Fig. 3.

Phosphorylation does not override the requirement for PIP₃. (A) Phosphorylation state analysis of Akt1^3P after coculture with A-443654 and okadaic acid. Tandem mass spectrometry of GluC digest. Additional substoichiometric phosphorylation of S477 in 4P species indicated in red. (B) Liposome pelleting assay for 0% and 5% PIP₃ liposomes indicating the binding of Akt1^3P and Akt1^1P to PIP₃ in the kinase assay shown in F. (C) Akt1 kinase assay ± PIP₃ liposomes, ± 10 μM MK-2206. Akt1^3P (37.5 nM), black bars; Akt1^1P (750 nM), gray bars. (D) Kinase assay of Akt1^3P in the presence of liposomes containing increasing concentrations of PIP₃. Left axis, and blue lines correspond to kinase activity. Right axis and black lines correspond to % PIP3 binding (determined by a liposome pelleting assay). Squares, Akt1^3P; circles, Akt1^3P preincubated for 10 min with 10 μM MK-2206. PIP₃ binding and PIP₃-dependent increase in kinase activity were fit to one-site binding models, taking into account basal Akt1^3P activity and nonspecific binding in the presence of 0 mol % PIP₃ liposomes respectively. (E) Kinase assay of Akt1^3P and Akt1^3P R144A with increasing substrate concentration in the presence of liposomes containing 0 or 5 mol% PIP₃. Error bars indicate the SD of three independent measurements.