JAB1/CSN5 and the COP9 signalosome. A complex situation

The Jun activating binding protein (JAB1) specifically stabilizes complexes of c-Jun or JunD with AP-1 sites, increasing the specificity of target gene activation by AP-1 proteins. JAB1 is also known as COP9 signalosome subunit 5 (CSN5), which is a component of the COP9 signalosome regulatory complex (CSN). Over the past year, JAB1/CSN5 has been implicated in numerous signaling pathways including those that regulate light signaling in plants, larval development in Drosophila, and integrin signaling, cell cycle control, and steroid hormone signaling in a number of systems. However, the general role of the CSN complex, and the specific role of JAB1/CSN5, still remain obscure. This review attempts to integrate the available data to help explain the role of JAB1/CSN5 and the COP9 signalosome in regulating multiple pathways (in this review, both JAB1 and CSN5 terminologies are used interchangeably, depending on the source material).     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

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)     

Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation.     

Clusterin and DNA repair: a new function in cancer for a key player in apoptosis and cell cycle control

The glycoprotein clusterin (CLU), has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, while a secretory form (sCLU) is pro-survival. Both forms are implicated in various cell functions, including DNA repair, cell cycle regulation, and apoptotic cell death. CLU expression has been associated with tumorigenesis and the progression of various malignancies. In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms, while simultaneously stimulating energy-expensive cell cycle checkpoints that delay the cell cycle progression to allow more time for DNA repair. This review summarizes our current understanding of the role of clusterin in DNA repair, apoptosis, and cell cycle control and the relevance.     

CSN5/JAB1 Interacts with the Centromeric Components CENP-T and CENP-W and Regulates Their Proteasome-mediated Degradation

The CENP-T·CENP-W complex is a recently identified inner centromere component that plays crucial roles in the formation of a functional kinetochore involved in cell division during mitosis. Using yeast two-hybrid screening, we identified an interaction between CENP-T and CSN5, the fifth component of the COP9 signalosome and a key modulator of the cell cycle and cancer. Co-immunoprecipitation revealed that CSN5 directly interacts with both CENP-T and CENP-W. Ectopically expressed CSN5 promoted the ubiquitin- and proteasome-dependent degradation of CENP-T·CENP-W. The formation of a CENP-T·CENP-W complex greatly enhanced the stabilities of the respective proteins, possibly by blocking CSN5-mediated degradation. Furthermore, dysregulation of CSN5 induced severe defects in the recruitment of CENP-T·CENP-W to the kinetochore during the prophase stage of mitosis. Thus, our results indicate that CSN5 regulates the stability of the inner kinetochore components CENP-T and CENP-W, providing the first direct link between CSN5 and the mitotic apparatus, highlighting the role of CSN5 as a multifunctional cell cycle regulator.     

COP9 Signalosome Component JAB1/CSN5 Is Necessary for T Cell Signaling through LFA-1 and HIV-1 Replication

To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.     

Cdk5: a new player at synapses

Cdk5 is a member of the cyclin-dependent kinase family. Unlike other conventional Cdks that are major regulators of eukaryotic cell cycle progression, Cdk5 displays diverse functions in neuronal as well as non-neuronal tissues. In particular, accumulating evidence points to the roles of this kinase in CNS development and other cellular processes. In this article, we summarize the functional roles of Cdk5 pertaining to the formation and functions of synapse, a specialized structure for the fundamental functions of neurons.     

Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif

Macrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50-65) and MPN are sufficient to mediate MIF-JAB1 interaction, respectively. Finally, endogenous CoIP of MIF-CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF-JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.     

Endothelin-1/Endothelin A receptor-mediated biased signaling is a new player in modulating human ovarian cancer cell tumorigenesis

The endothelin-1 (ET-1)/endothelin A receptor (ET_AR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and β-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ET_AR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ET_AR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ET_AR-targeted OC therapy, we investigated the role of other G protein subunits such as Gα_s in the ET_AR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ET_AR axis is well-characterized, Gαs signaling inhibits ET_AR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ET_AR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ET_AR-mediated β-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ET_AR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/β-arrestin-mediated oncogenic pathways, to improve the targeting of the ET_AR axis in OC.     

UVRAG: a new player in autophagy and tumor cell growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies(1) have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, under-scoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3-mediated autophagy and tumor cell control.     

