Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites

A novel peptide with multiple phosphorylation sites, which we designated as multide, was developed to detect a wide variety of protein kinases in crud e cell extracts. Multide, KKRKSSLRRWSPLTPRQMSFDC, has been designed to cont ain consensus sequences for various Ser/Thr protein kinases including cAMP- dependent protein kinase, protein kinase C, MAP kinases, and Ca2+/calmoduli n-dependent protein kinases in a single peptide. In-gel protein kinase assa y using multide was found to be very useful for analyzing the activities of protein kinases that are altered in response to various extracellular stim uli. The substrate specificities of the protein kinases thus detected were further determined by using five multide analogs with different phosphoryla tion sites.