Figure 3. The PTEN phosphorylation sites Thr³⁶⁶ and Ser³⁷⁰ do not affect phosphatase activity in vitro or in cells.

(A) Bacterially expressed PTEN proteins were assayed in vitro against phosphatidylcholine/PtdIns(3,4,5)P₃ vesicles (100 ng of enzyme) and against the soluble substrate Ins(1,3,4,5)P₄ (500 ng of enzyme). PTEN 2A and 2D refer to double mutants with either alanine or aspartic acid replacements respectively, of both Thr³⁶⁶ and Ser³⁷⁰. PTEN D3 refers to an aspartic acid triple replacement mutant of Ser³⁸⁰, Thr³⁸² and Thr³⁸³. Results are shown for each substrate as the mean±S.E.M. activity from triplicate samples and also the ratio of these two activities, shown as the mean±S.E.M. ratio from these paired triplicate samples. (B, C) U87MG cells were infected with viruses encoding the indicated PTEN proteins. PTEN A3 refers to an alanine triple replacement mutant of Ser³⁸⁰, Thr³⁸² and Thr³⁸³. At 24 h after infection, cells were lysed and the activity (B) and phosphorylation (C) of endogenous Akt/PKB was determined by immunoprecipitate kinase assay and Western blotting respectively. WT, wild-type.