FIGURE 3.

LKB1 activity is necessary for suppression of PAK1 activity. A, the wt LKB1, but not the kinase-dead mutant (KD), inhibits PAK1 activity. HEK293T cells were co-transfected with the indicated constructs. At 24 h post-transfection, immunoprecipitates were prepared with an anti-Myc antibody. The precipitates were used for in vitro kinase assays using GST-VASP-(158–277) as a substrate. The kinase activities were visualized using BAS-5000 Bio-imaging Analyzer. Cellular levels of Myc-PAK1 and HA-LKB1 were determined by Western blotting with anti-Myc or anti-HA antibody. B, recombinant LKB1 complex (LKB1-STRAD-MO25) directly inhibits PAK1 activity in vitro in a dose-dependent manner. The LKB1 complex (LKB1-STRAD-MO25) was preincubated with recombinant PAK1 and ATP in the absence of any PAK1 substrates, followed by addition of GST-VASP. The phosphorylated VASP was detected using an anti-phospho-VASP (Ser²³⁹) antibody. Coomassie Brilliant Blue was used for detection of GST-VASP-(158–277). Similar results were obtained in three independent experiments.