Abstract
Ligation of the T cell receptor (TCR) and costimulatory receptors leads to cytokine secretion and clonal expansion, whereas ligation of TCR alone leads to anergy. We have previously determined that anergic cells express Tob, a member of the novel APRO gene family, which inhibits T cell activation. The precise molecular mechanisms via which Tob mediates its effects in T cells are not fully understood. Tob functions as transcriptional coactivator and enhances DNA binding of Smads. Therefore, Tob may regulate de novo mRNA synthesis or gene transcription. To identify genes that are induced by Tob, Jurkat T cells that lack endogenous Tob, were transfected with Tob cDNA or empty vector and differential gene expression was determined by suppression subtractive hybridization. TRIM36 was one of the genes induced by Tob. TRIM36 is a RING finger E3 ubiquitin ligase. It belongs to a recently identified tripartite motif (TRIM) gene family which also includes Pyrin/Marenosrtin, MID1, MUL, PML, RFP and TIF1, proteins implicated in familial human diseases and cancer. E3 proteins confer substrate specificity to the ubiquitin system. Previous studies have shown that the trancriptional profile of anergic cells includes the E3 ubiquitin ligases Cbl-b, GRAIL and Itch. Therefore, the finding that Tob, a transcriptional regulator expressed in anergic cells, induces expression of TRIM36 E3 ubiquitin ligase is very intriguing. Northern blot analysis confirmed that TRIM36 mRNA was selectively upregulated in anergic T cells. To determine the role of TRIM36 on IL-2 gene transcription, Jurkat T cells were transfected with full-length TRIM36 cDNA along with the IL-2 promoter/enhancer cDNA (2kb) linked to the luciferase gene. TRIM36 inhibited CD3+CD28-mediated IL-2 transcription by 90%. Interestingly, when cells were stimulated with PMA+Ionomycin, which bypass the TCR proximal signals, IL-2 transcription was almost unaffected. These results prompted us to search for candidate ubiquitination substrates among signaling molecules that have a critical role on TCR-mediated T cell activation and IL-2 transcription. Previous studies have shown that among T cell signaling molecules, TCRζ, ZAP70, PLC-γ1 and PKC-𝛉 undergo ubiquitin-targeted degradation. For this reason, we investigated whether any of these proteins might be substrates for TRIM36-mediated ubiquitination. V5-tagged TRIM36 or empty vector was expressed in Jurkat T cells followed by stimulation with anti-CD3+anti-CD28 mAbs in the presence of ubiquitin aldehyde that prevents substrate deubiquitination. Immunoblot with antibodies specific for TCR ζ, ZAP70, PLC-γ1 and PKC-𝛉 showed that expression of PLC-γ1 and PKC-𝛉 was selectively reduced in the presence of TRIM36. Immunoprecipitation with V5 mAb followed by immunoblot with substrate-specific antibodies revealed that PLC- γ1 and PKC-𝛉 coprecipitated with TRIM36. Immunoblot with ubiquitin-specific antibody revealed that PLC-γ1 and PKC- 𝛉 were substrates for ubiquitination by TRIM36. Our results show that at least one molecular mechanism via which Tob mediates its inhibitory effect on T cell activation involves the induction of TRIM36 ubiquitin ligase, which mediates degradation of two key signaling proteins, PLC- γ1 and PKC-𝛉. Moreover, these results suggest that TRIM36 may represent a novel target of molecular intervention for induction of transplantation tolerance.
The E3 ubiquitin ligase MARCH1 regulates antimalaria immunity through interferon signaling and T cell activation
