Molecular programming of MAPK signaling by temporal dynamic site-specific phosphorylation

• Applicant: Dr. Stefan Jacob
• Subject Area: Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 549085121

The fundamental molecular mechanisms of how exactly multiple different signals are transmitted and encrypted from environment to the target site in the cell are still enigmatic, particularly because of the limited number of signaling proteins and a multitude of transitions inside the living cell. Protein phosphorylation is one of the hallmarks enabling transmission and encryption of signals whereby it is often induced by mitogen activated protein kinases (MAPK). In TEDYPHOS, we use the phytopathogenic fungus Magnaporthe oryzae to study the MAPK MoHog1p comprehensively with unprecedented precision. MoHog1p is a key regulator in the HOG pathway, well conserved in eukaryotes and classified as a p38-type MAPK. Signal transduction at this MAPK is achieved by dual phosphorylation of a conserved TxY-motif, but it is not known how various stimuli are individually encrypted and transmitted. We aspire to assemble a dynamic multidimensional model of the individual time- and stimulus-dependent intensity of T, Y and T/Y phosphorylation to unravel the molecular MAPK programming in vivo. The multiple dimensions comprise (1) the intensity of phosphorylation at T, Y and T/Y, (2) variation of stimuli, (3) different concentrations of stimuli, (4) time course and (5) different (transcriptional) responses. We recently were able to pinpoint key differences in the degree of the intensity of phosphorylated T, Y and T/Y over time after activation of the signaling pathway upon salt stress and found this pattern individually different as compared to the pattern upon fludioxonil fungicide stress. Consequently, we set up the hypothesis, that temporal dynamic site-specific phosphorylation at the single amino acids of the TxY-motif is the molecular mechanism to encrypt and encode multiple signals at MAPKs. In order to address this hypothesis, TEDYPHOS will overcome the limitations of current assay systems and comprehensively monitor data for all five dimensions by combining two worlds: Molecular fungal genetics and cutting-edge label-free biophysical protein analytics. The model organism used is easy to handle, fast growing and molecular manipulation is straight forward. The possibility of implementing reproducible assays in pure water and synchronizing millions of individuals facilitates the application of high-precision mass spectrometry-based phospho-analytics. Therefore, it can be concluded that the TEDYPHOS project provides an excellent opportunity to unravel signal encryption, which will contribute novel insights for basic research in cellular signaling.

• DFG Programme: Research Grants