synthesized nelfe phospho-peptide  (GenScript corporation)

Journal: Nature Communications

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

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: UV-light-induced phosphorylation of NELFE by MK2 leads to 14-3-3 binding. a Identification of p38-dependent NELFE interaction partners after UV light. SILAC-labeled U2OS cells expressing GFP-NELFE were mock-treated or irradiated with UV light. Cells were lysed and protein extracts were incubated with GFP Trap agarose. Enriched proteins were resolved on SDS-PAGE and digested in-gel into peptides. Peptides were extracted from gel and analyzed by LC-MS/MS. The scatter plot shows the logarithmized SILAC ratios of proteins quantified in the pull down. The color coding indicates the density. b NELFE interaction with 14-3-3 after UV light is p38- and MK2/3/5-dependent. U2OS cells expressing Flag-Strep-14-3-3 or an empty vector were mock-treated, irradiated with UV light or pretreated with the p38 or MK2/3/5 inhibitor, and then irradiated with UV light. Cells were lysed and protein extracts were incubated with StrepTactin sepharose. Enriched proteins were resolved by SDS-PAGE and selected proteins were detected with the indicated antibodies. c NELFE interaction with GST-14-3-3 is abolished in p38 and MK2 knockdown cells. U2OS cells were transfected with non-targeting, p38, or MK2-targeting siRNA and then irradiated with UV light. Cells were lysed and protein extracts were incubated with recombinant GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and NELFE was detected using a specific antibody. d NELFE is phosphorylated after UV light on a 14-3-3-binding motif. GFP-NELFE was pulled down using GFP Trap agarose. Phosphorylation of NELFE was detected using antibodies recognizing the 14-3-3 motif. NELFE knockdown was used as control. e NELFE interacts with 14-3-3 after inhibition of P-TEFb. U2OS cells were treated with the p38 inhibitor or P-TEFb inhibitor 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. f Dynamics of NELFE interaction with 14-3-3 after UV light. U2OS cells were exposed to UV light and left to recover for the indicated time points. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. g NELFE interaction with 14-3-3 is partially dependent on the NER machinery. U2OS cells were transfected with a non-targeting siRNA or siRNA targeting XPC or CSB and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: Phospho-proteomics, Binding Assay, Multiplex sample analysis, Labeling, Expressing, Irradiation, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Knockdown, Transfection, Recombinant, Control, Inhibition, Lysis

Journal: Nature Communications

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

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: NELFE phosphorylation on S115 is required for the interaction with 14-3-3. a Schematic representation of NELFE domain organization and phosphorylation sites that were identified by phosphoproteomics. The SILAC ratios quantified for phosphorylation sites on NELFE after UV light and p38 inhibition are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. b The table shows all phosphorylation sites identified on NELFE by phosphoproteomics. The position, SILAC ratios, 14-3-3 binding prediction and sequence window are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. c Serine 115 phosphorylation is required for the interaction of NELFE and 14-3-3. U2OS cells expressing GFP-tagged wild-type NELFE or NELFE serine-to-alanine mutants were irradiated with UV light. Protein extracts were incubated with GST-14-3-3 and enriched proteins were resolved on SDS-PAGE. d Mass spectrometric parent ion scan of the peptide SISADDDLQESSR corresponding to S115 in NELFE. The SILAC triplet shows the relative abundance and mass to charge (m/z) of the phosphorylated peptide in mock-treated cells and cells irradiated with UV light without or with pretreatment with the p38 inhibitor. e Absolute occupancy of serine 49, 51, 115, and 251 phosphorylation in NELFE in undamaged cells and after UV light was determined by MS. f NELFE S115A mutant does not bind to 14-3-3. SILAC-labeled cells overexpressing GFP-tagged wild-type NELFE or NELFE S115A mutant were irradiated with UV light. UV-light-irradiated U2OS cells overexpressing GFP alone were used as control. Cells were lysed and protein extracts were incubated with GFP Trap agarose. The scatter plot shows the logarithmized SILAC ratios of quantified proteins. The color-coding indicates the density. g Recombinant 14-3-3 binds to phosphorylated NELFE peptide. Biotinylated phosphorylated NELFE peptide corresponding to serine 115 was bound to NeutrAvidin agarose. Phosphorylated and dephosphorylated peptide were incubated with purified 14-3-3. h Structure of 14-3-3 epsilon in complex with NELFE phosphorylated peptide QPFQRSI(p)SADDDLQE. Structure of the 14-3-3 epsilon in cartoon representation (Yellow and Cyan) and NELFE phosphorylated peptide in ball and stick model (Green). The inset on the right shows the 14-3-3 epsilon–NELFE phosphorylated peptide interaction

