Figure 4
TQ induces early upregulation of ERK1/2 and the formation of a PAK1-ERK1/2 complex. A. TQ treated cells were assessed for ERK1/2 activity. Data is presented as fold activity normalized to control activity. Each value is the mean ± SD of two independent experiments each done in duplicates. B. Cells pretreated for 1 hour with 10 μM IPA-3 were incubated with 40 μM TQ and cell pellets were collected after 1, 3, 6 and 24 hours. Lysates were immunoblotted for pERK1/2 and total ERK1/2. GAPDH was used as loading control. C. Box plot analysis of the percentage of tumor cells expressing nuclear pERK1/2 in mouse xenografts after TQ treatment. n represents the number of investigated mouse tissues in each group. *5 corresponds to a sample outlayer. D. Cell lysates stimulated with 60 μM TQ for 10, 45 minutes, 1 and 24 hours were immunoprecipitated for PAK1 (upper panel) and ERK1/2 (lower panel) and blotted against total PAK and ERK1/2. IgG bands were used as loading control. Data shown are representative of two independent experiments. E. Cells transfected with the PAK1wt vector and the T423E mutant vector were exposed to 24 hours of 100 μM TQ. 30 μg of proteins was immunoblotted against pERK1/2. *indicates the omission of intermediate samples. F. Cells transfected or not with T212A and T212E mutants of PAK1 were treated with 100 μM TQ. Lysates were immunoblotted against pPAKThr423. Data shown are representative of two independent experiments. G. Model of PAK1 activation (in dark blue) and catalytic loop (purple) presenting the different amino acids of interest. The kinase domain in PAK1 (residues 249–545) is where T423 resides. H. Model of PAK1 activation (in dark blue) and catalytic loop (purple) in the presence of TQ showing rearrangements in the main activation loop under TQ. There are additional hydrogen bonds in the catalytic loop region involving residues Arg388 and Arg421 which are known to interact with Thr423 for the catalytic activity.