Jun Activation Domain-binding Protein Research Articles

Jun activation domain–binding protein 1 binds Smad5 and inhibits bone morphogenetic protein signaling

Bone morphogenetic proteins (BMPs) play an important role in the development and the homeostasis and pathology of cartilage tissue, particularly in the differentiation and anabolic activity of chondrocytes. The present study was undertaken to identify binding partners of the Smad proteins, the intracellular mediators of BMP activity, which might actively modify BMP signaling in chondrocytes. Yeast 2-hybrid technology was used to screen a complementary DNA library, constructed from human adult articular cartilage, for molecular binding partners of Smad5, a major intracellular mediator of BMP signaling. Primary interaction partners were verified by coimmunoprecipitation, and the relevance of the interactions to BMP signaling was evaluated by transcriptional reporter assay. Additionally, messenger RNA expression analysis (conventional and quantitative polymerase chain reaction) and immunostaining were performed in adult normal and osteoarthritic articular cartilage. We identified a novel Smad5 interactor, Jun activation domain-binding protein 1 (Jab1), expressed in adult cartilage. The interaction was confirmed in coimmunoprecipitation experiments. Overexpression of Jab1 resulted in an attenuation of BMP-dependent transcriptional responses, suggesting that Jab1 acts as an inhibitor of BMP signaling. Jab1 is a newly identified intracellular (negative) modulator of BMP signaling in chondrocytes and other cells. Jab1 represents an interesting molecule for understanding anabolic signaling in chondrocytes, as well as a potential therapeutic target for anabolic activation. Most interestingly, Jab1 appears to crosslink the BMP and interleukin-1 pathways.

Adapter protein NRBP associates with Jab1 and negatively regulates AP-1 activity

Jun activation domain-binding protein 1 (Jab1) is a coactivator of activating protein-1 (AP-1) and is the fifth component of the COP9 signalosome complex. It interacts with a variety of proteins and plays important roles in diverse signaling pathways and cellular function including oncogenesis. We show here that Jab1 interacts in vivo with nuclear receptor binding protein (NRBP), an evolutionarily conserved adapter protein with a kinase-like domain. We further show that NRBP inhibits Jab1-induced phosphorylation of c-Jun and AP-1 activation. Finally, overexpression of NRBP in mammalian cells specifically inhibits AP-1 activation by various stimuli. Taken together, our data suggest that NRBP may be an important negative regulator of Jab1-mediated functions such as gene transcription and tumor progression.

Mediators of endoplasmic reticulum stress‐induced apoptosis

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

Potential Role of Jun Activation Domain–Binding Protein 1 as a Negative Regulator of p27kip1 in Pancreatic Adenocarcinoma

Reduced expression of p27 has been associated with poor prognosis in most human cancers, including pancreatic adenocarcinoma. Jun activation domain-binding protein 1 (JAB1), an activator protein (AP-1) coactivator, previously implicated in p27 degradation, is overexpressed in various tumors and correlates with low p27 expression. We examined JAB1 and p27 in normal and neoplastic pancreatic tissues. Increased JAB1 expression was seen in pancreatic carcinoma samples but not in paired normal pancreatic tissues. Immunohistochemical analysis using tissue microarrays showed that JAB1 was overexpressed in all 32 (100%) pancreatic adenocarcinoma samples tested, predominantly nuclear in 23 (72%) samples and predominantly cytoplasmic in 9 (28%) tumors. When 10% was used as a cutoff for p27 positivity, p27 was expressed in 11 (34%) of tumors; however, p27 expression was localized in the nuclei of tumor cells in only 4 (13%) of the samples. Overexpression of the JAB1 in the pancreatic carcinoma cell lines Panc-1, Mia PaCa-2, and Panc-28 resulted in decreased p27 expression. Conversely, down-regulation of JAB1 by short interfering RNA substantially increased p27 expression and inhibited progression from G(1) to S phase of the cell cycle. Interestingly, JAB1-mediated p27 degradation was not impaired when S-phase kinase-interacting protein 2 (Skp2), an F-box protein required for the ubiquitination and consequent degradation of p27, was silenced. Thus, JAB1 may have an Skp2-independent p27 degradation mechanism in pancreatic cancer cells. These findings suggest that JAB1 overexpression is involved in the pathogenesis of pancreatic cancer through JAB1-mediated p27 degradation and that control of JAB1 expression is a novel therapeutic target in patients with pancreatic adenocarcinomas.

