New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells
Figure 9
Mutating the gatekeeper amino acid sensitized PKD1 to the inhibition of 1-NA-PP1.
A.Alignment of the primary sequences containing the gatekeeper amino acid in PKD. Arrow indicates the consensus gatekeeper amino acid “Methionine” (M) in a shaded rectangle. B. Expression of wild-type and mutant PKDs. HEK293 cells were transfected with wild-type and two gatekeeper mutants of Flag-PKD1 (Flag-PKD1M659G and Flag-PKD1M659A). Two days after transfection, cells were lysed and subjected to Western blotting for PKD1 and tubulin (loading control). C. 1-NA-PP1 concentration-dependently inhibited PMA-induced activation of Flag-PKD1 and Flag-PKD1M659G. HEK293 cells transfected with Flag-PKD1 and Flag-PKD1M659G were serum-starved for 24 h and pre-treated with 1-NA-PP1 at increasing concentrations in serum-free medium for 45 min, followed by stimulation with PMA at 10 nM for 20 min. The cells were harvested and subjected to immunoblotting for p-S916-PKD1, PKD1, and tubulin. The experiment was repeated three times and representative images from one experiment are shown.