Fig. 2
AKT3-Q60H is expressed at higher levels than wild-type AKT3 but exhibits reduced catalytic activity. A The indicated constructs were used to transduce spontaneously immortalized lung fibroblasts isolated from Akt1fl/flAkt2−/−Akt3−/− mice. Following this, the cells were transduced with MigR1-Cre to ablate the endogenous Akt1. The overall expression of AKT3-Q60H is higher than the expression of wild-type AKT3, but its phosphorylation at both Thr305 and Ser472 is reduced. B An in vitro kinase assay, using GSK3 as the kinase substrate, confirmed that AKT3-Q60H exhibits reduced catalytic activity. C The activation of AKT3-Q60H by IGF1 is impaired. Triple-AKT-knockout lung fibroblasts were transduced with the indicated constructs. Following serum starvation, the cells were treated with IGF1. In vitro kinase assays, using GSK3 as the kinase substrate, revealed that the activation of AKT3-Q60H by IGF1 is impaired. D The activation of AKT1-Q61H by IGF1 is also impaired. Upper panel. Triple-Akt-knockout lung fibroblasts were transduced with the indicated constructs. Following serum starvation, the cells were treated with IGF1. Probing Western blots of the cell lysates with antibodies recognizing the phosphorylated activation loop (Thr308) or the phosphorylated C-terminal hydrophobic motif revealed that the activation of AKT1-Q61H by IGF1 is also impaired. Lower panel. In vitro kinase assays, using GSK3 as the kinase substrate, confirmed the low enzymatic activity of AKT1-Q61H in IGF1-treated cells. The retroviral vector for the constructs used in these experiments was pBabe-puro. Quantification of the Western blot bands was performed using ImageJ software. For the kinase assays (panels B, C, and lower part of D), we calculated the ratio of phosphorGSK3 to the corresponding wild-type Myc-AKT3 or Myc-AKT1, and we gave these ratios the value of 1. The phosphor/total ratios of the mutant forms were calculated relative to the ratios of the wild-type forms. For wild-type AKT3 and AKT1 phosphorylated at Ser472/Ser473 or Thr305/Thr308, the ratios of phosphorAKT3/myc-AKT3 and phosphorAKT3/AKT3 (panel A) or phosphorAKT1/pan-AKT (upper part of panel D) were calculated and given the value of 1. The ratios of the mutant forms of AKT3 and AKT1 were again calculated relative to the ratios of the wild-type forms. The experiments in Figs. 2A and 2D were performed twice. The experiment in Fig. 2C was in part a repeat of the experiment in 2B. Importantly, the kinase experiments also confirmed the results of the Western blotting experiments, and the high expression of the AKT3-Q60H mutant provided additional strong evidence for the impaired enzymatic activity of the mutant