Protein kinase Cα (PKCα) is a critical component of pathways that govern cancer-related phenotypes such as invasion and proliferation. Proteins that serve as immediate substrates for PKCα offer potential targets for anticancer drug design. To identify specific substrates, a mutant of PKCα (M417A) was constructed at the ATP binding site such that it could bind a sterically large ATP analogue derivatized through the N6 amino group of adenosine ([γ-32P]-N6-phenyl-ATP). Because this analogue could be utilized by the mutant kinase but not by wild-type PKCα (or presumably other protein kinase) to phosphorylate peptide or protein substrates, 32P-labeled products were the direct resu... More
Protein kinase Cα (PKCα) is a critical component of pathways that govern cancer-related phenotypes such as invasion and proliferation. Proteins that serve as immediate substrates for PKCα offer potential targets for anticancer drug design. To identify specific substrates, a mutant of PKCα (M417A) was constructed at the ATP binding site such that it could bind a sterically large ATP analogue derivatized through the N6 amino group of adenosine ([γ-32P]-N6-phenyl-ATP). Because this analogue could be utilized by the mutant kinase but not by wild-type PKCα (or presumably other protein kinase) to phosphorylate peptide or protein substrates, 32P-labeled products were the direct result of the mutant PKCα. Kinetic analysis with [γ-32P]-N6-phenyl-ATP revealed that the mutant retained undiminished affinity for the peptide substrate (Km = 12.4 μM) and a Vmax value (10.3 pmol/min) that was only 3-fold lower than that exhibited by the wild-type enzyme with natural ATP. However, with [γ-32P]ATP, the mutant had a somewhat lower affinity (Km = 82.8 μM) than the wild-type enzyme (Km = 9.3 μM) in vitro but was competent in causing aggressive motility in nonmotile MCF-10A human breast cells (with endogenous ATP), as previously described for wild-type PKCα. The FLAG-tagged PKCα mutant was expressed in MCF-10A cells and used to co-immunoprecipitate high-affinity substrates from lysates. Immunopellets were reacted with [γ-32P]-N6-phenyl-ATP, and radiolabeled products were analyzed by SDS-PAGE and autoradiography. Mass spectrometry of selected bands identified several known substrates of PKC, thereby validating the methods used in these studies. These findings provide a foundation for future applications of this traceable PKCα mutant.
