Itch, a novel E3 Ub ligase, is absent in the non-agouti-lethal 18H, or Itchy, mice. These mutated mice develop immunological and inflammatory diseases, including inflammation in the lung and stomach, hyperplasia of lymphoid organs, and constant itching in the skin, suggesting that Itch is involved in the regulation of immune responses. Itch contains an amino-terminal protein kinase C-related C2 domain, four WW protein-interaction domains and carboxy-terminal HECT ligase domain. However, the biological pathways regulated by Itch E3 ligase remained unclear. We proposed that, first, Itch targets protein substrates for degradation through the interaction of the four WW domains; second, Itch regulates signal transduction pathways via the ubiquitination of its protein substrates; and, third, Itch-deficient mice have a dysregulation of protein ubiquitination and of intracellular signal transduction.
We examined the T cell function in Itchy mice and found that Itch-/- T cells display increased cell proliferation and express cell surface activation markers in aging mice. More interestingly, these T cells tend to differentiate into T cell helper type 2 (Th2) cells, with enhanced IL-4 and IL-5 cytokine production in both in vitro and in vivo. Consistent with this notion, there are more IgG1 and IgE antibodies in the sera of Itchy mice. Molecularly, we identified that Jun-B/c-Jun is the target molecule for Itch E3 ligase. Itch-/- T cells have more Jun-B in the nuclear fraction as well as increased Jun-B DNA-binding activity. Since it was previously shown that T cells harboring Jun-B transgene preferentially produce Th2 cytokines, we conclude that Itch regulates T cell differentiation via promoting Jun-B ubiquitination.
Protein ubiquitination has been implicated in the regulation of transforming growth factor (TGF)-β signaling, particularly via the Ub conjugation to and subsequent degradation of, the Smad signaling mediators or their binding proteins. We examined the involvement of Itch in TGF-β signaling, and found that Itch-/- fibroblasts display reduced response to TGF-β treatment. Instead of the proteasome-dependent degradation as previously demonstrated in transient transfection studies, we observed that Itch in fact modulates the interaction between Smad2 and the TGF-β receptor and the subsequent Samd2 phosphorylation. The study points out a novel function of Itch in the regulation of TGF-β signaling via modulating protein-protein interactions.
Although those studies suggest that Itch is important in modulating critical signaling pathways by targeting specific substrates for ubiquitination, the mechanisms by which Itch-induced protein ubiquitination is regulated remain largely unclear. A recent study may shed light on this aspect, in which a MEKK1-JNK-mediated signaling pathway controls the turnover of Jun proteins via the phosphorylation of Itch and its subsequent activation. This new finding distinguishes from previous observations, in that the upstream kinases do not induce the phosphorylation of the substrates, rather they directly modulate the E3 Ub ligases for the substrates.
We continued the study of the regulation of Itch E3 ligase by tyrosine phosphorylation. It was found that Fyn kinase induces the tyrosine phosphorylation of Itch at tyrosine residue 371. Strikingly, unlike JNK-induced serine/threonine phosphorylation, Fyn-mediated tyrosine phosphorylation does not affect Itch ligase activity. Rather, it negatively modulates the association between Itch and its substrate JunB. The results suggest that Itch function is tightly controlled by upstream kinases via counterbalancing serine/threonine vs. tyrosine phosphorylation.