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. 2010 Mar 9;5(3):e9616. doi: 10.1371/journal.pone.0009616

Figure 3. Akt phosphorylates Thr117 of MST2 in vivo and the pMST2-T117 regulates autophospho-MST2-T180.

Figure 3

(a) Characterization of anti-pMST-T117 antibody. HEK293 cells were transfected with constitutively active Akt and Flag-MST2 or Flag-MST2-T117A and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-pMST2-T117 antibody (top). Middle and bottom panels show expression of the transfected plasmids. (b) Akt phosphorylates endogenous MST2-T117. Following transfection of HEK293 cells with indicated different Akt, cells were lysed and immunoblotted with anti-pMST2-T117 (top), -MST2 (panel 2), -HA (panel 3) and -actin (bottom) antibodies. (c) IGF1 induces pMST2-T117 and represses pMST2-T180. After serum starvation for 12 h, HEK293 cells were stimulated with IGF1 for 1 h and then immunoblotted with indicated antibodies. (d) Re-expression of Akt in Akt1-/- MEF increases pMST2-T117 and reduces pMST2-T180 level. Akt1-null MEFs were infected with adenovirus expressing Akt or control vector and immunoblotted with indicated antibodies. (e) Knockdown of Akt decreases pMST2-T117 and increases pMST2-T180. MDA-MB-468 cells were transfected with AKT1/shRNA and control shRNA. After 72 h incubation, cells were lysed and immunoblotted with indicated antibodies. (f) Phospho-Thr117 regulates MST2 autophosphorylation of Thr180. HEK293 cells were transfected with indicated plasmids and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-pMST2-T180 (top) and -Flag (bottom) antibodies.