TITLE:
A Highly Sensitive Detection Method, Phos-tagTM Affinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in Arabidopsis
AUTHORS:
Di Xi, Leping He, Lei Zhang
KEYWORDS:
Calcium-Dependent Protein Kinase (CDPK), CDPK-Related Protein Kinase (CRK), WD-Repeat Protein (WDRP), Protein Phosphorylation, Phos-tagTM
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.9 No.8,
July
31,
2018
ABSTRACT: Phosphorylation
of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very
important to evaluate diverse biological
processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based
on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC)
further to study and visualize the interaction between CRK5 and WDRP in living
cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His
antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might
be phosphorylated by CRK5 in vitro. Site
mutation analysis suggested that serine-70 might be the amino acid
phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used
to analyze the kinase reaction using recombinant WDRP as substrate. These
results demonstrated that WDRP was phosphorylated by cell extracts and that there
may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of
phosphorylation in plants.