Fig. 5.

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