig. 3. IRAP-GFP is a good surrogate marker for GLUT4 vesicles in 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with a plasmid encoding IRAP-GFP. 16 hours later the cells were serum starved for 2 hours and then incubated in the absence (A,C) or presence (B) of transferrin-Alexa568 for 30 minutes. In C, the cells were incubated for 30 minutes in the absence or presence of insulin, as indicated. The cells were then fixed and stained with anti-GLUT4 antibodies (A, as indicated with GLUT4) as required. The images show the distribution of IRAP-GFP (visualised as GFP fluorescence), GLUT4 immunofluorescence or Tf-Alexa labelling, as indicated. A merged image is also provided.

View larger version (45K): [in a new window]   Fig. 4. PKB[KD]-GLUT4 and Myr-PKB[KD] act as dominant-negative inhibitors of insulin-stimulated IRAP-GFP translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with plasmids encoding IRAP-GFP (A-C) and either kinase-inactive PKB (A; PKB[KD]), kinase-inactive PKB fused to the N-terminus of GLUT4 (B; PKB[KD]-Glut4) or kinase-inactive myristoylated PKB (C; Myr-PKB[KD]). 16 hours later the cells were serum starved for 2 hours and then incubated in the presence of insulin at 100 nM for 30 minutes. The cells were then fixed and stained with anti-HA antibodies to visualise the PKB constructs (as indicated). Bars, 10 µm. The arrow in C indicates a cell that expresses IRAP-GFP but little detectable Myr-PKB[KD] and which still responds to insulin, unlike its neighbour which co-expresses significant amounts of Myr-PKB[KD].

View larger version (16K): [in a new window]   Fig. 5. Quantitative analysis of the dominant-negative effect of PKB[KD]-GLUT4 and Myr-PKB[KD]. In A and C, cells were scored for the visual presence of IRAP-GFP in the plasma membrane by double blind analysis by two independent workers as discussed in the Materials and Methods. The results shown represent means±s.d. for three independent experiments with each condition being represented by a minimum of 50 cells. *P<0.01 versus the vector only injected cells incubated in the presence of insulin. In B the amount of IRAP-GFP residing in the plasma membrane was calculated as a percentage of the total expressed IRAP-GFP in that cell for each condition. The results are displayed as means±s.d. for a minimum of 10 cells for each condition, and they were collected from at least three separate experiments. *No significant difference from the control without insulin incubation. In C, adipocytes were microinjected with plasmids encoding IRAP-GFP and either kinase-inactive PKB (PKB[KD]), kinase-inactive PKB lacking the PH domain (PH-PKB[KD]) or kinase-inactive PKB fused to the N-terminus of GLUT4 (PKB[KD]-GLUT4). 16 hours later the cells were serum starved for 2 hours and then incubated in the absence or presence of insulin for 30 minutes. The cells were then scored for the presence of IRAP-GFP in the plasma membrane as discussed in the Materials and Methods. The results shown represent means±s.d. for three independent experiments, with each condition being represented by a minimum of 50 cells. *P<0.01 versus the PKB[KD]-injected cells incubated in the presence of insulin.

View larger version (24K): [in a new window]   Fig. 6. Lack of a dominant-negative effect of PKB[KD]GLUT4 on insulin-stimulated Forkhead translocation out of the nucleus of 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with a plasmid encoding a GFP-tagged human FKHR in the absence (A) or presence (B) of the PKB[KD]-GLUT4 plasmid. After 24 hours the cells were serum starved and treated without (left-hand panels) or with (right-hand panels) insulin for 30 minutes as indicated. The cells were fixed and examined for the distribution of the FKHR-GFP by confocal microscopy. In C, the amount of nuclear FKHR was quantified as described in the Materials and Methods. The results shown represent means±s.d. for at least three independent experiments with each condition represented by a minimum of 11 cells. *P<0.001 versus control incubated without insulin and no significant difference from the insulin-stimulated cells expressing PKB[KD]-GLUT4. #No significant difference from control in the absence of PKB[KD]-GLUT4.

View larger version (27K): [in a new window]   Fig. 7. The effect of constitutively active PKB constructs on IRAP-GFP translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with plasmids encoding IRAP-GFP (A-C) and either constitutively active PKB (A; PKB[DD]), constitutively active PKB fused to the N-terminus of GLUT4 (B; PKB[DD]-Glut4) or a constitutively active myristoylated PKB (C; Myr-PKB). 16 hours later the cells were serum starved for 2 hours and then fixed and stained with anti-HA antibodies to visualise the PKB constructs (as indicated). Panels A-C show the distribution of the HA-tagged PKB constructs and GFP fluorescence resulting from the expression of the IRAP-GFP construct. Bars, 10 µm. In D, cells were scored for the presence of IRAP-GFP in the plasma membrane after incubation in the absence or presence of insulin for 30 minutes as discussed in the Materials and Methods. The results shown represent means±s.d. for three independent experiments with each condition being represented by a minimum of 50 cells. *P<0.01 versus the vector only injected cells incubated in the presence of insulin.