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 Cα (PKC-α) in nontransformed human breast MCF-10A cells. Here, a nonradioactive variation of this method compared the phosphoprotein profiles of three traceable PKC isoforms (α, δ, and ζ) 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-α and -δ were similar and differed markedly from that of PKC-ζ. 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-α, -δ, and -ζ 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-α, -δ, and -ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and -δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms.
Department of Chemistry and Biochemistry, Queens College, The City University of New York, 65-30 Kissena Boulevard, Flushing, NY 11367, USA.
Xiangyu
X
Chen
Xin
X
Zhao
Thushara P
TP
Abeyweera
Susan A
SA
Rotenberg
D000595:Amino Acid Sequence; D001940:Breast / Q000473:pathology*; D045744:Cell Line, Tumor; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics
D007527:Isoenzymes; D034741:RNA, Small Interfering; D011493:Protein Kinase C