Mapping proteome-wide targets of protein kinases in plant stress responses

Abstract

Protein kinases are major regulatory components in almost all cellular processes in eukaryotic cells. By adding phosphate groups, protein kinases regulate the activity, localization, protein–protein interactions, and other features of their target proteins. It is known that protein kinases are central components in plant responses to environmental stresses such as drought, high salinity, cold, and pathogen attack. However, only a few targets of these protein kinases have been identified. Moreover, how these protein kinases regulate downstream biological processes and mediate stress responses is still largely unknown. In this study, we introduce a strategy based on isotope-labeled in vitro phosphorylation reactions using in vivo phosphorylated peptides as substrate pools and apply this strategy to identify putative substrates of nine protein kinases that function in plant abiotic and biotic stress responses. As a result, we identified more than 5,000 putative target sites of osmotic stress-activated SnRK2.4 and SnRK2.6, abscisic acid-activated protein kinases SnRK2.6 and casein kinase 1-like 2 (CKL2), elicitor-activated protein kinase CDPK11 and MPK6, cold-activated protein kinase MPK6, H2O2-activated protein kinase OXI1 and MPK6, and salt-induced protein kinase SOS1 and MPK6, as well as the low-potassium-activated protein kinase CIPK23. These results provide comprehensive information on the role of these protein kinases in the control of cellular activities and could be a valuable resource for further studies on the mechanisms underlying plant responses to environmental stresses.

Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold

“…The other individually enriched motifs were [SXpS], [LXR/KXXpS/pT], [pS/pTF], [pS/pTG], and [pSXP]. Most of these motif sequences are well annotated to their up-stream kinases in the literature and databases [ 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ]. Proline-directed motifs [pS/pTP] are a potential substrate for MAPK, cyclin-dependent protein kinase (CDK), CDK-like, and glycogen synthase kinase-3/shaggy-like kinase (GSK3/SLK).…”

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus

“…We asked whether ABA signaling could affect host antiviral RNA-silencing components at a post-translational level. Public data from A. thaliana phosphoproteomes were analyzed ( Umezawa et al, 2013 ; Wang et al, 2013 , 2020 ; Mergner et al, 2020 ). We considered a subset of proteins redundantly identified in at least two independent phosphoproteomic datasets from plants treated with ABA or subjected to osmotic stress ( Supplemental Data 1C ).…”

“…If ABA signaling does not regulate the transcript abundance of RNA-silencing genes, what is the functional role of ABA in antiviral immunity? A network of protein phosphatases and kinases mediates ABA signaling and plant responses to osmotic stress ( Cutler et al, 2010 ; Umezawa et al, 2013 ; Wang et al, 2013 , 2020 ), and this network might post-translationally regulate host proteins that are directly or indirectly necessary for plant immunity or susceptibility. Translation inhibition, RNA maturation and turnover, and their regulation by phosphorylation events have critical roles in the antiviral immune responses of human cells ( Narayanan and Makino, 2013 ; McCormick and Khaperskyy, 2017 ; Boudreault et al, 2019 ).…”

“…The datasets of A. thaliana proteins with significantly altered phosphorylation status in response to ABA or dehydration stress (ABA_DH), ABA (ABA_1, ABA_2), mannitol, or NaCl have been reported ( Umezawa et al, 2013 ; Wang et al, 2013 , 2020 ). Wild-type and snrk2.2/2.3/2.6 triple-mutant results were considered, and protein lists are included in Supplemental Data 1D .…”

FERONIA cytoplasmic domain: node of varied signal outputs

“…Whether FER has different functions that are dependent on or independent of kinase activity is unclear. Studying kinase activity and phosphorylation sites of FER triggered by different RALFs, and systematic identification of FER kinase substrates using quantitative phosphoproteomics analysis (Wang et al 2020b;Wang et al 2013) will help in clarifying the basis of varied signaling outputs that are caused by the FER cytoplasmic domain.…”