Figure 5. Sesn2 activates AMPK and induces TSC2 phosphorylation.

(A) Wt, but not N-terminally truncated, Sesn2 activates AMPK. HEK293 cells were cotransfected with AMPKα1F expression vector along with GFP, Sesn2F and Sesn2F-ΔN expression vectors. After 48 hrs the cells were lysed and AMPKα1F immune complexes were isolated and assayed for their ability to phosphorylate a TSC1:TSC2 complex isolated by immunoprecipitation from lysates of transiently transfected HEK293 cells. The amounts of AMPKα1F, Sesn2 and TSC2 in the kinase reaction were examined by immunoblotting (IB). (B) Sesn2 enhances TSC2 phosphorylation. HEK293 cells were co-transfected with TSC1Myc and TSC2F expression vectors along with either wt Sesn2F, N-terminally truncated Sesn2F or GFP expression vectors. After 48 hrs cells were metabolically labeled with ³²P-orthophosphate and the TSC1:TSC2 complex was immunoprecipitated 4 hrs later, gel separated and autoradiographed. TSC2 and Sesn2 expression were examined by immunoblotting (IB). (C) Sesn2-associated AMPKα is more extensively phosphorylated than AMPK that is not associated with Sesn2. Sesn2F or GFP were induced by doxycycline withdrawl in MCF7-tet cells. After 24 hrs cells were lysed and the lysates were subjected to immunoprecipitation with anti-Flag antibody. The supernatant of the immunoprecipitation reaction, the immune complexes and the total infractionated lysate were examined for AMPKα phosphorylation and content by immunoblotting.