CDPK Activation in PRR Signaling
Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme’s apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.
Key wordsCDPK Signal transduction In-gel kinase assay Electrophoretic band shift Transient expression Protoplast Phosphorylation target
This work was supported by the Agence Nationale de la Recherche to M.B. (grant number ANR-15-CE20-0003-01) and by German Science Foundation DFG within SFB 973 and FOR 964 to T.R. The IPS2 benefits from the support of the LabEx Saclay Plant Sciences-SPS (grant number ANR-10-LABX-0040-SPS).
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