These results suggest that Itch and Cyld cooperatively switch K63-linked to K48-linked ubiquitination. We also tested whether additional deubiquitinases, A20 and Cezanne38, 39, which deubiquitinate the signaling molecule RIP1, likewise regulate Tak1 ubiquitination. system6. Production of pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin 6 (IL-6) and IL-1, by TAMs not only trigger pro-survival signals in tumor cells, but also support their growth and metastasis6, 7. However, the molecular mechanisms that regulate inflammation and tumor progression remain unclear. Post-translational modifications Azilsartan medoxomil monopotassium mediated by ubiquitin conjugation have emerged as a key regulatory mechanism in the immune cells8, 9. The ubiquitin signal is interpreted based on the number of attached ubiquitin chains and the topology of their linkage. A polyubiquitin chain is formed when one of the seven lysines within ubiquitin is linked to the C-terminal glycine of another ubiquitin. Although ubiquitin contains seven lysine Azilsartan medoxomil monopotassium residues, linkage generally occurs either K48 or Azilsartan medoxomil monopotassium K63. K48-linked polyubiquitination predominantly targets proteins for proteasomal degradation, whereas K63-linked polyubiquitination results in non-proteasomal modifications, such as subcellular localization or protein-protein interactions8. We and others have demonstrated HECT type E3 ligase Itch as a critical regulator of inflammation10, 11. On a mouse C57BL/6 background, Itch deficiency results in the development of a late onset lymphoproliferation disorder and chronic pulmonary inflammation12, 13. Itch possesses four WW domains that recognize the Pro-rich PPXY (PY) consensus sequence in their substrate targets14. Although it is clear that Itch regulates inflammation, no systematic study has linked Itch to tumorigenesis. Similar to phosphorylation, protein ubiquitination is reversible, and removal of ubiquitin molecules is mediated by de-ubiquitinating (DUB) enzymes, such as Cyld15. Cyld is encoded by a NF-B-inducible gene16, inhibits NF-B and the kinase JNK by removing K63-linked polyubiquitin chains from signaling pathway Azilsartan medoxomil monopotassium molecules such as Bcl-3, TRAF2, TRAF6 and Nemo (also known as IKK-)17C21. Cyld through its deubiquitinating function has been shown to regulate various types of human cancers, including lung, hepatocellular, colon and cervical cancers15. Cyld was also shown to negatively regulate tumor-promoting cytokine secretion by inflammatory cells22. Despite these important observations, the molecular mechanisms regulating Cyld function and its role in inflammation remain largely unknown. Here, we demonstrate that Itch interacts with Cyld and cooperatively regulates Tak1 and inflammation. RESULTS Augmented tumor growth in tail vein. Both the WW-PPXY motifs We previously reported that Itch regulated TNF-induced NF-B signaling by modulating cFLIPL turnover27. Cyld deubiquitin activity was shown earlier to interfere with the ubiquitination of TRAF molecules and Tak1, which are involved directly in the TNF-associated NF-B signaling pathway15. Since Itch and Cyld can regulate similar pathways, we tested whether these enzymes can cooperate to terminate NF-B signaling and inflammatory response during tumor growth and metastasis. To determine whether Itch and Cyld can directly interact with each other, we performed sequence analysis and found a highly conserved Cyld PPXY motif (Fig. 2a) by which it could interact with Itch WW domains. We tested this interaction by transiently transfecting 293T cells with Flag-Itch and Myc-Cyld. Cell lysates were immunoprecipitated using control mouse IgG or antibodies against Flag or Myc. Antibody against Myc immunoprecipitated Flag-Itch and interaction through PPXY motif. (a) Sequence alignment of PPXY motif of Cyld which is conserved across species. Y485 critical for interaction with Itch is indicated (*). (b) 293T cells transiently co-transfected with expression vectors for Flag-Itch and Myc-Cyld. The cell lysate was immunoprecipitated using mouse IgG, antibodies against Flag and Myc. The immunoprecipitates were blotted using antibodies against Flag and Myc. (c) Immunoblot of His-Cyld precipitated using GST or GST-Itch purified from the PPXY motif, we generated Y to A mutant Cyld (Cyld(Y485A)) by site-directed mutagenesis. The Cyld(Y485A) mutation disrupted Itch-Cyld interaction (Fig. Azilsartan medoxomil monopotassium 2e). Taken together these data collectively indicate that Mmp2 Itch forms a complex with Cyld its interaction through PPXY motif. Itch targets Tak1 for K48-linked ubiquitination Next, we sought to understand the role of Itch-Cyld complex in the regulation of inflammatory response. Inflammatory stimuli driven signaling converge at the IB kinase (IKK) complex,.
These results suggest that Itch and Cyld cooperatively switch K63-linked to K48-linked ubiquitination