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. Author manuscript; available in PMC: 2010 Jan 30.
Published in final edited form as: Mol Cell. 2009 Jan 30;33(2):237–247. doi: 10.1016/j.molcel.2008.12.026

Figure 5.

Figure 5

Phosphorylation of LKB1 on Ser325 and Ser428 is involved in the regulation of AMPK activation by LKB1.

(A) Mutation of Ser325 or Ser428 of LKB1 to Ala enhances its activity on AMPK activation. Lkb1-/- MEFs were infected with retrovirus containing the vector control, WT LKB1, S325A, S428A or S325A/S428A LKB1, and treated with 1 mM AICAR for 1 hr. Cell lysates were used for western blotting with indicated antibodies.

(B) Expression of LKB1 S325A/S428A mutant stimulates AMPK in SK-MEL-28 cells. SK-MEL-28 cells were infected with retrovirus containing WT LKB1, S428A, S325A or S325A/S428A (AA) LKB1 mutants. Cell lysates were used for western blotting analysis with indicated antibodies.

(C) Mutation of Ser325 and Ser428 to Ala enhances the ability of LKB1 to associate with AMPK, but not STRAD and MO25. HEK293 cells were transfected with GST-AMPKα1, Omni-STRAD, FLAG-MO25 together with WT or S325A/S428A (AA) mutant of HA-LKB1. Cell lysates were immunoprecipitated with indicated antibodies or incubated with GSH agarose beads for 2hr followed by western blotting with indicated antibodies.

(D) Mutation of Ser325 and Ser428 to alanines or U0126 treatment enhances the ability of LKB1 to associate with endogenous AMPK in SK-Mel-28 cells. SK-Mel-28 cells stably expressing FLAG-LKB1 WT or AA mutant were treated with DMSO or 20 μM of U0126 for 2 hr. Cell lysates were incubated with M2 anti-FLAG agarose beads for 2 hr followed by Western blotting with indicated antibodies.

(E) Knockdown of B-RAF expression enhances the ability of LKB1 to associate with endogenous AMPK in SK-Mel-28 cells. Sk-Mel-28 cells stably expressing FLAG-LKB1 WT were infected with retrovirus containing two different shRNA constructs in pSUPER-retro against B-RAF or pSUPER-retro empty vector. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads followed by western blotting using indicated antibodies.