In vivo Protein Interaction during Cell Signaling

The ability to adapt to a continuously changing environment is an essential property of every organism. This adaptation depends on proper responses to environmental signals perceived by cell receptors and integrated via a network of mitogen-activated protein kinases (MAPKs). In Arabidopsis MAPKs have been identified in regulation of stress and developmental signalling, however mechanisms which help define MAPK signalling specificity remain unclear. Studies of mammalian MAPK signalling demonstrate that localisation of signalling components is important for signaling outcome and that MAPK phosphatases control signalling outcome in cells by inactivation of MAPKs as well as by spatiotemporal regulation. We have identified Arabidopsis PP2C-type protein phosphatases AP2Cs as MAPK phosphatases, which interact with and inactivate MAPKs. Specific AP2Cs control plant immunity or development. Moreover, we found that localization of AP2Cs strongly influences the MAPK signaling outcome. Lessons from mammals and evidence in plants suggest our working hypothesis that plant MAPK phosphatases inactivate MAPK in different cell compartments and direct the outcome of the signaling by their localization and interaction with MAPKs in cells. Knowing intracellular localization of enzymes could help to specify their functions and access to substrates thus assist in defining specificity of cellular signalling in plants. Here, we propose to study the localization and in vivo interaction of MAPK phosphatases with their substrate MAPKs in Arabidopsis by taking advantage of recently developed advanced imaging technologies. To investigate where/how negative regulators AP2Cs interact with MAPKs, we will study the dynamic localisation and interaction between the phosphatases and MAPKs in vivo under normal and stress conditions by combination of imaging with molecular biology and biochemical techniques. Taken together, this work will substantially advance understanding of regulation of MAPK signal transduction in plants and highlight the cell-autonomous mechanisms of plasticity plants display in order to adapt to environmental changes.

Signal transduction describes the perception, transmission and procession of (environmental) signals perceived from the cell, which lead to changes e.g. in gene expression and altered metabolism. The family of mitogen-activated protein kinases (MAPKs) plays a major role within these signaling processes, as their sequential phosphorylation enables a rapid and specific cellular response. A major question in basic and applied research is the identification of high specificity of MAPK cascades, as a plethora of described pathways channels only through the same few MAPKs, although leading to precise output. These results are additionally puzzling as MAPKs are in general ubiquitously expressed. MAPK activation occurs transiently, the essential inactivation by dephosphorylation is performed by protein phosphatases (MAPK phosphatases). This project aimed to approach the question, if signaling specificity in plants could be explained by analysing the intracellular protein distribution and protein interactions between MAPK and MAPK phosphatases by using advanced microscopy technologies. We indeed showed, that after MAPK activation and application of inhibitors that interfere with protein localization, a MAPK phosphatase shuttles between intracellular organelles, while the investigated MAPKs did not significantly change their localizations. Further, we identified a domain within the phosphatase as putatively responsible for these translocations. This study provides a seminal contribution to dissect the regulation of MAPK signal specificity, even beyond plant research.