Overexpression of jun activation domain‐binding protein 1 in nonsmall cell lung cancer and its significance in p27 expression and clinical features

Decreased expression of p27, which is an inhibitor of cyclin-dependent kinase, is associated with cancer aggressiveness. It is believed that Jun activation domain-binding protein 1 (Jab1) plays a role in p27 degradation in a manner that is independent from the role played by S-phase kinase-associated protein 2 (Skp2). To examine the clinical significance of Jab1 in nonsmall cell lung cancer (NSCLC), the protein expression of Jab1 in tumor tissues was investigated with regard to the expression of p27 and Skp2. The clinicopathologic features and immunohistochemical expression levels of Jab1, p27, and Skp2 proteins were studied in 138 specimens from patients who underwent surgical resection for NSCLC. Survival analyses were performed by using the Kaplan-Meier method and Cox regression models. High Jab1 expression (>50% of cancer cell nuclei stained) was observed in 81 specimens (59%). In Skp2-negative specimens (n = 81), an inverse correlation between the protein expression of p27 and Jab1 was observed both with the chi-square test (P = .02) and with the Mann-Whitney U test (P = .02). High Jab1 expression levels were related to poor outcomes in 118 patients who underwent complete resection, with a 5-year overall survival rate of 43.9% for patients who had high Jab1 expression and 63.1% for patients who had low Jab1 expression (P = .01). This difference was greater in patients who had Skp2-negative specimens (51.9% vs. 79.3% (P = .02), but it was not significant in patients who had Skp2-positive specimens. The multivariate analysis revealed that Jab1 expression was an independent prognostic factor for survival in patients with NSCLC (relative risk, 2.247; P = .01). The current results showed for the first time that high Jab1 protein expression is related to poor outcome in patients with NSCLC and that this protein may be a target of therapy in NSCLC.

Jab1, a Novel Protease-activated Receptor-2 (PAR-2)-interacting Protein, Is Involved in PAR-2-induced Activation of Activator Protein-1

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.

CSN5/Jab1 inhibits cardiac L-type Ca2+ channel activity through protein–protein interactions

L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.

The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

Jun activation domain-binding protein 1 is required for mitotic checkpoint activation via its involvement in hyperphosphorylation of 53BP1

p53-binding protein 1 (53BP1), a participant in the DNA damage response pathway, has also been implicated in the cellular response to mitotic stress conditions. Here, we sought to broaden our understanding of the protein network surrounding 53BP1 by identifying and characterizing a 53BP1-interacting protein. Yeast two-hybrid screening was performed to identify possible binding partners of 53BP1. To investigate the functional meaning of the interaction, knock-down cells were established by introduction of antisense construct or siRNA into HeLa cells. The hyperphosphorylation of 53BP1 after treatment with nocodazole, a microtubule-interfering agent, was monitored by immunoblotting. And the cell cycle arrest at mitotic phase was measured by flow cytometry after staining with phospho-(Ser10)-histone H3 antibody. Jun activation domain-binding protein 1 (Jab1) was identified as a 53BP1-binding protein, and the interaction between them was confirmed to occur in mammalian cells. We found that nocodazole-induced 53BP1 hyperphosphorylation was abolished in Jab1 knock-down cells. In addition, ectopic overexpression of Jab1 augmented 53BP1 hyperphosphorylation. On the cellular level, Jab1 knock-down cells exhibited reduced mitotic arrest upon exposure to nocodazole, resulting in cellular resistance to the drug. Taken together, these results suggest that Jab1 is required for the hyperphosphorylation of 53BP1 upon mitotic stress conditions and is involved in proper activation of mitotic checkpoint mechanism. Our study also suggests the possibility that modulation of Jab1 activity may be an intriguing approach for enhancing the efficacy of microtubule-interfering anticancer drugs.

