Fumiyo Ikeda

Atypical ubiquitin chains: new molecular signals

Ubiquitin (Ub) is a small protein modifier that regulates many biological processes, including gene transcription, cell‐cycle progression, DNA repair, apoptosis, virus budding and receptor endocytosis. Ub can be conjugated to target proteins either as a monomer or as Ub chains that vary in length and linkage type. The various types of Ub modification are linked to distinct physiological functions in cells. MonoUb, for example, regulates DNA repair and receptor endocytosis, whereas lysine 48‐linked Ub chains label proteins for proteasomal degradation. More recently, the importance of chains conjugated through the other six lysines in Ub, known as atypical Ub chains, has been revealed. Atypical chains can be homotypic, sequentially using the same lysine residue in Ub for conjugation; mixed‐linkage, utilizing several distinct lysines to connect consecutive Ub moieties; or heterologous, connecting Ub with other Ub‐like modifiers. Here, we describe recent progress in the understanding of atypical Ub chain assembly and their recognition by Ub‐binding domains, and we discuss further their functional roles in vivo.

Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a ‘pill’ that awakens the innate immune system to kill cancer metastases.

What Determines the Specificity and Outcomes of Ubiquitin Signaling

Ubiquitin signals and ubiquitin-binding domains are implicated in almost every cellular process, but how is their functionality achieved in cells? We assess recent advances in monitoring the dynamics and specificity of ubiquitin networks in vivo and discuss challenges ahead.

Inflammatory cardiac valvulitis in TAX1BP1‐deficient mice through selective NF‐κB activation

Nuclear factor kappa B (NF‐κB) is a key mediator of inflammation. Unchecked NF‐κB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1‐binding protein 1 (TAX1BP1) is a negative regulator of TNF‐α‐ and IL‐1β‐induced NF‐κB activation and that binding to mono‐ and polyubiquitin by a ubiquitin‐binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF‐κB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age‐dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF‐α and IL‐1β. TAX1BP1−/− cells are more highly activated for NF‐κB than control cells when stimulated with TNF‐α or IL‐1β. Mechanistically, TAX1BP1 acts in NF‐κB signalling as an essential adaptor between A20 and its targets.

Core-binding factor α1 (Cbfa1) induces osteoblastic differentiation of C2C12 cells without interactions with Smad1 and Smad5

Core-binding factor alpha(1) (Cbfa1) is an essential transcription factor for osteoblastic differentiation and osteogenesis. Bone morphogenetic protein (BMP) is also a powerful inducer of differentiation of pluripotent mesenchymal cells to osteoblast lineage and bone formation. Recent studies suggest that Cbfa1 plays a critical role during BMP-induced osteoblastic differentiation through association with cytoplasmic BMP signaling molecules, Smads. However, other studies have suggested that Cbfa1 may exhibit its osteogenic function without interaction with Smads. Therefore, it remains unclear whether association with Smad is essential for Cbfa1 function. In this study we examine the effects of Cbfa1 on osteoblastic differentiation in the presence or absence of interactions with Smad1 or Smad5 using C2C12 undifferentiated mesenchymal cells. Cbfa1 expression was induced upon stimulation with BMP-2 in C2C12 cells. Introduction of Cbfa1 into C2C12 cells induced osteoblastic differentiation and promoted transactivation of osteocalcin gene promoter without forming the complex with Smad1 or Smad5. Furthermore, in C2C12 cells in which the association of Cbfa1 with Smad1/Smad5 was prevented by the overexpression of the natural antagonist, Smad6, Cbfa1 still induced osteoblastic differentiation and transactivated osteocalcin gene promoter, regardless of BMP-2 stimulation. These results suggest that the interactions with Smad1 or Smad5 are not essential for Cbfa1 to demonstrate its osteogenic actions. However, interactions with Smad1/Smad5 enhance these osteogenic actions of Cbfa1. Of note, BMP-2-induced or Smad-induced osteoblastic differentiation was inhibited by dominant-negative Cbfa1, suggesting that the function of Cbfa1 is critical for BMP-2-induced osteoblastic differentiation. Our results suggest that Cbfa1 is essential and also sufficient to induce osteoblastic differentiation in undifferentiated mesenchymal cells, and establishment of an association with Smad1/Smad5 enhances the osteogenic actions of Cbfa1. On the other hand, Cbfa1 expression requires the activation of Smad1/Smad5 by BMP-2.

