Peptide Assay of Protein Kinases and Use of Variant Peptides to Determine Recognition Motifs

It is now clear that the major mechanism by which cellular function is switched from one state to another in eukaryotic cells, including the response to cellular stress (1 ), is via changes in phosphorylation of key proteins. This is usually achieved by modulation of the activity of pre-existing protein kinases (and/or protein phosphatases), rather than by changes in expression at the transcriptional or translational level: most protein kinases are in fact constitutively expressed. Assays of kinase expression levels (e.g., by Western blotting) are therefore not sufficient. In some cases (e.g., MAP kinases), activating phosphorylation events may produce a shift in mobility of the kinase on a Western blot which can be used as an index of activation. However, since nonactivating phosphorylation events can also produce mobility shifts, this method can be unreliable and there remains a requirement to develop direct assays of protein kinase activity. In many cases, phosphorylation of synthetic peptide substrates provides the simplest assay for routine use.