Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells by the Traceable Kinase Method

A previous report [Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370] described the application of the traceable kinase method in identifying substrates of protein kinase C alpha (PKC-alpha) in nontransformed human breast MCF-10A cells. Here, a nonradioactive variation of this method compared the phosphoprotein profiles of three traceable PKC isoforms (alpha, delta, and zeta) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high-affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N-6-phenyl-ATP, and the resulting phosphoproteins were analyzed by Western blotting with an antibody that recognizes the phosphorylated. PKC consensus site. Phosphoprotein profiles generated by PKC-alpha and -delta were similar and differed markedly from that of PKC-zeta. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-alpha, -delta, and -zeta isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 +/- 0.5 (moles of phospho-CEP4 per mole of CEP4). Following knockdown with isoform-specific shRNA-encoding plasmids, the level of phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC-alpha, -delta, and -zeta), whereas testing of kinase-dead mutants supported a role for only PKC-alpha and -delta in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms.