Figure 4. HGK directly phosphorylates TRAF2 at serine 35 that mediates TRAF2 degradation.

(a) Co-immunoprecipitations (IP) of endogenous HGK with TRAF2 in lysates of mouse primary splenic T cells treated with chloroquine. NS, normal serum. IB, immunoblotting. (b) In vitro binding assays of purified HGK-Myc and Flag-TRAF2 proteins. (c) Signals of the interaction between HGK-Myc and TRAF2-Flag in lysates of HEK293T cells determined by amplified luminescent proximity homogeneous assays (α). Means±s.e.m. are shown. (d) In vitro kinase assays using purified HGK-Myc and Flag-TRAF2 proteins. (e) Mass spectrometry (MS)/MS fragmentation spectra of the tryptic peptides of TRAF2 contain the phosphorylation of Ser35. In vitro phosphorylated Flag-tagged TRAF2 was isolated, digested with trypsin and subjected to LC-MS/MS analyses. (f) Immunoblotting analyses of indicated molecules in lysates of HEK293T cells transfected with WT TRAF2 or TRAF2 mutants in the presence or absence of HGK plasmids. (g) IL-6 production in the mouse primary splenic T cells. T cells were transfected with GFP-TRAF2 short hairpin RNA (shRNA) and a control GFP vector. The transfected T cells were stimulated with PMA plus ionomycin for 3 h and then determined by flow cytometry at day 3 after transfection. Data show the percentages of IL-6 producing T cells (green fluorescent protein (GFP)-gated) and presented as mean±s.e.m. from triplicate experiments. WT, littermate controls (HGK^f/f mice); HGK cKO, T-HGK cKO mice. Data shown are representatives of three (a–d,g) and two (e,f) independent experiments. *P-value<0.05; **P-value<0.01 (two-tailed Mann–Whitney U-test).