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: Phospho-proteomics, Multiplex sample analysis, Inhibition, Labeling, Binding Assay, Sequencing, Expressing, Irradiation, Incubation, SDS Page, Mutagenesis, Control, Recombinant, Purification

Journal: Nature Communications

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

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: p38-dependent phosphorylation of NELFE promotes its dissociation from chromatin. a Chromatin-associated proteins were extracted from untreated, UV-light-treated, and p38i, UV-light-treated U2OS cells, and analyzed by SILAC-based quantitative mass spectrometry. The bar plot shows the GO-BP (biological process) terms associated with proteins specifically enriched on or removed from chromatin after UV light (40 J/m 2 , 1 h recovery). The significance of the enrichment of a specific term was determined using Fisher’s exact test. P -values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. b The bar plot shows selected proteins associated with DNA repair and cell cycle that are significantly recruited or removed from chromatin after UV light. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance. c The NELF complex subunits are removed from chromatin in a UV light and p38-dependent manner. The bar plot shows selected proteins whose removal from chromatin after UV light is dependent on p38. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance. d NELFE dissociates from chromatin after UV light. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies (left). The levels of NELFE on chromatin were quantified from three replicate experiments and normalized to MCM7 levels (right). The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance (*** p -value < 0.001, ** p -value < 0.01). e Dynamics of NELFE removal from chromatin. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance (** p -value < 0.01). f Knockdown of NELFE reduced the ability of U2OS cells to form colonies after UV light. The error bars show the mean and SD of results obtained in three replicate experiments performed in three technical replicates. Two-sided Student’s t -test was used to assess the significance (*** p -value < 0.001, ** p -value < 0.01)

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: Phospho-proteomics, Multiplex sample analysis, Mass Spectrometry, SDS Page, Western Blot, Knockdown

Journal: Nature Communications

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

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: UV light leads to an increase in transcriptional elongation. a Metagene analysis showing total RNA pol II occupancy measured by ChIP-seq in mock-treated U2OS cells and cells irradiated with UV light (40 J/m 2 , 1 h recovery). All TSSs bound by RNA pol II in untreated cells and after UV light exposure were used for the analysis. Metagene analysis shows an average of two independent replicate ChIP-seq experiments. b Exposure of U2OS cells with UV light (40 J/m 2 , 1 h recovery) promotes the release of RNA pol II into downstream regions of genes. The box plot shows the calculated RNA pol II release ratios (PRRs) in untreated cells and in cells irradiated with UV light. The average PRRs were calculated from two independent replicate experiments. The lower and upper hinges represent the first and third quartiles (25th and 75th percentiles, respectively). The line in the center of the box corresponds to the median of the data range. P -value was calculated using the Wilcoxon’s rank-sum test with continuity correction. c REACTOME terms significantly enriched among genes with UV light upregulated PRRs. The bar plot shows significantly overrepresented REACTOME terms associated with genes containing upregulated PRR compared with all RNA pol II bound genes. The significance of the enrichment of a specific term was determined using a hypergeometric test. P -values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. d UCSC Genome Browser tracks displaying the density of RNA pol II around the PLK3 and RPL11 gene in untreated cells and after exposure of cells with UV light. e Model for the NELF complex regulation by p38-MK2. Exposure of human cells to UV light leads to rapid activation of p38 and its downstream effector kinase MK2. MK2 triggers widespread phosphorylation of RNA-binding proteins, including the NELF complex subunit NELFE. Site-specific NELFE phosphorylation on S115 induces its transient interaction with 14-3-3. NELFE phosphorylation leads to dissociation of NELFE from chromatin that is accompanied by RNA pol II elongation

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: ChIP-sequencing, Irradiation, Activation Assay, Phospho-proteomics, RNA Binding Assay