Basic investigation on the development of molecular targeting therapy against cyclin-dependent kinase inhibitor p27Kip1 in head and neck cancer cells.

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 is a diagnostic and prognostic marker of various malignancies. Low expression of p27Kip1 reflects poor prognosis, and an inverse correlation between the expression of p27Kip1 and degree of tumor malignancy has been reported. Because p27Kip1 mutation is extremely rare in human tumors, expression of p27Kip1 protein is thought to be controlled by post-transcriptional mechanism involved in inactivator, S-phase kinase associated protein 2 (Skp2) and Jun activation domain-binding protein 1 (Jab1). In this study, we investigated the role of gene therapy targeting p27Kip1 in human head and neck cancer cells. Human head and neck cancer (HNt and HSY) cells expressed p27Kip1, Skp2 and Jab1. To determine the function of p27Kip1, Skp2 and Jab1, we transfected head and neck cancer cells with pcDNA3.1-p27Kip1 wt, pcDNA3.1-p27Kip1 mt or treated with Skp2 antisense oligonucleotide (AS) or Jab1 AS. The transfections or treatments inhibited the growth of HNt and HSY cells. The growth inhibition mediated by pcDNA3.1-p27Kip1 mt or Skp2 AS or Jab1 AS specifically due to a significant induction of apoptosis characterized by an increase in fragmentation of nuclei and activation of caspase-3. The transfection of pcDNA3.1-p27Kip1 mt or treatment with Skp2 AS and Jab1 AS induced a strong growth inhibition of xenograft tumors. These findings suggest that p27Kip1 mt, Skp2 AS and Jab1 AS have the potential to become a novel and powerful gene therapy tool, and that stability of p27Kip1 protein may improve therapeutic benefits to patients with head and neck cancer.

Expression of Jun activation domain-binding protein 1 and p27 (Kip1) in thyroid medullary carcinoma

p27 is a prominent regulator of cell proliferation by universally inhibiting the cell cycle, while Jun activation domain-binding protein 1 (Jab1), a multifunctional cell signaling protein, contributes to carcinoma progression by degrading p27. In this study, we investigated the expression of these proteins in medullary thyroid carcinoma. We immunohistochemically examined Jab1 and p27 expression in 64 medullary thyroid carcinomas. Of the 64 cases examined, decreased p27 expression was observed in 38 cases (59.4%). The p27 expression level was inversely linked to tumour size as well as plasma calcitonin level. Jab1 expression level was generally high, and 46 cases (71.9%) were classified as overexpressing Jab1. The incidence was higher than those in papillary and follicular carcinomas, which were previously reported. Jab1 expression level was inversely linked to that of p27, and all five cases with only cytoplasmic but not nuclear staining of p27 overexpressed Jab1. These findings suggest that (1) decrease in p27 expression may contribute to local tumour growth; (2) Jab1 expression is related to the progression of medullary carcinoma by decreasing the amount of p27 in the cell and accelerating its degradation; and (3) Jab1 may play a more vital role in the pathogenesis of medullary carcinoma than papillary and follicular carcinomas.

