Journal: The Journal of Biological Chemistry

Article Title: Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

doi: 10.1016/j.jbc.2021.101097

Figure Lengend Snippet: The roles of phosphatases PTPN2 and PTPN22 in restraining GITR signaling. A , splenic T cells isolated from littermate control (LMC) or T-Traf3 −/− mice were pretreated with DMSO ( upper-left ), inhibitors (5 μM) of PTPN2 (SF1670, upper-right ), PTPN22 (LTV-1, bottom-left ) or ERK (U0126) ( bottom-right ) before being stimulated via GITR for the indicated times. Whole cell lysates were subjected to SDS-PAGE and Western blot analysis. Representative blots of four similar experiments are shown. B–D , quantification of four independent experiments including representative blots shown in A . Curves represent mean ± SEM of relative band intensities, where statistical significance was determined by two-way ANOVA. ∗ p < 0.05; ∗∗ p < 0.01; n.s indicates no significant difference. E , subclones of HuT28.11 or Traf3 −/− HuT28.11 T cells expressing hCD40-GITR (FLAG-tagged) were stimulated via anti-hCD40 Ab-conjugated protein G beads (see ) for times indicated and the hCD40-GITR signaling complex was immunoprecipitated. IPs ( left ) were subjected to SDS-PAGE and Western blotting for TRAF3, PTPN2 and PTPN22. Blots of whole cell lysates are shown on the right . Ctrl = IP with protein G beads conjugated with isotype control mAb. Positions of TRAF3 and PTPN2 bands are indicated by arrows . ∗ indicates the heavy chain of the IP Ab remaining in the IP sample. Blots in E are representative of ≥ 3 similar experiments. Quantification of band intensity is summarized in Fig. S4 .

Article Snippet: The small-molecule inhibitors for PTPN2 (SF1670) and PTPN22 (LTV-1) were purchased from Tocris Bioscience.

Techniques: Isolation, Control, SDS Page, Western Blot, Expressing, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

doi: 10.1016/j.jbc.2021.101097

Figure Lengend Snippet: Model: TRAF3 suppresses GITR expression and signaling in T cells. GITR transcription is stringently regulated by activity of NFκB2. In normal T cells ( left panel ), the major upstream kinase for activation of NFκB2, NIK is constitutively polyubiquitinated (represented as chain of black dots attached to NIK) and targeted for degradation by an E3 ubiquitination complex containing TRAF2, TRAF3, and cIAP. Engagement of GITR with GITRL leads to recruitment of TRAF3 and disassembly of the TRAF2/TRAF3/cIAP E3 complex, which releases NIK and activates downstream NFκB2. In addition, phosphatases PTPN2 and PTPN22 attenuate the signaling strength of the GITR complex independent of TRAF3. Negative regulation of GITR signaling by TRAF3 may also involve competition with TRAF2 and TRAF5 ( yellow and blue ovals ), both of which can promote GITR signaling . In the absence of TRAF3 ( right panel ), NFκB2 is constitutively active and upregulates GITR transcription. Increased levels of the GITR receptor and lack of TRAF3 regulation of the GITR signaling cascade lead to elevated GITR signaling in TRAF3-deficient T cells.

Article Snippet: The small-molecule inhibitors for PTPN2 (SF1670) and PTPN22 (LTV-1) were purchased from Tocris Bioscience.

Techniques: Expressing, Activity Assay, Activation Assay, Ubiquitin Proteomics