Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases

BMC Plant Biology

Research Article

Ulrich Schwaneberg¹  Marco Bocola¹  Uwe Conrath²  Franz Leissing²  Gerold J. M. Beckers²  Mika Nomoto³  Yasuomi Tada⁴ 

[1] Department of Biotechnology, Aachen Biology and Biotechnology, RWTH Aachen University, 52056, Aachen, Germany;Department of Plant Physiology, Aachen Biology and Biotechnology, RWTH Aachen University, 52056, Aachen, Germany;Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, 464-8602, Nagoya, Aichi, Japan;Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, 464-8602, Nagoya, Aichi, Japan;The Center for Gene Research, Division of Biological Science, Nagoya University, Furo-cho, Chikusa-ku, 464-8602, Nagoya, Aichi, Japan;

关键词: Mitogen-activated protein kinase;    (thio-)phosphorylation;    MPK3/4/6;    Arabidopsis thaliana;    Analog-sensitive kinase;   

DOI  :  10.1186/s12870-016-0731-6

 received in 2015-10-27, accepted in 2016-02-06,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundMitogen-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 in Arabidopsis thaliana, and we disclosed their phosphorylation sites.ResultsWe 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.ConclusionOur work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.

【 授权许可】

CC BY   
© Leissing et al. 2016