Teruaki Nomura

Role of PML and PML-RARα in Mad-Mediated Transcriptional Repression

Abstract Fusion of the promyelocytic leukemia (PML) protein to the retinoic acid receptor-α (RARα) generates the transforming protein of acute promyelocytic leukemias. PML appears to be involved in multiple functions, including apoptosis and transcriptional activation by RAR, whereas PML-RARα blocks these functions of PML. However, the mechanisms of leukemogenesis by PML-RARα remain elusive. Here we show that PML interacts with multiple corepressors (c-Ski, N-CoR, and mSin3A) and histone deacetylase 1, and that this interaction is required for transcriptional repression mediated by the tumor suppressor Mad. PML-RARα has the two corepressor-interacting sites and inhibits Mad-mediated repression, suggesting that aberrant binding of PML-RARα to the corepressor complexes may lead to abrogation of the corepressor function. These mechanisms may contribute to events leading to leukemogenesis.

Increased susceptibility to tumorigenesis of ski-deficient heterozygous mice

The c-ski proto-oncogene product (c-Ski) acts as a co-repressor and binds to other co-repressors N-CoR/SMRT and mSin3A which form a complex with histone deacetylase (HDAC). c-Ski mediates the transcriptional repression by a number of repressors, including nuclear hormone receptors and Mad. c-Ski also directly binds to, and recruits the HDAC complex to Smads, leading to inhibition of tumor growth factor-β (TGF-β) signaling. This is consistent with the function of ski as an oncogene. Here we show that loss of one copy of c-ski increases susceptibility to tumorigenesis in mice. When challenged with a chemical carcinogen, c-ski heterozygous mice showed an increased level of tumor formation relative to wild-type mice. In addition, c-ski-deficient mouse embryonic fibroblasts (MEFs) had increased proliferative capacity, whereas overexpression of c-Ski suppressed the proliferation. Furthermore, the introduction of activated Ki-ras into c-ski-deficient MEFs resulted in neoplastic transformation. These findings demonstrate that c-ski acts as a tumor suppressor in some types of cells. The level of cdc25A mRNA, which is down regulated by two tumor suppressor gene products, Rb and Mad, was upregulated in c-ski-deficient MEFs, whereas it decreased by overexpressing c-Ski in MEFs. This is consistent with the fact that c-Ski acts as a co-repressor of Mad and Rb. These results support the view that the decreased activities of Mad and Rb in ski-deficient cells at least partly contribute to enhanced proliferation and susceptibility to tumorigenesis. Human c-ski gene was mapped to a region close to the p73 tumor suppressor gene at the 1p36.3 locus, which is already known to contain multiple uncharacterized tumor suppressor genes.

Requirement of the co-repressor homeodomain-interacting protein kinase 2 for ski-mediated inhibition of bone morphogenetic protein-induced transcriptional activation.

Multiple co-repressors such as N-CoR/SMRT, mSin3, and the c-ski proto-oncogene product (c-Ski) mediate the transcriptional repression induced by Mad and the thyroid hormone receptor by recruiting the histone deacetylase complex. c-Ski also binds directly to Smad proteins, which are transcriptional activators in the transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signaling pathways, and inhibits TGF-β/BMP-induced transcriptional activation. However, it remains unknown whether other co-repressor(s) are also involved with Ski in the negative regulation of the TGF-β/BMP signaling pathways. Here, we report that the co-repressor homeodomain-interacting protein kinase 2 (HIPK2) directly binds to both c-Ski and Smad1. HIPK2 efficiently inhibited Smad1/4-induced transcription from the Smad site-containing promoter. A dominant negative form of HIPK2, in which the ATP binding motif in the kinase domain and the putative phosphorylation sites were mutated, enhanced Smad1/4-dependent transcription and the BMP-induced expression of alkaline phosphatase. Furthermore, the c-Ski-induced inhibition of the Smad1/4-dependent transcription was suppressed by a dominant negative form of HIPK2. The HIPK2 co-repressor activity may be regulated by an uncharacterized HIPK2 kinase. These results indicate that HIPK2, together with c-Ski, plays an important role in the negative regulation of BMP-induced transcriptional activation.

Fbxw7 acts as an E3 ubiquitin ligase that targets c-Myb for nemo-like kinase (NLK)-induced degradation.

The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-β-activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7α directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7α, suggesting that the c-Myb/Fbxw7α interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7α and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7α-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7.

A B-Myb complex containing clathrin and filamin is required for mitotic spindle function.

