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-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.
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