p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage
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Abstract
Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in DNA, resulting in the activation of the DNA damage response. This has a global effect on transcription and splicing. The components and regulatory mechanisms of DNA repair are relatively well established. However, an understanding of the signaling pathway that orchestrates complex changes in transcription and RNA metabolism after UV-light-induced DNA damage is only beginning to emerge. The p38 mitogen-activated protein kinases (MAPK) is a key transducer of cellular stress signaling and is activated by a number of stress-inducing agents, including UV-light. The activation leads to the phosphorylation of other serine/threonine (S/T) kinases, namely MK2, MK3, and MK5 (MK2/3/5). These kinases, in turn, phosphorylate a number of substrates that affect the functionality of diverse cellular processes, such as cell cycle progression, transcription, translation, splicing, and protein trafficking. Phosphorylation by S/T kinases can be recognized by 14-3-3 proteins. The crosstalk between p38 MAPK activation and 14-3-3 recognition has been demonstrated for a few proteins but has not yet been established as a mode of signaling.
Here, we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems-wide view on protein phosphorylation patterns induced by UV-light and uncover the dependencies of phosphorylation events on canonical DNA damage signaling by the S/T kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) and the p38 MAPK pathway. Our data provide evidence that the activation of the p38 MAPK pathway is independent of the ATM/ATR pathways and regulates a different subset of proteins after low dosages of UV-C light. We detect the phosphorylation of p38 MAPK as well as its downstream kinases, MK2/3, shortly after radiation and observe it for up to one-hour. The p38 MAPK acts primarily through the MK2/3 kinase, which recognizes the LXRQX[ST] motif on the substrates. The same motif, when it is phosphorylated, is also recognized by the 14-3-3 family. We identify RNA-binding proteins as primary substrates and 14-3-3 as a direct “reader” of p38-MK2-dependent phosphorylation induced by UV-light. We mechanistically demonstrate that MK2 phosphorylates the RNA-binding subunit of the NELF complex, NELFE, on serine 49 (S49), S51, S115, and S251. NELFE phosphorylation promotes the recruitment of 14-3-3. Further analysis has determined that S115 plays the crucial role in 14-3-3 binding. This interaction between NELFE and 14-3-3 leads to the rapid dissociation of the NELF complex from chromatin. Aligned with this finding, we discover that the transient knockdown of NELFE results in an increased sensitivity of cells to UV-light. Our ChIP-seq analysis demonstrates that the NELF release is accompanied by RNA polymerase II elongation. Altogether, these events seem to promote cell survival during the response to UV-light DNA damage.