Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography

Abstract

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

The Arabidopsis Transcription Factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 Is a Direct Substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and Regulates Immunity

“…In addition, AtERF5 and AtERF6 transcription factors have also been reported as direct substrates of MPK6 and/or MPK3 that regulate plant immunity against fungal and bacterial pathogens, whereas their function in PTI is uncertain (Moffat et al, 2012;Son et al, 2012;Meng et al, 2013). Proteomic and phosphoproteomic approaches have been used to identify putative substrates for MAPKs in vivo; however, their biological significance requires further investigation (Peck et al, 2001;Merkouropoulos et al, 2008;Hoehenwarter et al, 2013). In this study, we show that BES1 is a direct substrate of MPK6 in PTI.…”

Phosphorylation of an ERF Transcription Factor by Arabidopsis MPK3/MPK6 Regulates Plant Defense Gene Induction and Fungal Resistance

“…For instance, phosphorylation of WRKY33 by MPK3/MPK6 not only activates the expression of camalexin biosynthetic genes, driving the induction of camalexin biosynthesis in response to pathogen invasion, it is also is involved in the transcriptional activation of ACS2 and ACS6 genes, which contributes to the highlevel ethylene induction by fungal pathogen (Li et al, 2012). In a recent survey using tandem metal oxide affinity chromatography followed by tandem mass spectrometry analysis, more than 30 novel putative MPK3/MPK6 substrates were identified (Hoehenwarter et al, 2013). Detailed function analysis of these substrates and their crosstalk will allow us to have a better understanding of MPK3/ MPK6 cascade in plant signaling.…”

Phototropism: Growing towards an Understanding of Plant Movement

“…Conversion of NPH3 DS to NPH3 LS requires the presence of phot1, and prevention of this conversion through either genetic or pharmacological means abrogates the development of phototropic responses, suggesting that NPH3 LS is necessary for normal phototropic signaling. It appears that a type 1 protein phosphatase mediates the dephosphorylation of NPH3 DS (Pedmale and Liscum, 2007), while a mitogen-activated protein kinase 3/6 complex may regulate phosphorylation of NPH3 in darkness (Hoehenwarter et al, 2013).…”