Figure 4.
Biological significance of AMPK-α1 cleavage
(A) Human α1β2γ1 complexes (WT or D529A or STREP-stop mutants) were expressed in E. coli; following purification on Ni2+-agarose (WT or D529A mutant) or Ni2+-agarose and Strep-Tactin columns (STREP-stop mutant), duplicate samples were analyzed by western blots probed with the indicated antibodies.
(B–E) Bacterially expressed, unphosphorylated human α1β2γ1 complexes (WT, D529A, or STREP-stop) were incubated in duplicate with the indicated amounts of either (B and C) human LKB1:STRAD:MO25 complex or (D and E) CaMKK2. Western blots and AMPK activity were measured.
(F) Purified rat liver AMPK was first depleted of α2-containing complexes by immunoprecipitation, and the remaining α1 complexes were incubated for 2 h with the amounts of bacterially expressed caspase-3 shown in Figure S4C. Samples were assayed for AMPK activity in the presence or absence of AMP (200 μM) or A-769662 (10 μM). Results are mean ± SEM (n = 3); significant differences from controls without AMP or A-769662 are indicated (∗∗∗p < 0.001).
(G) Jurkat cells were treated with indicated concentrations of anti-Fas. Nuclear and cytoplasmic extractions were prepared and western blot analysis carried out.
(H) HeLa cells were transiently transfected with the indicated GFP-tagged full-length (GFP-WT-AMPK-α1) or truncated (GFP-cl-AMPK-α1 and GFP-ACTP) proteins. Following transfection, cells were visualized by confocal microscopy with nuclei stained using Hoechst 33342 (scale bars, 10 μm).
(I) mCherry-tagged full-length (mCherry-WT-AMPK-α1, red) and truncated (GFP-cl-AMPK-α1, green) AMPK-α1 were co-transfected into HeLa cells and cells visualized by confocal microscopy with nuclei stained with Hoechst 33342 (scale bars, 10 μm).
(J–L) Jurkat cells expressing indicated constructs were exposed to etoposide (10 μM for 6 h). Cytotoxicity, caspase activity, and western blots were carried out; data shown are mean ± SD (n = 3; ∗∗p < 0.01 and ∗p < 0.05).