Identification of MAPK Substrates Using Quantitative Phosphoproteomics

Abstract

Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

Post-translational regulation of plant immunity

“…Although phosphorylation is a prominent posttranslational mechanism for propagating rapid and transient immune responses, identifying the kinases upstream of a known substrate remains a major challenge. The use of targeted, quantitative proteomics and the application of proximity-dependent affinity labeling of protein complexes in vivo show promise for the discovery of unknown kinases [62,63••,64••]. Additionally, advances in the sensitivity of proteomics instrumentation is rapidly increasing our ability to detect PTMs that occur in low stoichiometry, and therefore will open the door to understanding how these modifications contribute to immune responses.…”

Metabolomics of Early Stage Plant Cell–Microbe Interaction Using Stable Isotope Labeling

“…Recently, significant progress has been made to understand the intricate plant defense networks that involve the generation of reactive oxygen species ( Camejo et al, 2016 ), activation of protein kinases ( Zhang et al, 2017 ), induction of pathogenesis-related ( PR ) gene expression, synthesis of phytoalexin ( Piasecka et al, 2015 ) and activation of programmed cell death ( Teh and Hofius, 2014 ). However, the contribution of primary metabolites to plant immune response is not well-understood.…”

Proteomics data of SNF1-related protein kinase 2.4 interacting proteins revealed by immunoprecipitation-mass spectrometry

“…Analysis of IP-MS data was performed as previously described [8 , 10] . All raw data were searched with MaxQuant [11 , 12] against the TAIR10 protein database (35,386 entries, downloaded on October 2017).…”