Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes

摘要

Background: Although numerous non-radioactive methods are in use to measure the catalytic activity of proteinkinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection ofendogenous kinase activity in biological samples. Kinases of the DYRK family have important functions indevelopmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed todevelop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues orcells from diverse sources.Results: Phosphorylation-site specific antibodies can be used to monitor the accumulation of the phosphorylatedproduct in kinase assays. We present a modified configuration of an enzyme-linked immunosorbent assay (ELISA)-based kinase assay by using the phosphospecific antibody as the capture antibody. This assay format allowed thedetection of small amounts of phosphopeptide in mixtures with an excess of the unphosphorylated substratepeptide (10 fmol phosphorylated peptide over a background of 50 pmol unphosphorylated peptide).Consequently, low substrate turnover rates can be determined. We applied this method to the measurement ofendogenous DYRK1A activity in mouse heart tissue by immunocomplex kinase assay. Furthermore, we detectedDYRK1-like kinase activity in Xenopus laevis oocytes and identified this kinase as a DYRK1 isoform distinct from theXenopus DYRK1A ortholog.Conclusion: We present a non-radioactive and highly sensitive method for the measurement of endogenousactivities of DYRKs in biological samples. Xenopus laevis oocytes contain an active DYRK1-related protein kinasemore similar to mammalian DYRK1B than DYRK1A.