Fig. 5
CBAP is crucial for Akt-mediated TSC2 phosphorylation and cellular Rheb-GTP levels. a CBAP is required for Akt-mediated TSC2 phosphorylation. Various plasmids were transfected into HEK293T cells (Ctrl or CBAP-KO). Flag-TSC2 was immunoprecipitated with anti-Flag antibody and then the immunoprecipites were subjected to immunoblotting. The input levels of proteins are shown. Black and white arrows indicate exogenous and endogenous Akt proteins, respectively. N = 2. b In vitro Akt kinase activity on TSC2 is increased in the presence of recombinant GST-CBAP protein. An in vitro Akt kinase assay was established using in vitro-translated Flag-TSC2 protein as the substrate. Phospho-Akt (S473) and non-phospho-Akt were immunopurified from CBAP-KO Jurkat cells treated with or without MK2206. Quantification of the levels of phosphorylated TSC2 normalized to total TSC2 protein is indicated above the lanes. One representative picture from two independent experiments was shown. c Absence of CBAP is correlated with low levels of Rheb-GTP in Jurkat cells. Endogenous Rheb-GTP levels were evaluated by immunoprecipitation using an antibody that selectively recognizes Rheb-GTP. Lysates were treated with GTPγS or GDP, which were included as positive and negative controls, respectively. d CBAP is essential for the Akt-induced increase in Rheb-GTP levels. Ctrl and CBAP-KO HEK293T cells were transfected with the indicated plasmids before Rheb-GTP levels were determined in the lysates as described in (c). Expression levels of the transfected plasmids were determined by immunoblotting using tag-specific antibodies. N = 2
