Combined 15N-Labelling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis

ABSTRACT: Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the large dynamic range of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the mass spectrometry measurement and accurate quantification of low abundant, transiently phosphorylated peptides. Since tandemMOAC is not biased towards the enrichment of acidophilic, basophilic or proline-directed kinase substrates, the method is applicable to identify targets of members of all three classes of protein kinases. Using this phosphoproteomics approach, we identified several mitogen-activated protein kinase (MPK) substrates downstream of the MKK7-MPK3/6 phosphorylation cascade of Arabidopsis. The MKK7-MPK3/6 module is involved in the regulation of plant development and plant basal and systemic immune responses but little is known about downstream cascade components. The identification and validation of dynamin-related protein (DRP) 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Seedling

DISEASE(S): Disease Free

SUBMITTER: Wolfgang Hoehenwarter  

LAB HEAD: Wolfgang Hoehenwarter

PROVIDER: PXD008200 | Pride | 2018-01-02

REPOSITORIES: Pride