The major focus of research in this laboratory is to analyze the function of signaling molecules in intracellular biochemical events initiated by the T cell receptor (TCR) and cytokine receptors, with particular emphasis on the mechanisms that turn off signal transduction through protein ubiquitination. Ubiquitin (Ub)-dependent proteolysis has been implicated in a variety of cellular processes, including cell-cycle control, signal transduction, transcriptional regulation, DNA repair, receptor down-regulation, antigen presentation, and apoptosis. Abnormalities in the Ub system have been shown to cause pathological responses, including malignant transformation, and several genetic diseases. Ubiquitination of protein substrates involves a cascade of enzymatic reactions: first, Ub, a highly conserved 76-amino acid polypeptide, is activated by Ub-activating enzyme, or E1, leading to a ATP-dependent formation of high energy thiol-ester bond between the C-terminus of Ub and E1; the activated Ub is then transferred to E2s (Ub-conjugating enzymes or Ubcs). Ub-protein ligases, or E3s, are responsible for substrate recognition and for promoting Ub ligation to the substrate. A substrate may be multiply ubiquitinated by sequential linkage of additional Ub molecules to form a poly-Ub chain, which marks the protein substrate for the recognition and consequent degradation by the 26S proteasome. Based on amino acid sequences for E2 binding, E3 ligases are generally classified into two families: RING (really interesting new gene)-type E3 ligases, and HECT (homologous to the E6-associated protein C-terminus)-type E3 ligases. We are currently studying Cbl and Cbl-b RING-type E3s and Itch HECT-type E3.

Although protein ubiquitination has been considered to be a means of garbage disposal, i.e., protein degradation, works from many labs including my own have convincingly demonstrated that it also represents a novel means of protein modification, such as affecting protein-protein interaction, or protein phosphorylation.

1) Functional analysis of Cbl proteins in T cell activation and tolerance

We originally discovered that Cbl acts as RING-type E3 by recruiting Ubc E2 conjugating enzyme via its RING-finger domain and at the same time, binds to the activated receptor tyrosine kinases via its N-terminal tyrosine kinase binding domain, which results in the Ub transfer from E2 to the substrate (in this case, the tyrosine kinase) and the subsequent downmodulation of the receptor. We then shifted our efforts from biochemical studies to more physiologically relevant analysis using gene-targted T cells. Particularly, we showed that Cbl-b interacts with, and induces Ub conjugation to, the p85 regulatory subunit of PI-3 kinase. More importantly, we showed that ubiquitination of p85 does not lead to its degradation, but affects its association with upstream molecules such as CD28. This study presented a novel concept on protein ubiquitination in the functional modification of protein substrates.

We then focused on the study of an essential role of Cbl-b in T cell anergy induction. The Cbl-b E3 Ub ligase is implicated in setting the activation threshold of mature T cell and in regulating the autoimmune response. However, the exact mechanisms underlying Cbl-b-mediated T cell tolerance were unclear. Here we used mouse models of autoimmune diseases to investigate the effect of Cbl-b deficiency on T cell tolerance induction and the development of autoimmunity. Ablation of Cbl-b resulted in exacerbated diseases, which were accompanied by the hyper-responsiveness and augmented transcriptional activation of Cbl-b-/- T cells. Mechanistically, it was found that loss of Cbl-b resulted in resistance to T cell anergy induction, whereas the other potential tolerance mechanisms like the generation of natural killer T or T regulatory cells, and the peripheral T cell deletion, were relatively normal. Moreover, loss of Cbl-b largely rescued the reduced calcium mobilization of anergic T cells. In conclusion, our results point out a critical role for Cbl-b in regulating the peripheral tolerance by contributing to T cell anergy induction.

2) The E3 ligase Itch in T cell differentiation

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.