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BMC Biochem
2010 May 20;11:20. doi: 10.1186/1471-2091-11-20.
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Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes.
Lilienthal E
,
Kolanowski K
,
Becker W
.
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BACKGROUND: Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources.
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.
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.
Figure 1. Direct sandwich ELISA format for the detection of the phosphorylated DYRK substrate peptide tau207-219. A, Scheme illustrating the principle of the assay. Bio, biotin; TMB, tetramethylbenzidine; HRP, horseradish peroxidase (coupled to streptavidin). B and C, Titration of phosphorylated and unphosphorylated tau207-219. The wells were coated with 100 ng anti tau(pT212) and loaded with dilution series of either phosphorylated or unphosphorylated tau207-219. The background signal from wells loaded only with the buffer was subtracted from all values. A representative experiment of three is shown. Panel C presents the same data as in panel B with a linear x-axis to visualize the linear range of the ELISA. The inset shows an enlargement of lower range. D, Titration of phosphorylated and non-phosphorylated tau207-219 on streptavidin-coated wells. Detection was performed with primary anti-tau(pT212) antibody and secondary goat anti-rabbit antibody coupled to HRP. The graph is representative of two experiments. E, Detection of phosphorylated tau207-219 in the presence of excess unphosphorylated peptide. Different amounts of phosphorylated tau207-219 (0.01 pmol, 0.1 pmol, 1 pmol) were mixed with a dilution series of unphosphorylated tau207-219 (12.5 - 100 pmol). Signals obtained in wells loaded only with the same amount of the unphosphorylated peptide were subtracted from the read-out of the mixtures. The graph is representative of two experiments. In B-E, error bars indicate the difference between duplicate wells.
Figure 2. Assay sensitivity. A, In vitro-kinase reactions were performed with 50 μM tau207-219, 100 μM ATP and variable concentrations of GST-DYRK1A-ΔC or GST-DYRK2 for 30 min at 30°C. Reactions were stopped by addition of EDTA and peptide phosphorylation was analysed by the ELISA method. Panels B and C show enlarged views of the lower kinase concentration range. Error bars indicate the difference between duplicate measurements.
Figure 3. Immunoprecipitation of xDYRK1B. A, C-terminal sequences of human and Xenopus DYRK1A and DYRK1B. Amino acids identical with the immunogenic peptide used to raise the DYRK1B antibody are highlighted in bold. B, Immunoprecipitation of xDYRK1B. HeLa cells were transiently transfected with expression plasmids for xDYRK1B, GFP-DYRK1B or the empty vector (control). After immunoprecipitation with DYRK1B-specific antiserum (αD1B) or preimmune serum (Pre), bound proteins were subjected to Western blot analysis with a phosphotyrosine-specific antibody (P-Tyr) and the DYRK1B antiserum as indicated. Detection of IgG is shown as a loading control. The asterisk marks an unspecific band. C, Specificity of the DYRK1B antiserum. COS7 cells were transiently transfected with expression plasmids for the GFP fusion proteins of mammalian DYRK1A or DYRK1B as indicated on top of the lanes. Aliquots of the whole cell lysates (input) or proteins eluted from the immunoprecipitates (αD1B, Pre) were subjected to Western blot analysis with anti GFP and anti DYRK1B antibodies as indicated. Detection of IgG is shown as a loading control.
Figure 4. Immunocomplex kinase assay of xDYRK1B from Xenopus laevis oocytes. Xenopus oocytes were lysed and the lysate was subjected to immunoprecipitation with either DYRK1B antiserum or preimmune serum (αD1B, Pre). Aliquots from immunocomplex kinase reactions (100 μM ATP, 50 μM tau207-219, 30°C, total volume of 50 μL) were taken at different times and the amounts of phosphorylated tau207-219 were determined by the ELISA method with the help of a standard curve. Error bars represent the difference between the duplicate measurements.
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