Involvement of the ubiquitin-like domain of TBK1/IKK-i kinases in regulation of IFN-inducible genes.

TANK‐binding kinase 1 (TBK1/NAK/T2K) and I‐κB Kinase (IKK‐i/IKK‐ε) play important roles in the regulation of interferon (IFN)‐inducible genes during the immune response to bacterial and viral infections. Cell stimulation with ssRNA virus, dsDNA virus or gram‐negative bacteria leads to activation of TBK1 or IKK‐i, which in turn phosphorylates the transcription factors, IFN‐regulatory factor (IRF) 3 and IRF7, promoting their translocation in the nucleus. To understand the molecular basis of activation of TBK1, we analyzed the sequence of TBK1 and IKK‐i and identified a ubiquitin‐like domain (ULD) adjacent to their kinase domains. Deletion or mutations of the ULD in TBK1 or IKK‐i impaired activation of respective kinases, failed to induce IRF3 phosphorylation and nuclear localization and to activate IFN‐β or RANTES promoters. The importance of the ULD of TBK1 in LPS‐ or poly(I:C)‐stimulated IFN‐β production was demonstrated by reconstitution experiments in TBK1‐IKK‐i‐deficient cells. We propose that the ULD is a regulatory component of the TBK1/IKK‐i kinases involved in the control of the kinase activation, substrate presentation and downstream signaling pathways.

A CCAAT/Enhancer Binding Protein β Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes

ABSTRACT Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein β (C/EBPβ), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPβ, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPβ, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPβ isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

Signal transduction and transcriptional regulation during mesenchymal cell differentiation

Although bone appears to be an apparently simple tissue, it is in reality a unique and very complex tissue composed of a variety of types of cells, including osteoblasts, osteoclasts, osteocytes, chondrocytes, adipocytes, immune cells, and hematopoietic cells [1]. Mesenchymal cells contribute to this diversity of bone tissue because mesenchymal cells are multipotent to differentiate into osteoblasts, chondrocytes, and adipocytes [2,3]. Differentiation processes of mesenchymal cells are harmoniously and dynamically controlled by specifi c signal transduction and transcription factors. In the past decade, transcription factors that specifi cally control the differentiation program of mesenchymal cells have been identifi ed. Genetic studies clearly demonstrate that Runx2 (Cbfa1/Pepb2aA) and Osterix (Sp7) are indispensable transcription factors for osteoblast development [4–6] (Fig. 1). Chondrocyte differentiation requires Sox family members in the early stage and Runx2 in the late stage [7–9] (Fig. 1). C/EBP family members and PPAR-γ play critical roles in adipocyte differentiation from mesenchymal cells [10,11] (Fig. 1). Moreover, recent studies have further advanced our understanding of the molecular basis by which these transcription factors regulate each differentiation program. In particular, biochemical studies have revealed how the expression and function of these transcription factors are controlled or modulated by coactivators, co-repressors, and other transcriptional regulators that assemble large complexes with the transcription factors. The functional roles of these transcription factors are also strictly regulated by signal transduction that links extracellular changes with the nucleus through the cytoplasm. Several cytokines and hormones such as bone morphogenetic protein (BMP), transforming growth factor-β (TGFβ), Wnt, hedgehog, fi broblast growth factors, estrogen, and androgen are involved in the regulation of mesenchymal cell differentiation by stimulating intracellular signaling pathways [1,9]. Specifi c intracellular signaling molecules are activated through phosphorylation, ubiquitination, protein– protein interaction, and conformational change in response to the ligand stimulation. The activated signaling molecules elicit the specifi c transcription factors by upregulating their transcriptional activity and/or translocation into the nucleus. These signaling pathways also engage in cross-talk, forming a complex network system. In this review article, we describe recent progress in describing molecular mechanisms that conduct the differentiation of mesenchymal cells into osteoblasts, chondrocytes, and adipocytes. First, we illustrate the role of BMP, TGF-β, Wnt, and Indian hedgehog (Ihh) signaling in mesenchymal cell differentiation. Second, we introduce the transcriptional regulation associated with the differentiation program of mesenchymal cells.