B‐Myb is one member of the vertebrate Myb family of transcription factors and is ubiquitously expressed. B‐Myb activates transcription of a group of genes required for the G2/M cell cycle transition by forming the dREAM/Myb–MuvB‐like complex, which was originally identified in Drosophila. Mutants of zebrafish B‐myb and Drosophila myb exhibit defects in cell cycle progression and genome instability. Although the genome instability caused by a loss of B‐Myb has been speculated to be due to abnormal cell cycle progression, the precise mechanism remains unknown. Here, we have purified a B‐Myb complex containing clathrin and filamin (Myb–Clafi complex). This complex is required for normal localization of clathrin at the mitotic spindle, which was previously reported to stabilize kinetochore fibres. The Myb–Clafi complex is not tightly associated with the mitotic spindles, suggesting that this complex ferries clathrin to the mitotic spindles. Thus, identification of the Myb–Clafi complex reveals a previously unrecognized function of B‐Myb that may contribute to its role in chromosome stability, possibly, tumour suppression.

Structure and Function of the Proteins Encoded by the myb Gene Family

The nuclear proto-oncogene c-myb is the cellular homologue of the v-myb gene carried by the chicken leukemia viruses avian myelobastosis virus (AMV) and E26, which transform avian myeloid cells in vitro and in vivo (for review, see ref. 1). c-myb expression is linked to the differentiation state of the cell, since expression is down-regulated during terminal differentiation of hemopoietic cells (2) and constitutive expression of introduced c-myb blocks the induced differentiation of erythroleukemia (3). In addition, antisense oligonucleotides to c-myb appear to impede in vitro hematopoiesis (4) and homozygous c-myb mutant mice displayed a specific failure of fetal hepatic hematopoiesis (5). These results all indicate a role for c-myb in maintaining the proliferative state of hematopoietic progenitor cells. Both c-Myb and v-Myb are transcriptional activators (6–9). The v-myb proteins (v-Myb) encoded by AMV and E26 are amino (N)- and carboxyl (C)-terminally truncated versions of c-Myb. In this report, we shall address the structure and function of each functional domain in c-Myb, and also the relationship between the retroviral-transforming v-myb genes and its cellular homologue, the c-myb gene, to ask whether changes in the control of transcription may account for the generation of the transformation phenotype.

Intracellular Localization of the Ret Finger Protein Depends on a Functional Nuclear Export Signal and Protein Kinase C Activation

The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Cα (PKCα) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP.

Inhibition of the Nuclear Import of Cubitus Interruptus by Roadkill in the Presence of Strong Hedgehog Signal

Hedgehog (Hh) signalling plays an important role in various developmental processes by activating the Cubitus interruptus (Ci)/Glioblastoma (Gli) family of transcription factors. In the process of proper pattern formation, Ci activity is regulated by multiple mechanisms, including processing, trafficking, and degradation. However, it remains elusive how Ci distinctly recognizes the strong and moderate Hh signals. Roadkill (Rdx) induces Ci degradation in the anterior region of the Drosophila wing disc. Here, we report that Rdx inhibited Ci activity by two different mechanisms. In the region abutting the anterior/posterior boundary, which receives strong Hh signal, Rdx inhibited the nuclear import of Ci by releasing importin α3 from Ci. In this region, Rdx negatively regulated the expression of transcription factor Knot/Collier. In farther anterior regions receiving moderate levels of Hh signal, Rdx induced Ci degradation, as reported previously. Thus, two different mechanisms by which Rdx negatively regulates Ci may play an important role in the fine-tuning of Hh responses.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

Two regions in c-myb proto-oncogene product negatively regulating its DNA-binding activity

The c‐myb proto‐oncogeneproduct (c‐Myb) is a transcriptional regulator that binds to the specific DNA sequence. Deletion of the negative regulatory domain (NRD) in the carboxyl‐proximal region of c‐Myb results in both increased trans‐activating capacity and oncogenic activation. One possible mechanism to modulate c‐Myb activity is a regulation of DNA‐binding activity. However, it is not known whether any region in NRD affects the in vivo DNA‐binding activity of c‐Myb. Using the highly transfectable cell line 293T, we developed a system to precisely measure the DNA‐binding activity of Myb expressed in mammalian cells. Using this system, two regions in NRD were shown to repress DNA‐binding activity. These results suggest that DNA‐binding activity of c‐Myb is independently regulated by multiple mechanisms through these subdomains.