MK2/3-dependent mechanisms of RIPK1 phosphorylation-driven regulation of inflammation and cell death

• Applicant: Professor Dr. Matthias Gaestel, since 5/2019
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 405953283

TNF-receptor-interacting kinase RIPK1 is a master regulator of cell-fate decisions during infection and inflammation. Complex posttranslational modifications (PTMs) determine the effects of RIPK1 on cell fate, in response to TNF and pathogen associated molecular patterns like lipopolysaccharide (LPS). We recently identified a crosstalk between the stress- and cytokine-activated p38 MAPK-MK2/3 pathway and RIPK1 signaling, wherein MK2 directly phosphorylates RIPK1 suppressing RIPK1-activation, autophosphorylation and integration into cytosolic death-inducing complexes. Consistent with a negative role for MK2 in regulating RIPK1-kinase activity, it was observed that smac-mimetics (SM)-induced TNF production and subsequent leukemia cell death was enhanced by p38/MK2 inhibitors. In the current project we will investigate the mechanism of MK2-mediated control of RIPK1 functions in cell death and inflammation. MK2-dependent changes in the PTMs, kinase-activation, subcellular localization, oligomerization, and interactions of RIPK1 will be analyzed in MK2/3-deficient fibroblasts and macrophages and control cells by using phosphorylation site- and modification-specific antibodies as well as candidate- and discovery-LC-MS/MS-based approaches. CRISPR/Cas9 knock-in methods will be utilized to generate cell lines harboring phospho/ubiquitination-site mutants of RIPK1 and analyze the interplay of the PTMs in defining the outcome of TNF/LPS signaling. While MK2 positively regulates LPS-induced TNF production, a process independent of RIPK1 activity, it is a negative regulator of RIPK1activity-dependent cytokine production induced by SM. Combining LPS treatment with caspase inhibition leads to RIPK1-activity-dependent cytokine production. We will investigate this stimulus-dependent dichotomy of MK2 function in inflammation by a comparative analysis of these stimuli in primary and immortalized macrophage models. The candidate PTMs and interaction partners identified in the screens will be used to further delineate the mechanisms controlling cell and stimulus specific outcome of MK2-RIPK1 signaling. Because of its essential role in biosynthesis of TNF, MK2 is considered a promising target for anti-inflammatory therapy. Understanding the new, RIPK1-dependent role of MK2 in inflammation will be a key to the success of future therapeutic strategies. In addition, deciphering the mechanism of SM-induced TNF production will provide ways to circumvent SM-resistance in anti-cancer therapy. Hence, the successful completion of the well-defined and interlinked goals in the proposed project will provide both insights into the basic mechanisms of cell death control by MK2/RIPK1 and therapeutically relevant data for targeting this pathway in anti-inflammatory and cancer therapy.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Privatdozent Dr. Manoj Balakrishna Menon, until 4/2019