Design and characterization of a traceable protein kinase Cα

Protein kinase C alpha (PKC alpha) is a critical component of pathways that govern cancer-related phenotypes such as invasion and proliferation. Proteins that serve as immediate substrates for PKC alpha offer potential targets for anticancer drug design. To identify specific substrates, a mutant of PKC alpha (M417A) was constructed at the ATP binding site such that it could bind a sterically large ATP analogue derivatized through the N-6 amino group of adenosine ([gamma-P-32]-N-6-phenyl-ATP). Because this analogue could be utilized by the mutant kinase but not by wild-type PKC alpha (or presumably other protein kinase) to phosphorylate peptide or protein substrates, P-32-labeled products were the direct result of the mutant PKC alpha. Kinetic analysis with [gamma-P-32]-N-6-phenyl-ATP revealed that the mutant retained undiminished affinity for the peptide substrate (K-m = 12.4 mu M) and a V-max value (10.3 pmol/min) that was only 3-fold lower than that exhibited by the wild-type enzyme with natural ATP. However, with [gamma-P-32]ATP, the mutant had a somewhat lower affinity (K-m = 82.8 mu M) than the wild-type enzyme (K-m = 9.3 mu M) 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 PKC alpha. The FLAG-tagged PKC alpha mutant was expressed in MCF-10A cells and used to co-immunoprecipitate high-affinity substrates from lysates. Immunopellets were reacted with [gamma-P-32]-N-6-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 PKC alpha mutant.