Human Homologue of Maid Is a Useful Marker Protein in Hepatocarcinogenesis

Human homologue of maid (HHM) is a helix-loop-helix (HLH) transcriptional regulatory protein that is involved in the hepatic stem cell development and differentiation. We analyzed the potential involvement of HHM in hepatocarcinogenesis. We analyzed HHM expression in the choline-deficient L-amino acid defined (CDAA) diet model of rat hepatocarcinogenesis and in human adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC) biopsy samples. We assessed the effects of HHM on cell proliferation. We screened proteins that bind to HHM protein using a yeast 2-hybrid screen. High HHM expression was seen in foci and HCC induced in the rat CDAA diet model. HHM protein was expressed in 23 of 32 AH samples (72%), 19 of 28 well-differentiated HCC samples (68%), and 9 of 18 poorly-moderately differentiated HCC samples (50%). Over-expressed HHM enhanced the S phase. HHM interference RNA significantly inhibited cell proliferation. A yeast 2-hybrid screen identified Jun activation domain-binding protein 1 (Jab1) as a binding partner for HHM. We confirmed HHM and Jab1 binding by immunoprecipitation and immunofluorescent histochemistry. The expression of Jab1 was found in human AH and HCC samples. We found an association between levels of expression of HHM and those of Jab1 in AH and HCC tissues examined (P = .027 by chi2 test). High-level HHM expression was found from the very early stages of hepatocarcinogenesis, suggesting that HHM may be a useful marker protein to detect.

Prognostic Significance of Jab1 Expression in Laryngeal Squamous Cell Carcinomas

Jun activation domain-binding protein 1 (Jab1) is known as a coactivator of AP1 transcription factor, which contributes to tumor progression by degrading the p27kip1 protein. The purpose of this study is to investigate whether Jab1 expression is correlated with p27kip1 level and cell proliferation, as well as whether Jab1 expression is associated with clinicopathologic variables and prognosis of laryngeal squamous cell carcinoma (LSCC). Immunohistochemical and/or Western blot analysis was done in HEp-2 cells and 102 cases of LSCCs. Jab1 expression was negatively associated with p27kip1 expression and was positively associated with cell proliferation both in HEp-2 cells and LSCCs. Jab1 overexpression was detected in 51% LSCCs and was significantly associated with unfavorable clinicopathologic variables. Survival analysis revealed that Jab1 overexpression is significantly associated with short disease-free and overall survival (P = 0.0036 and P = 0.0001, respectively). When Jab1 and p27kip1 are combined, patients with Jab1(+)/p27kip1(-) revealed poor disease-free and overall survival (P= 0.0008 and P < 0.0001, respectively). When Jab1 expression and lymph node status are combined, patients with Jab1(+)/lymph node(+) revealed poorer disease-free andoverall survival than others (P < 0.0001 and P < 0.0001, respectively). Furthermore, patients with the phenotype of Jab1(+)/p27kip1(-)/lymph node(+) revealed the worst disease-free and overall survival (P < 0.0001 and P < 0.0001, respectively). Multivariate analysis revealed that Jab1 protein is an independent prognostic indicator for overall survival. These findings suggested that Jab1 protein may contribute to the tumor progression and represent a novel prognostic indicator for LSCCs.

Akt/PKB Activation-Induced Cytoplasmic Mislocalization of p27Kip1 Protein Is Mediated by Jab1 Shuttling in Human Myeloid Leukemia Cells.

We previously demonstrated the cytoplasmic mislocalization of p27Kip1 protein was highly correlated with constitutive Akt/PKB phosphorylation and poor prognosis in primary acute myeloid leukemia. However, mechanism involved in the Akt/PKB-mediated nuclear export of p27Kip1 in leukemia cells is not fully understood. Jun activation domain-binding protein 1 (Jab1)/CSN5 is the fifth component of the COP9 signalosome complex, which is involved in modulating signal transduction, ubiquitin-proteasome pathway, and nuclear transport. We examined the effects of Akt/PKB activation on the phosphorylation and localization of p27Kip1 protein in relation to Jab1 shuttling in K562 leukemia cells. Akt/PKB activation did not affect the phosphorylation at threonine 187 residue (T187) of p27Kip1 protein in K562 cells. However, the phosphorylation of p27Kip1 at the serine 10 (S10) and threonine T157 (T157) residue was markedly increased in the constitutively active Akt/PKB cDNA-transfected K562 (Myr-Akt/PKB-K562) cells. In the Myr-Akt/PKB-K562 cells, the S10- and T157-phosphorylated form of p27Kip1 localized almost exclusively to the cytoplasm. Leptomycin-B (LMB) pretreatment of Myr-Akt/PKB-K562 cells led to restoration of S10- and T157-phosphorylated p27Kip1 to the nucleus, indicating that Akt/PKB activation-induced cytoplasmic translocation of S10- and T157-phosphorylated p27Kip1 was mediated by CRM1-dependent pathway. We found that Akt/PKB activation did not alter the total cellular levels of Jab1 protein. However, Jab1 protein was exclusively localized to the cytoplasm in the Myr-Akt/PKB-K562 cells, whereas it was preferentially localized to the nucleus in the dominant negative Akt/PKB cDNA-transfected K562 cells. In Myr-Akt/PKB-K562 cells pretreated with LMB, the localization of the Jab1 was essentially nuclear. Jab1 silencing led to an accumulation of S10- and T157-phosphorylated p27Kip1 to the nucleus in the Myr-Akt/PKB-K562 cells, which was accompanied by a prominent decrease in the cytoplasmic level of p27Kip1. Taken together, we demonstrate that Akt/PKB activation-induced cytoplasmic mislocalization of S10- and T157-phosphorylated p27Kip1 protein is mediated by Jab1 shuttling in human myeloid leukemia cells. Apart from our understanding of the mechanism implicated in the regulation of phosphorylation and localization of p27Kip1 protein in leukemia cells, all these data might suggest possibilities for designing leukemia therapy since it should be possible to restore the function of a misplaced p27Kip1 protein.

