Ubiquitination of receptor protein-tyrosine kinases (RPTKs) terminates signaling by marking active receptors for degradation. c-Cbl, an adapter protein for RPTKs, positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and RING finger domains. Ubiquitin-protein ligases (or E3s) are the components of ubiquitination pathways that recognize target substrates and promote their ligation to ubiquitin. The c-Cbl protein acted as an E3 that can recognize tyrosine-phosphorylated substrates, such as the activated platelet-derived growth factor receptor, through its SH2 domain and that recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain. These results reveal an SH2-containing protein that functions as a ubiquitin-protein ligase and thus provide a distinct mechanism for substrate targeting in the ubiquitin system.

The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

The turnover of Jun proteins, like that of other transcription factors, is regulated through ubiquitin-dependent proteolysis. Usually, such processes are regulated by extracellular stimuli through phosphorylation of the target protein, which allows recognition by F box-containing E3 ubiquitin ligases. In the case of c-Jun and JunB, we found that extracellular stimuli also modulate protein turnover by regulating the activity of an E3 ligase by means of its phosphorylation. Activation of the Jun amino-terminal kinase (JNK) mitogen-activated protein kinase cascade after T cell stimulation accelerated degradation of c-Jun and JunB through phosphorylation-dependent activation of the E3 ligase Itch. This pathway modulates cytokine production by effector T cells.

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 PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.

Itch is an E3 ubiquitin ligase that is disrupted in nonagouti-lethal or itchy mice. Itch deficiency leads to severe immune and inflammatory disorders and constant itching of the skin. Here we show that Itchminus sign/minus sign T cells show an activated phenotype and enhanced proliferation. Production of the type 2 T helper (TH2) cell cytokines interleukin 4 (IL-4) and IL-5 by Itchminus sign/minus sign T cells was augmented upon stimulation, and the TH2-dependent serum concentrations of immunoglobulin G1 (IgG1) and IgE in itchy mice were also increased. Molecularly, Itch associated with and induced ubiquitination of JunB, a transcription factor that is involved in TH2 differentiation. These results provide a molecular link between Itch deficiency and the aberrant activation of immune responses in itchy mice.

The Chk1 kinase is a major effector of S phase checkpoint signaling during the cellular response to genotoxic stress. Here, we report that replicative stress induces the polyubiquitination and degradation of Chk1 in human cells. This response is triggered by phosphorylation of Chk1 at Ser-345, a known target site for the upstream activating kinase ATR. The ubiquitination of Chk1 is mediated by E3 ligase complexes containing Cul1 or Cul4A. Treatment of cells with the anticancer agent camptothecin (CPT) triggers Chk1 destruction, which blocks recovery from drug-induced S phase arrest and leads to cell death. These findings indicate that ATR-dependent phosphorylation of Chk1 delivers a signal that both activates Chk1 and marks this protein for proteolytic degradation. Proteolysis of activated Chk1 may promote checkpoint termination under normal conditions, and may play an important role in the cytotoxic effects of CPT and related anticancer drugs.

CD8+ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8+ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8+ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8+ T cells. Mechanically, Mn2+ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8+ T cell differentiation, activation and NK cell activation, and increased memory CD8+ T cells. Combining Mn2+ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn2+ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.