Autophagy: a new player in hepatic stellate cell activation

Hepatic stellate cell (HSC) activation, the transition from a resident quiescent HSC to a myofibroblastic collagen-producing HSC, is a fundamental feature of liver fibrosis. Autophagy has been implicated in major liver pathologies, such as HCV infection and hepatocarcinoma. However, its role in HSC biology is largely unknown. Recently, we were able to demonstrate that HSC activation is followed by an increased autophagic flux and that its inhibition can partially inhibit the HSC myofibroblastic transition. These results point to autophagy as a possible target in the prevention of HSC activation.     

Roles of VRK1 as a new player in the control of biological processes required for cell division

Cell division, in addition to an accurate transmission of genetic information to daughter cells, also requires the temporal and spatial coordination of several biological processes without which cell division would not be feasible. These processes include the temporal coordination of DNA replication and chromosome segregation, regulation of nuclear envelope disassembly and assembly, chromatin condensation and Golgi fragmentation for its redistribution into daughter cells, among others. However, little is known regarding regulatory proteins and signalling pathways that might participate in the coordination of all these different biological functions. Such regulatory players should directly have a role in the processes leading to cell division. VRK1 (Vaccinia-related kinase 1) is an early response gene required for cyclin D1 expression, regulates p53 by a specific Thr18 phosphorylation, controls chromatin condensation by histone phosphorylation, nuclear envelope assembly by phosphorylation of BANF1, and participates in signalling required for Golgi fragmentation late in the G2 phase. We propose that VRK1, a Ser-Thr kinase, might be a candidate to play an important coordinator role in these cell division processes as part of a novel signalling pathway.     

