MK2/3-dependent mechanisms of RIPK1 phosphorylation-driven regulation of inflammation and cell death
Biochemistry
Final Report Abstract
The signaling events of the TNF receptor complex are subject of intensive and competitive international research. The recent findings that the protein kinase MK2 phosphorylates RIPK1 at S321 and S336 and inhibits its proapoptotic activity in various cell types was the starting point for this project, which aims to identify molecular mechanisms and signaling processes involved. To reach this goal, the MK2-dependent subcellular localization and interacting proteins of RIPK1. Apart from this, the effect of phosphorylation of RIPK1 on the TNF-receptor mediated cytokine biosynthesis should be analyzed. While we focused on MK2-dependent mechanisms during this project, a wide variety of additional PTMs of RIPK1 – about 30 ubiquitinylations and 40 phosphorylations including auto-phosphorylations – have been identified. Also, further protein kinases besides MK2 and IKKs have been found to modify RIPK1 partially at the same residues: TAK1, TBK1, IKKε and ULK1. Therefore, the analysis of MK2-specific phosphorylation of RIPK1 became extremely difficult and, because of interference with the other newly identified kinases, phosphorylation-specific antibodies against pS320/321 generated by us were not able to detect an MK2-dependent subcellular translocation of RIPK1 and RIPK1-S321A knock-in cells generated by us were not suitable to identify MK2-specific effects. We then put our emphasis on identification of MK2-dependent interaction partners of RIPK1 by SILAC experiments. Of these, the arginine-methyltransferase PRMT5 was further analyzed and demonstrated to modify RIPK3 covalently within the pro-necroptotic RIPK1/RIPK3 complex. In addition, we analyzed RIPK1/MK2-dependent cytokine expression and made the interesting observation that RIPK1-dependent expression of Csf3 (G- CSF) is strongly increased in the absence of MK2. Finally, we screened a kinase inhibitor library in MK2-rescued and - deficient cells for sensitizing these cells to TNF induced cell and identified 5-Iodotubericidin (5-ITu) as an inhibitor which strongly induces RIPK1-dependent cell death only in the absence of MK2. These findings could form the basis for further roads to the understanding of MK2’s role in the complex scenario of PTM-mediated function of the TNF receptor complex.
Publications
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5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling. Cell Death Discovery, 9(1).
Chauhan, Chanchal; Kraemer, Andreas; Knapp, Stefan; Windheim, Mark; Kotlyarov, Alexey; Menon, Manoj B. & Gaestel, Matthias
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PRMT5-mediated regulatory arginine methylation of RIPK3. Cell Death Discovery, 9(1).
Chauhan, Chanchal; Martinez-Val, Ana; Niedenthal, Rainer; Olsen, Jesper Velgaard; Kotlyarov, Alexey; Bekker-Jensen, Simon; Gaestel, Matthias & Menon, Manoj B.
