A phosphorylation assay using [γ-32P]ATP: A highly sensitive detection of protein kinase C

Abstract

The involvement of protein kinase C (PKC) in many biological processes such as development, memory, cell differentiation and proliferation, and carcinogenesis has been demonstrated. Using the mep45 gene encoding the 45-kDa major envelope protein (Mep45) of Selenomonas ruminantium, a protein-fused substrate (neurogranin-Mep45, MFS-PKC) was cloned, which is a highly selective substrate for PKC. The recombinant protein-fused substrate can be constantly produced in reasonable quantities with a small outlay. In this study, a suitable strategy for the detection of the phosphorylation of a peptide-type substrate and a Mep45-fused substrate catalyzed by PKC by using a sensitive radiodetection is described. This strategy can be applicable to the development of protein microarray, which can be a useful tool for high-throughput screening in biological and medical research.