Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Leissing, Franz; Nomoto, Mika; Bocola, Marco; Schwaneberg, Ulrich; Tada, Yasuomi; Conrath, Uwe; Beckers, Gerold
• Is Part Of: BMC plant biology. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 11
• Publication Date: 2016-12
• Subjects: Mitogen-activated protein kinase -- (thio-)phosphorylation -- MPK3/4/6 -- Arabidopsis thaliana -- Analog-sensitive kinase; Plant molecular biology -- Periodicals; Botany -- Periodicals; 580.5
• DOI: 10.1186/s12870-016-0731-6
• Languages: English
• ISSNs: 1471-2229

Abstract

Abstract Background Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates inArabidopsis thaliana, and we disclosed their phosphorylation sites. Results We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases. Conclusion Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.