Figure 3.

The T-loop Thr is the major site of LKB1 phosphorylation on the AMPK-related kinases. Wild-type (WT) and T-loop Thr to Ala (Thr → Ala) mutants of the indicated GST-AMPK-related kinases were incubated with the LKB1:STRAD:MO25 complex in the presence of Mg²⁺ and [γ³²P]ATP. Phosphorylation of protein substrates was determined by electrophoresis on a polyacrylamide gel and subsequent autoradiography of the Coomassie blue-stained bands corresponding to each substrate. An aliquot of each incubation was also analysed by Western blotting probing with an anti-HA antibody to ensure equal loading of wild-type and mutant AMPK-related kinases (which all possess an HA epitope tag). All proteins migrated with the expected mobility, taking into account the epitope tags. Similar results were obtained in three separate experiments.