Identifycation of myb77 transcription factor as a novel substrate of mpk3/6 in vitro
Keywords:Arabidopsis, MYB77, MPK3/6, phosphorylation, substrate, transcription factor.
Mitogen-activated protein kinase (MPK) cascades have emerged as a major signal transduction mechanism that connects diverse receptors or sensors to cellular and nuclear responses in eukaryotes. Although function and activation of some MPKs in response to stimuli have been studied in details, but substrates of these kinases were still poorly understood. Here, we provide that Arabidopsis MYB77 transcription factor, a R2R3-MYB protein, is a substrate of MPK3/6 in vitro. The MPK3, MPK6 and MYB77 proteins from Arabidopsis were expressed in Escherichia coli. Using in vitro phosphorylation assays we have identified that MYB77 was phosphorylated by recombinant MPK3/6. To identify the phosphorylation sites of MYB77 by MPK, purified MYB77 protein was phosphorylated by MPK, desalted and passed over TiO2 for improved phosphopeptide detection. This sample has been used to analysis for mass spectrometry. Seven phosphopeptides of MYB77 contained predictive MPKs phosphorylation sites (S-P motif) were analyzed by mass spectrometry. As results we identified four putative phosphorylation sites originated from 7 phosphopeptide peaks. Our results suggest that MYB77 is a novel substrate of MPK3/6 containing four phosphorylation sites in Arabidopsis.