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Summary: Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-ζ (TCR-ζ) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-ζ was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the ζ chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-ζ was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-ζ and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction. [Copyright &y& Elsevier]

Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCγ-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells. [Copyright &y& Elsevier]

Summary: The molecular basis of T cell anergy is not completely understood. We show that in antigen-primed anergic murine CD4+ T cells the linker for activation of T cells (LAT) is hypophosphorylated upon CD3/CD28 restimulation. Signaling events downstream of LAT (PLCγ1 phosphorylation and p85 [PI3-K] association) were impaired, whereas upstream events (CD3ζ and ZAP-70 phosphorylation) remained intact. LAT recruitment to the immunological synapse and its localization in detergent-resistant membrane (DRM) fractions were defective in anergic T cells. These defects resulted from impaired palmitoylation of LAT and were selective since the DRM localization and palmitoylation of Fyn were intact. This LAT defect was independent of Cbl-b and did not reflect enhanced LAT degradation. These results identify LAT as the most upstream target of anergy induction; moreover, they suggest that regulation of the amount of LAT in the immunological synapse and DRM by posttranslational palmitoylation contributes to the induction of T cell anergy. [Copyright &y& Elsevier]

Summary: Conjugation of ubiquitin (Ub) to a protein substrate targets the substrate for degradation or functional modification, which is tightly controlled by diverse mechanisms including phosphorylation of the substrate. An emerging mechanism involves regulation of the E3 Ub ligase, for example, the JNK-dependent phosphorylation and activation of Itch E3 ligase, which controls the turnover of Jun proteins and T cell differentiation. Here we show that Itch is also modulated by an Src kinase Fyn via tyrosine phosphorylation at the Tyr371 residue. Fyn associates with Itch, and loss of Fyn results in reduced Itch phosphorylation. Importantly, tyrosine phosphorylation of Itch appears to reduce its interaction with its substrate JunB. The turnover of JunB is accelerated in Fyn-deficient T cells, which is further reconstituted by Itch Tyr371 mutation. Thus, in contrast to the activation pathway mediated by serine/threonine phosphorylation, tyrosine phosphorylation of Itch plays a negative role in modulating Itch-promoted ubiquitination. [Copyright &y& Elsevier]