Phosphorylation of Akt at threonine 308 or serine 473 are not required
for the activation of Akt by GIP. A, illustration of HA-Akt1
constructs and the respective point mutations used for this study.
PH, pleckstrin homology domain; HM, hydrophobic motif.
B, Western blot showing representative levels of
HA-Akt1T308A/S473A protein in transfected cells relative to
endogenous Akt in untransfected cells, as well as the purity of
Akt1T308A/S473A immunoprecipitates. C, INS-1 cells
transfected with HA-Akt1308/473 or HA-Akt1179 and 36 h
later treated with ±10 nm GIP or 10 nm IGF-I for
15 min. Western analysis was performed on Akt KA assays with indicated
antibodies. D and E, shown is the mean change ± S.E.
in Akt activity (P-GST-GSK3/Akt) relative to Basal (n = 4) for INS-1
cells transfected with HA-Akt1308/473 (D) or
HA-Akt1179 (E). #, p < 0.05 versus
Basal. Anti-GST (GST-GSK3) blots were internal controls.