Jun-ya Kato

Mammalian COP9 signalosome.

The COP9 signalosome (CSN) complex is highly conserved from yeast to human. Although the plant CSN was first identified as a negative regulator of photomorphogenesis, the mammalian CSN is linked to different biological responses such as checkpoint control, signal transduction, development and the cell cycle. Frequent over‐expression of the CSN subunit in a variety of human cancers suggests its involvement in cell transformation and tumorigenesis. The best‐known biochemical function associated with the CSN is the control of protein stability via the ubiquitin–proteasome system through regulation of cullin‐RING‐E3 ubiquitin ligase activity by deneddylation, by controlling the activity of COP1 E3 ligase, or by counteracting ubiquitin‐mediated degradation through a CSN‐associated deubiquitinating enzyme. In addition to affecting the stability of transcription factors, the CSN may regulate gene transcription by directly associating with chromatin. This review summarizes recent findings and discusses the physiological role and the cellular function of the mammalian CSN in terms of the regulation of cell proliferation.

Shuttling Imbalance of MLF1 Results in p53 Instability and Increases Susceptibility to Oncogenic Transformation

ABSTRACT Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cells susceptibility to oncogenic transformation.

CSN5 specifically interacts with CDK2 and controls senescence in a cytoplasmic cyclin E-mediated manner.

The fifth component (CSN5) of the mammalian COP9 signalosome complex plays an essential role in cell proliferation and senescence, but its molecular mediator remains to be determined. Here, we searched for interactors among various cell cycle regulators, and found that CSN5, but not the CSN holo-complex, bound to CDK2 in vivo and in vitro. Depletion of CSN5 enhanced phosphorylation of CDK2 by Akt, resulting in cytoplasmic accumulation of CDK2 together with cyclin E in a leptomycin B-resistant manner, and impaired phosphorylation of the retinoblastoma protein. Additional knockdown of CDK2, which reduced the expression of cyclin E to the normal level, did not restore cell proliferation, but significantly suppressed senescence in CSN5-depleted cells. Enforced expression of cytoplasmic cyclin E induced premature senescence in immortalized cell lines. These results show that CSN5 functions through CDK2 to control premature senescence in a novel way, depending on cyclin E in the cytoplasm.

Stable Form of JAB1 Enhances Proliferation and Maintenance of Hematopoietic Progenitors

Overexpression of JAB1 is observed in a variety of human cancers, but how JAB1 is involved in tumor development remained to be investigated. Here we analyzed mice with modified Jab1 expression. Mice ectopically expressing a more stable form of JAB1 protein under the control of a constitutive promoter were rescued from the embryonic lethality caused by the Jab1–/– allele and developed a myeloproliferative disorder in a gene dosage-dependent manner. Hematopoietic cells from the bone marrow of Jab1 transgenic mice had a significantly larger stem cell population and exhibited higher and transplantable proliferative potential. In contrast, Jab1+/– mice, which express ∼70% as much JAB1 protein as their wild-type littermates, showed inefficient hematopoiesis. Expression of the tumor suppressor p16INK4a was inversely correlated with that of JAB1, and the oncoprotein SMYD3, a newly identified JAB1 interactor, suppressed transcription of p16 in cooperation with JAB1. Thus, the expression and function of JAB1 are critical for the proliferation and maintenance of hematopoietic progenitors.

The COP1 E3-ligase interacts with FIP200, a key regulator of mammalian autophagy

BackgroundThe ubiquitin ligase COP1, COnstitutively Photomorphogenic 1, functions in many biological responses in mammalian cells, but its downstream pathway remains unclear.ResultsHere, we identified FIP200, a key regulator of mammalian autophagy, as a novel COP1-interacting protein by yeast two-hybrid screening. The interaction was confirmed by a GST-pulldown assay. Split-GFP analysis revealed that interaction between COP1 and FIP200 predominantly occurred in the cytoplasm and was enhanced in cells treated with UV irradiation. Different forms of FIP200 protein were expressed in cultured mammalian cells, and ectopic expression of COP1 reduced one of such forms.ConclusionsThese data suggest that COP1 modulates FIP200-associated activities, which may contribute to a variety of cellular functions that COP1 is involved in.

CSN5/Jab1 controls multiple events in the mammalian cell cycle

MINT‐8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)

Small mitochondrial ARF (smARF) is located in both the nucleus and cytoplasm, induces cell death, and activates p53 in mouse fibroblasts.

The ARF transcript produces two proteins, the full‐length ARF, p19ARF, and a short mitochondrial version, smARF. To explore the functional difference between the two, we generated GFP‐fused expression vectors for each protein and introduced them into NIH3T3 murine fibroblasts, which sustains a global deletion in the INK4a locus but contains a functional p53 gene. GFP‐p19ARF was located within the nucleolus as previously reported, whereas GFP‐smARF was detected mainly in the nucleoplasm. GFP‐smARF induced cell death although to a slightly lesser extent than p19ARF. GFP‐smARF stabilized p53 thereby inducing expression of the target genes, MDM2 and p21. We suggest that smARF has functions other than mitochondria‐mediated autophagy, and induces p53 expression and cell death via a novel mechanism.

Isolation and characterization of cytoplasmic cyclin D1 mutants.

Cdk4 (uniprotkb:P30285) physically interacts (MI:0218) with Cyclin D1 (uniprotkb:P24385) by anti bait coimmunoprecipitation (MI:0006)

Depletion of CSN5 inhibits Ras-mediated tumorigenesis by inducing premature senescence in p53-null cells.

The mammalian COP9 signalosome (CSN) complex is involved in cell transformation, but its molecular mechanism remains undetermined. Here we show that disruption of the fifth component (CSN5) prevented the formation of tumors by p53‐null cells transformed with an active form of Ras in subcutaneously injected mice. Depletion of CSN5 suppressed cell proliferation, and induced premature senescence characterized by upregulation of senescence‐associated‐β‐galactosidase activity and increased expression of CDK inhibitors. CSN5‐depleted cells exhibited enhanced activation of the PI3 kinase–Akt pathway, and chemical inhibition of this pathway reduced the level of senescence. Thus, CSN5 is suggested to be a novel target in cancer therapy and for drugs against tumor cells harboring mutated p53.

New twist in the regulation of cyclin D1

Abstract Among the cell cycle-related mammalian cyclins, cyclin D1 is more closely connected with cell proliferation in response to extracellular signals than the cell cycle clock itself. Because both its mRNA and protein are labile, the intracellular abundance of cyclin D1 is thought to be largely regulated at the level of transcription. However, recent findings suggest that, in certain cell types, cyclin D1 is post-translationally regulated, and a disturbance of this regulatory mechanism induces aberrant entry into the cell cycle and proliferation, sometimes leading to diseases such as cancer. In this review, we summarize recent findings and discuss the physiological role and cellular function of the novel mechanism of regulation of cyclin D1 in terms of the control of cell proliferation.