JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003532/files/Ishikawa_Tama_TurboID_Ikaros_CD3CL_FusionIT.xlsxhttps://storage.jpostdb.org/JPST003532/files/144_Ishikawa_1_Tama_TurboID_Ikaros_CD3CL_minus_FusionIT.rawhttps://storage.jpostdb.org/JPST003532/files/150_Ishikawa_5_Tama_TurboID_Ikaros_CD3CL_plus_FusionIT.rawhttps://storage.jpostdb.org/JPST003532/files/152_Ishikawa_3_Tama_TurboID_Ikaros_CD3CL_minus_FusionIT.rawhttps://storage.jpostdb.org/JPST003532/files/148_Ishikawa_2_Tama_TurboID_Ikaros_CD3CL_minus_FusionIT.rawhttps://storage.jpostdb.org/JPST003532/files/146_Ishikawa_4_Tama_TurboID_Ikaros_CD3CL_plus_FusionIT.rawhttps://storage.jpostdb.org/JPST003532/files/154_Ishikawa_6_Tama_TurboID_Ikaros_CD3CL_plus_FusionIT.rawprimary200OKProteomicsHidetaka KosakoMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST003532Tokushima UniversityjPOSTDevelopment of invariant natural killer T (iNKT) cells in the thymus requires cell-cell interaction through invariant TCR (iTCR) and CD1d, which induces expression of the transcription factor, promyelocytic leukemia zinc finger (PLZF). However, the signaling pathway linking iTCR and PLZF remains unclear. Here, we report that a serine/threonine kinase, protein kinase D (PKD), plays a pivotal role in iNKT cell development. In T cell-specific PKD-deficient (Prkd2/3∆CD4) mice, PLZF induction and iNKT cell generation were severely impaired, which were rescued by introduction of a PLZF transgene. We identified the transcription factor Ikaros as a substrate of PKD upon iTCR stimulation. Knock-in mice carrying a phosphorylation-defective mutant Ikaros (Ikzf1S267/275A) exhibited an impairment of iNKT cell development, whereas conventional T cells were normal. In iNKT cells, Ikaros binds to the upstream region of the PLZF gene to induce its transcription. Mutant mice lacking the Ikaros-binding site (Zbtb16∆IBS) generated fewer iNKT cells than WT mice. These results suggest that PKD links iTCRs to PLZF induction through Ikaros, thereby mediating iNKT cell development.Invariant TCR-triggered protein kinase D activation mediates NKT cell development.Ishikawa Eri E, Kosako Hidetaka H, Motooka Daisuke D, Imasaka Mai M, Watarai Hiroshi H, Ohmuraya Masaki M, Yamasaki Sho SfalseProximity biotinylation of iNKT cells expressing TurboID-Ikaros with or without TCR stimulationIkaros cDNA fused to TurboID was cloned into a retroviral vector and transfected into Phoenix retrovirus packaging cells using PEI MAX (Polysceinces). Culture supernatant containing retroviruses was used for infection into DN32.D3 cells. Cells were cultured in 10%FBS/DMEM which does not contain biotin for more than three days and treated with 10 ug/ml of anti-CD3(2C11). After washing cells, 100 ug/ml of goat anti-hamster IgG was added to stimulate cells by TCR crosslinking in the presence of 50 uM D-biotin (Nacalai Tesque) and incubated at 37°C for 45 min. The cells were washed with ice-cold HEPES-saline and lysed in Guanidine-TCEP buffer　(8 M guanidine-HCl, 100 mM HEPES-NaOH pH7.5, 10 mM TCEP, 40 mM chloroacetamide). The lysates were dissolved by heating and sonication and then centrifuged at 20,000 × g for 15 min at 4 °C. The supernatants were recovered, and proteins were purified by methanol–chloroform precipitation and solubilized using PTS buffer (12 mM SDC, 12 mM SLS, 100 mM Tris-HCl, pH8.0). After sonication and heating, the protein solution was diluted 5-fold with 100 mM Tris-HCl, pH8.0 and digested with trypsin (MS grade, Thermo Fisher Scientific) at 37 °C overnight. The resulting peptide solutions were diluted 2-fold with TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Biotinylated peptides were captured on a 15 uL slurry of MagCapture HP Tamavidin 2-REV magnetic beads (FUJIFILM Wako) by incubation for 3 h at 4 °C. After washing with TBS five times, the biotinylated peptides were eluted with 100 uL of 1 mM biotin in TBS for 15 min at 37 °C twice. The combined eluates were desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 um × 150 mm; Nikkyo Technos) with a linear 4–32% ACN gradient for 0–60 min, followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. The MS1 spectra were measured with a resolution of 120,000, an AGC target of 4e5, and a mass range of 375–1500 m/z. HCD MS/MS spectra were acquired in a linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 200 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 10 s. Raw data were directly analyzed against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with the Mascot search engine. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages, (b) precursor mass tolerance of 10 ppm, (c) fragment mass tolerance of 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification, and (e) acetylation of protein N-terminus, oxidation of methionine, and biotinylation of lysine as variable modifications. Peptides were filtered at an FDR of 1% using the percolator node. Label-free quantification was performed based on the intensities of the precursor ions using the precursor ions quantifier node. Normalization was performed such that the total sum of the abundance values for each sample over all peptides was the same.Tue Sep 09 00:00:00 BST 2025PXD0592891009040965511