p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

UV-light-induced DNA damage affects RNA metabolism but the underlying signalling pathways are largely unexplored. Here, the authors show that UV light triggers p38-MK2-mediated phosphorylation of the NELF complex, promoting its release from chromatin and concurrent transcriptional elongation

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

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.UV-Strahlung induziert die Bildung von sperrigen Fotoprodukten in der DNA, was zur Aktivierung der DNA-Schadensfunktion führt. Dies hat einen globalen Einfluss auf die Transkription und das Spleißen. Die Komponenten und Regulationsmechanismen der DNA-Reparatur sind relativ gut etabliert. Ein Verständnis des Signalwegs, der komplexe Veränderungen in der Transkription und im RNA-Stoffwechsel nach UV-Licht-induzierten DNA-Schäden organisiert, beginnt sich jedoch erst zu entwickeln. Die p38 MAPK sind ein Schlüsselwandler für zelluläre Stresssignale und werden durch eine Reihe von Stress-induzierenden Substanzen aktiviert, darunter UV-Licht. Die Aktivierung führt zur Phosphorylierung anderer Serin/Threonin (S/T)-Kinasen, nämlich MK2, MK3 und MK5. Diese Kinasen wiederum phosphorylieren eine Reihe von Substraten, die die Funktionalität verschiedener Zellprozesse beeinflussen, wie z.B. Zellzyklusverlauf, Transkription, Translation, Spleißen und Proteinhandel. Die Phosphorylierung durch S/T-Kinasen kann an 14-3-3 Proteinen erkannt werden. Der Schnittpunkt zwischen p38 MAPK-Aktivierung und 14-3-3 Erkennung wurde für einige Proteine nachgewiesen, ist aber noch nicht als Signalisierungsmodus etabliert. Hier verwenden wir quantitative Phosphoproteomik und Proteinkinase-Hemmung, um einen systemweiten Überblick über die durch UV-Licht induzierten Proteinphosphorylierungsmuster zu erhalten und die Abhängigkeiten von Phosphorylierungsereignissen von kanonischen DNA-Schadenssignalen durch die S/T-Kinasen ATM und ATR und den p38 MAPK-Pfad zu entdecken. Unsere Daten liefern den Nachweis, dass die Aktivierung des p38 MAPK-Pfades unabhängig von den ATM/ATR-Pfaden ist und eine andere Teilmenge von Proteinen nach niedriger Dosierung von UV-C-Licht reguliert. Wir entdecken die Phosphorylierung von p38 MAPK sowie der nachgelagerten Kinasen MK2/3 kurz nach der Bestrahlung und beobachten sie bis zu einer Stunde lang. Die p38 MAPK wirkt hauptsächlich durch die MK2/3-Kinase, die das LXRQX[ST]-Motiv auf den Substraten erkennt. Das gleiche Motiv, wenn es phosphoryliert ist, wird auch von der Familie 14-3-3 erkannt. Wir identifizieren RNA-bindende Proteine als Primärsubstrate und 14-3-3 als direkten "Reader" der p38-MK2-abhängigen Phosphorylierung, die durch UV-Licht induziert wird. Wir zeigen mechanistisch, dass MK2 die RNA-bindende Untereinheit des NELF-Komplexes, NELFE, auf Serin 49 (S49), S51, S115 und S251 phosphoryliert. NELFE Phosphorylierung fördert die Rekrutierung von 14-3-3. Weitere Analysen haben ergeben, dass S115 die entscheidende Rolle bei der 14-3-3 Bindung spielt. Diese Interaktion zwischen NELFE und 14-3-3 führt zu einer schnellen Dissoziation des NELF-Komplexes vom Chromatin. Ausgehend von diesem Ergebnis stellen wir fest, dass der transiente Knock-down von NELFE zu einer erhöhten Empfindlichkeit der Zellen gegenüber UV-Licht führt. Unsere ChIP-seq-Analyse zeigt, dass die NELF-Freisetzung von der Verlängerung der RNA-Polymerase II begleitet wird. Insgesamtcheinen diese Ereignisse das Zellüberleben bei der Reaktion auf DNA-Schäden durch UV-Licht zu fördern

Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential

A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts

Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential

Ian E, Malko DB, Sekurova ON, et al. Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential. PloS one. 2014;9(5): e96719.A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts

HSP90 is necessary for the ACK1-dependent phosphorylation of STAT1 and STAT3

Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT? and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90 alpha/beta). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT? and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis

Enterocin biosynthesis gene clusters identified in the genomes of <i>Streptomyces</i> spp. PVA 94-07 and GBA 94-10.

<p>Genomic region of <i>S. albus</i> J1074 where the cluster has been inserted is shown in the middle.</p