FIGURE 6.

Effect of phosphorylation of Ser²³⁷ of Tbc1d1. A, the relative amount of HA-GLUT4-GFP at the cell surface in adipocytes expressing FLAG-tagged wild-type Tbc1d1 (WT), T596A Tbc1d1 (TA), or S237A Tbc1d1 (SA) in the presence of insulin (Ins) or AICAR plus insulin was determined as described in the legend to Fig. 5. The values are the averages ± S.E. for four independent experiments. They have been normalized to 1.0 for the wild type in the insulin state. For each Tbc1d1 construct the difference between insulin and AICAR plus insulin was significant at p < 0.05, whereas the difference between the AICAR plus insulin for SA compared with AICAR plus insulin for WT and TA was not significant at p < 0.05. B, 3T3-L1 adipocytes were electroporated with vector (V) or the plasmids for FLAG-tagged wild-type, T596A, or S237A Tbc1d1 and treated with insulin, AICAR, or AICAR plus insulin as described in the legend to Fig. 5. The samples were immunoblotted for phospho-Ser²³⁷, FLAG-tagged Tbc1d1 (anti-FLAG), peEF2, and, as a loading control, AMPK. The 1× load was 50 μg of protein. A replicate of this experiment gave similar results.