Thioredoxin modulates activator protein 1 (AP-1) activity and p27Kip1 degradation through direct interaction with Jab1.

Thioredoxin (Trx) is a cellular redox enzyme that plays multiple roles in regulating cell growth and apoptosis. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein 1 (AP-1) transcription and was also shown to promote degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Recently, Jab1 expression was associated with the progression and poor prognosis of pituitary, epithelial ovarian, and breast cancers, suggesting that it plays a role in oncogenesis. Here, we report that Trx specifically interacts with and modulates the function of Jab1. Fluorescence resonance energy transfer and co-immunoprecipitation studies revealed that Trx and Jab1 colocalize and directly interact with each other. Further, Trx negatively regulates two important Jab1-controlled signaling pathways, activation of AP-1 transcription and degradation of p27Kip1, probably through a direct interaction between Trx and C-terminal of Jab1. The negative effect of Trx on AP-1 activity is Jab1-dependent, as it disappears when Jab1 levels are suppressed by an antisense approach. In addition, Trx competes with p27Kip1 for Jab1 binding. Taken together, our results suggest that Trx may regulate cell cycle and growth through a novel modulation of Jab1-mediated proliferation signals, further indicating that Trx may have the ability to control tumor progression.

Macrophage migration inhibitory factor directly interacts with hepatopoietin and regulates the proliferation of hepatoma cell

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in growth factor-dependent cell proliferation, cell cycle, angiogenesis, and tumorigenesis. Several studies have documented MIF expression in the sera following hepatic resection or in the course of liver cancer progression, but there is a paucity of information regarding the effect of MIF on hepatoma cells and relating mechanisms. In this paper, by [ 3H] thymidine incorporation, we found that exogenously added MIF could promote the proliferation of HepG2 in a dose-dependent manner. Hepatopoietin (HPO), as a liver-specific regeneration augmenter, could be induced by the expression of MIF in hepatoma cells. The activity of HPO promoter was increased, and its levels were enhanced after MIF was overexpressed in hepatoma cells. The similarities between HPO and MIF in structure and action led us to investigate their interaction and the inducing biological significance. Using yeast two-hybrid identification, we found that HPO interacted with MIF in yeast cells, and their binding ability was higher than that between HPO and JAB1 (Jun activation domain binding protein) or MIF and JAB1 in yeast cells. Their interaction was further verified by His pull-down assay in vitro and coimmunoprecipitation experiment in vivo. They were colocalized in the cytoplasm. Both HPO and MIF could bind to JAB1 and modulate the AP-1 pathway. When HPO and MIF were cotransfected into HepG2 cells, the binding activity of MIF to JAB1 was reduced, and the activity of AP-1 was improved. In contrast, MIF overexpressed in HepG2 was unable to interfere with the binding activity of HPO to JAB1, but its potentiation on AP-1 activity was reduced significantly. Taken together, these results indicate that MIF plays an important role in the proliferation of hepatoma cells, and the effect of MIF is in concert with HPO.

Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity

Hepatopoietin (HPO)/augmenter of liver regeneration (ALR) is a specific hepatotrophic growth factor, which plays a key role in liver regeneration. Our previous study indicated that HPO executes its function by an inter-reactive network of the autocrine, paracrine and endocrine pathways. Recently, we have demonstrated that intracellular HPO interacts with Jun activation domain-binding protein 1 (JAB1) and leads to potentiation of activating protein-1 (AP-1) activity in a MAPK independent fashion. JAB1 is the fifth subunit of the COP9 signalosome (CSN), which is first identified as a suppressor of plant morphogenesis. A protein complex kinase activity associated with the CSN has been reported but not identified yet. In this report, we investigated further the association of HPO with the whole CSN. HPO exists in a complex with the eight-component CSN, both when purified from glycerol gradient centrifugation and when reciprocal immunoprecipitated from the lysates of transfected COS-7 cells. Intracellular HPO colocalizes with endogenous CSN in nucleus of hepatic cells. In addition, intracellular function of HPO that increases the phosphorylation of c-Jun leading to potentiate the AP-1 activity is inhibited by curcumin, a potent inhibitor of CSN-associated kinase. Taken together, these results elucidate a novel relationship of intracellular growth factor, HPO with large protein complex, CSN, which suggests a possible linkage between CSN and liver regeneration.

Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule.

The immunological and neuroendocrine properties of macrophage migration inhibitory factor (MIF) are diverse. In this article we review the known cellular, molecular and genetic properties of MIF that place it as a key regulatory cytokine, acting within both the innate and adaptive immune responses. The unexpected and paradoxical induction of MIF secretion by low concentrations of glucocorticoids is explored. The role of MIF as a locally acting modulator of glucocorticoid sensitivity within foci of inflammation is also discussed. MIF has no homology with any other pro-inflammatory cytokine and until recently lacked a recognised transmembrane receptor. MIF has also been shown to be directly taken up into target cells and to interact with intracellular signalling molecules, including the Jun activation domain-binding protein Jab-1.Comprehensive analysis of the MIF gene has identified important functional polymorphisms and a series of genetic studies has revealed both association and linkage of MIF with inflammatory diseases. Altered MIF regulation may therefore be pivotal to acquiring chronic inflammation following an innate immune response.

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer’s disease, Parkinson’s disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1α is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1α mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1α using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1α in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1α. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1α.

The Potentiation Role of Hepatopoietin on Activator Protein-1 Is Dependent on Its Sulfhydryl Oxidase Activity

Hepatopoietin (HPO) is a novel hepatotrophic growth factor that stimulates hepatocyte proliferation by two pathways. In the first, intracellular HPO specifically modulates the activator protein-1 (AP-1) pathway through JAB1 (Jun activation domain-binding protein 1), whereas in the second, extracellular HPO triggers the mitogen-activated protein kinase pathway by binding its specific receptor on the cell surface. In this report we demonstrate that HPO is a flavin-linked sulfhydryl oxidase, and the invariant CXXC (Cys-Xaa-Xaa-Cys) motif in HPO is essential for the enzyme activity of HPO but not for its dimerization nor for its binding ability with JAB1. Two intramolecular disulfides were identified in HPO by mass spectrometry, one of which is formed by the redox CXXC cysteine residues. HPO site-directed mutants (Cys/Ser) at active sites, which lost sulfhydryl oxidase activity, could not increase c-Jun phosphorylation and failed to potentiate JAB1-mediated AP-1 activation. However, the mutants still have mitogenic stimulation and mitogen-activated protein kinase activation effects on HepG2 cells. Thus, it can be concluded that the potentiation role of HPO on AP-1 is dependent on its sulfhydryl oxidase activity.