Background
Although numerous non-radioactive
Conclusion
We present a non-radioactive and highly sensitive method for the measurement of endogenous activities of DYRKs in biological samples. Xenopus laevis oocytes contain an active DYRK1-related protein kinase more similar to mammalian DYRK1B than DYRK1A.
Results
Phosphorylation-site specific antibodies can be used to monitor the accumulation of the phosphorylated product 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 the detection of small amounts of phosphopeptide in mixtures with an excess of the unphosphorylated substrate peptide (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 of endogenous DYRK1A activity in mouse heart tissue by immunocomplex kinase assay. Furthermore, we detected DYRK1-like kinase activity in Xenopus laevis oocytes and identified this kinase as a DYRK1 isoform distinct from the Xenopus DYRK1A ortholog.