Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition
Figure 3
The Lower Sensitivity of PKBγ for AKT Inhibitor VIII Can Be Partially Reversed by Creating a Chimera (PHα-KINγ)
(A) A dynamic model of the PH-in conformation of PKBγ after 10-ns run is shown in panels (a) and (b) (cross section at the level of Trp 79). NIH3T3 cells expressing GFP-PKBγ or a mutant in which Trp 79 was changed to an Ala (W79A) were treated for 30 min with different concentrations of AKT inhibitor VIII as indicated prior to 5-min PDGF stimulation. Thr 308 and Ser 473 phosphorylations were detected by western blot using phosphospecific antibodies, and the quantifications were done using the Odyssey/LI-COR fluorescence detection system. The data are presented in percentage of Thr 308 (left graph) or Ser 473 (right graph) phosphorylations upon PDGF stimulation for each construct.
(B) Two dynamic runs of the PH-in conformation of the chimera PHα-KINγ of 10 ns and 8 ns are presented in panels (a and b) and (c and d), respectively. The dynamic models show that the replacement of PH and linker of PKBγ by those of PKBα in the chimera PHα-KINγ lead to a partial reopening of the PH-induced cavity in the kinase domain KINγ of the chimera. NIH3T3 cells expressing GFP-PKBγ or GFP-CHIM PHα-KINγ were treated for 30 min at different concentrations of AKT inhibitor VIII as indicated prior to 5-min PDGF stimulation. Thr 308 and Ser 473 phosphorylations were detected by western blot using phosphospecific antibodies and the quantifications were performed using the Odyssey/LI-COR fluorescence detection system. The data are presented as a percentage of Thr 308 (left graph) or Ser 473 (right graph) phosphorylations upon PDGF stimulation for each construct. The p-values were calculated using an unpaired t-test with Welch's correction on a minimum of three independent experiments.
A single asterisk (*) and double asterisks (**) indicate p < 0.025 and p < 0.001, respectively. Error bars indicate the standard error of the mean.