Identification of phosphorylation site of Npas4 by LC-MS/MS
To identify the phosphorylation sites of Npas4, GST-Npas4-390-489 aa, GST-Npas4 490-597 aa or GST-Npas4 598-701 aa was expressed in COS7 cells with or without the coexpression of MAP2K1-CA. Cells were lysed in lysis buffer [20 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (PhosStop, Roche), pH 7.5], and sonicated 3 times for 5 sec. After centrifugation at 16,000×g at 4°C for 10 min, the soluble supernatant was incubated in 30 µl of glutathione-Sepharose 4B beads (GE Healthcare) for 1 hr at 4°C with rotation. The beads were then washed three times with lysis buffer and an additional three times with wash buffer (20 mM Tris/HCl, 1 mM EDTA, and 150 mM NaCl, pH 7.5). The bound proteins were extracted from the beads using urea solution, reduced via incubation in 5 mM dithiothreitol for 30 min, and alkylated using 10 mM iodoacetamide for 1 h in the dark. The proteins were digested with Trypsin/Lys-C (Promega) or Glu-C (Promega)/Asp-N (FUJIFILM Wako). Demineralization was performed using SPE c-tips according to the manufacturer’s instructions. The peptides were analyzed by LC−MS using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc). Dual-phosphorylated peptide DLVCTPPYTPHQPGGCAFLFSLHEPFQTHLPPPSSSLQE, containing T423 and T427 phosphorylation sites, was identified from digested fragments of GST-Npas4-390-489 aa; LPPSPSSPGNGDCTLLALAQLR, containing S577 and S580 phosphorylation sites, was identified from digested fragments of GST-Npas4 490-597 aa; and GLLTPEASPVKQSFFHYTEKE, containing T611 and S615 phosphorylation sites, was identified from digested fragments of GST-Npas4 598-701 aa. Selected ion monitoring (SIM) analysis revealed that the amount of these dual-phosphorylation was significantly increased by coexpression with MAP2K1-CA. These results suggest that Npas4 is phosphorylated by MAPK at T423, T427, S577, S580, T611 and S615 sites.