Entosis,a key player in cancer cell competition

Cell-in-cell structures, also referred to as ''entosis'', are frequently found in human malignancies, although their prognostic impact remains to be defined. Two articles recently published in Cell Research report the stimulation of entosis by one prominent oncogene, Kras, as well as by one class of tumor suppressors, namely epithelial cadherins E and P, illustrating the complex regulation of this biological process.A number of different terms have been used to describe live cell engulfments giving rise to cell-in-cell structures (CICS): entosis, emperipolesis, cannibalism and phagocytosis. Heterotypic live cell engulfment usually involves the ingestion of leukocytes by non-leukocytes (such as epithelial cells or fibroblasts). Homotypic live cell engulfment (among cells of the same type) mostly occurs in cancers, probably reflecting major alterations in cellular physiology that are associated with oncogenesis and tumor progression.CICS can be visualized by conventional hematoxylin-eosin staining and have been described to occur in many different human cancers¹. CICS produced as the result of entosis exhibit β-catenin localization patterns that are indicative of a cell junction-mediated mechanism of engulfment, and this polarized distribution of β-catenin can be taken advantage of to visualize CICS in vivo, in tumors¹. However, the prognostic impact of CICS is highly context-dependent. Thus, CICS are particularly frequent in high-grade, aggressive breast cancer with dismal prognosis². CICS are only found in castration-resistant, not in androgen-dependent, prostate cancer and hence correlate with poor prognosis in this particular malignancy³. In contrast, in pancreas adenocarcinomas, high levels of CICS correlate with a lower incidence of metastases⁴. These findings point to a complex role of CICS in cancer biology.Two papers by Sun et al.^5,6 recently published in Cell Research characterized one particular mechanism of homotypic live cell engulfment termed entosis. The first paper of this series⁵ provides evidence that one of the most prominent oncogenes, activated Kras, can stimulate entosis, while the second paper⁶ demonstrates that a prominent tumor suppressor, epithelial cadherin (E-cadherin), can increase entosis as well.Cancers are highly complex mixtures of cells in which the malignant population is genetically and epigenetically heterogeneous, reflecting a history of clonal selection. One particular type of competition among distinct cells may consist in the engulfment of one cell (the ''loser'') by another (the ''winner''), as demonstrated by Sun et al. in several cell culture models, as well as in human cancers that were xenografted into immunodeficient mice⁵. Importantly, co-culture of non-transformed cells with their malignant counterparts systematically leads to engulfment of the former by the latter, suggesting that oncogenic transformation is coupled to the ''winner'' status⁵. Indeed, competition by entosis leads to the physical elimination of the ''loser'' cells, which usually succumb to non-apoptotic cell death as soon as the phagosome enveloping the engulfed cell is decorated with LC3 and then fuses with lysosomes^1,7. What is then the difference between ''loser'' and ''winner'' cells? Sun et al.⁵ propose that one cardinal feature of ''winners'' is a high degree of mechanic deformability, as demonstrated by biophysical experiments and computer simulations. This is a highly provocative finding because human tumors are known to be more mechanically heterogeneous than normal tissues and that tumor progression is increased with an elevated mechanic deformability of the cancer cells. This reduction in cell stiffness may hence not only increase the metastatic potential of tumor cells⁸, but may also reflect an increased entotic activity⁵.Transfection-enforced expression of active KrasV12 was sufficient to confer winner status onto non-tumorigenic cells, correlating with an increase in mechanic deformability⁵. This effect of KrasV12 relied on Rac1, as demonstrated by the facts that knockdown of Rac1 suppressed the ''winner'' status conferred by KrasV12, expression of constitutively active Rac1 induced a ''winner'' phenotype and dominant-negative Rac1N17 imparted a ''loser'' status⁵. However, at this point it remains to be explored whether other pathways downstream of Kras such as the phosphatidylinositide 3-kinases (PI3K)/protein kinase B (PKB, best known as AKT)/mechanistic target of rapamycin (mTOR) pathway may contribute to ''winner'' status. Inhibition of mTOR interferes with degradation of engulfed cells⁹, suggesting that activation of the PI3K/AKT/mTOR axis might favor the manifestation of the ''winner'' phenotype as well. Similarly, it remains an open question as to whether other oncogenes than Kras may regulate entosis as well.Breast cancers cells engineered to express epithelal E- or P-cadherins (but not mesenchymal-type cadherins, such as N-cadherin and cadherin-11) re-establish epithelial junctions and engulf and kill non-transfected parental cells in transformed growth assays⁶. The induction of entosis by epithelial E- or P-cadherins is associated to the polarized distribution of RhoA and contractile actomyosin dependent on the p190A Rho-GTPase-Activating Protein (p190A RhoGAP) that is recruited to epithelial junctions⁶. Inhibition of RhoA by overexpression of RhoA-N19 or p190A RhoGAP was sufficient to impart winner status to cells mixed with controls, whereas overexpression of RhoA, ROCKI, or ROCKII had the opposite effect and hence created ''loser'' cells⁵. It has been known that Rho-GTPase and Rho-kinase are not required in engulfing cells but are required in internalizing cells¹, underscoring the idea that ''loser'' cells are not just passive ''victims'' of a cannibalistic attack but somehow contribute to their fatal fate. The ''loser'' status was accompanied by the ROCK-dependent accumulation of actomyosin⁶, and computer simulations suggest that actomyosin contractility within ''loser'' cells constitutes a critical driving force of entosis⁵. The levels of phosphorylated myosin light chain 2 at Ser19 (pMLC2), a readout of contractile myosin downstream of ROCKI/II, were also increased in ''loser'' cells as compared to ''winners''⁶. RhoA, ROCKI/II, MLC2, actin and myosins all accumulated at particularly high levels in ''losers'' at the cell cortex oriented away from cell-cell adhesions⁶.The aforementioned data support a dual implication of entosis in carcinogenesis (Figure 1). On one hand, entosis carried out by ''winner'' cells may constitute a competitive advantage of aggressive tumor cells, perhaps allowing the ''winners'' to retrieve amino acids and other building blocks for anabolic reaction from their cannibalistic activity⁹ or increasing their genomic instability subsequent to mitotic aberrations^2,10. In this context, pharmacological suppression of entosis by Y27632, a ROCKI/II inhibitor, abolished the competitive advantage of transformed cells over their non-transformed siblings in mixed culture experiments⁵. On the other hand, stimulation of entosis by re-expression of epithelal E- or P-cadherins reduced the clonogenic potential of breast cancer cells. In this context, Y27632 facilitated tumor cell growth in vitro⁶. These observations underscore the need of exploring the detailed mechanisms through which entosis may repress or favor oncogenesis and tumor progression.Open in a separate windowFigure 1A dual role for entosis in cancer. (A) Entosis as a pro-tumorigenic process. (B) Entosis as a tumor-suppressive mechanism.