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Renal fibrosis is controlled by profibrotic and antifibrotic forces. Exploring anti-fibrosis factors and mechanisms is an attractive strategy to prevent organ failure. Here we identified the JNK-associated leucine zipper protein (JLP) as a potential endogenous antifibrotic factor. JLP, predominantly expressed in renal tubular epithelial cells (TECs) in normal human or mouse kidneys, was downregulated in fibrotic kidneys. Jlp deficiency resulted in more severe renal fibrosis in unilateral ureteral obstruction (UUO) mice, while renal fibrosis resistance was observed in TECs-specific transgenic Jlp mice. JLP executes its protective role in renal fibrosis via negatively regulating TGF-β1 expression and autophagy, and the profibrotic effects of ECM production, epithelial-to-mesenchymal transition (EMT), apoptosis and cell cycle arrest in TECs. We further found that TGF-β1 and FGF-2 could negatively regulate the expression of JLP. Our study suggests that JLP plays a central role in renal fibrosis via its negative crosstalk with the profibrotic factor, TGF-β1., Qi Yan et al. find that JNK-associated leucine zipper protein (Jlp) counteracts the profibrotic effects of TGF-β1 and autophagy on renal tubular epithelial cells and that TGF-β1 and FGF-2 can negatively regulate the expression of Jlp. These findings provide insights into the role of Jlp in kidney fibrosis.

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

Cutaneous melanoma is the most aggressive form of skin cancer. This aggressiveness appears to be due to the cancer cells' ability to reversibly switch between phenotypes with non-invasive and invasive potential, and microphthalmia-associated transcription factor (MITF) is known to play a central role in this process. The transcription factor glioma-associated oncogene homolog 1 (GLI1) is a component of the canonical and noncanonical sonic hedgehog pathways. Although GLI1 has been suggested to be involved in melanoma progression, its precise role and the mechanism underlying invasion remain unclear. Here we investigated whether and how GLI1 is involved in the invasive ability of melanoma cells. Gli1 knockdown (KD) melanoma cell lines, established by using Gli1-targeting lentiviral short hairpin RNA, exhibited a markedly reduced invasion ability, but their MITF expression and activity were the same as controls. Gli1 KD melanoma cells also led to less lung metastasis in mice compared with control melanoma cells. Furthermore, the Gli1 KD melanoma cells underwent a mesenchymal-to-epithelial-like transition, accompanied by downregulation of the epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (EMT-TF) Snail1, Zeb1 and Twist1, but not Snail2 or Zeb2. Collectively, these results indicate that GLI1 is important for maintaining the invasive and mesenchymal-like properties of melanoma cells independent of MITF, most likely by modulating a subset of EMT-TF. Our findings provide new insight into how heterogeneity and plasticity are achieved and regulated in melanoma. © 2017 Japanese Cancer Association.

CD4

Selected vulnerability of neurons in Huntington's disease suggests that alterations occur in a cellular process that is particularly critical for neuronal function. Supporting this idea, pathogenic Htt (polyQ-Htt) inhibits fast axonal transport (FAT) in various cellular and animal models of Huntington's disease (mouse and squid), but the molecular basis of this effect remains unknown. We found that polyQ-Htt inhibited FAT through a mechanism involving activation of axonal cJun N-terminal kinase (JNK). Accordingly, we observed increased activation of JNK in vivo in cellular and mouse models of Huntington's disease. Additional experiments indicated that the effects of polyQ-Htt on FAT were mediated by neuron-specific JNK3 and not by ubiquitously expressed JNK1, providing a molecular basis for neuron-specific pathology in Huntington's disease. Mass spectrometry identified a residue in the kinesin-1 motor domain that was phosphorylated by JNK3 and this modification reduced kinesin-1 binding to microtubules. These data identify JNK3 as a critical mediator of polyQ-Htt toxicity and provide a molecular basis for polyQ-Htt-induced inhibition of FAT.

The mitogen-activated protein kinase (MAPK) cascades, including c-Jun N-terminal kinase (JNK), are composed of a MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Previously, we reported that JNK-binding protein 1 (JNKBP1) enhances JNK activation induced by the TGF-beta-activated kinase1 (TAK1) MAPKKK in transfected cells. We have investigated whether JNKBP1 functions as an adaptor protein for nuclear factor (NF)-kappaB activation mediated by TAK1 in COS-7 cells. Co-expression experiments showed that JNKBP1 interacted with not only TAK1, but also with its upstream regulators, TNF-receptor associated factors 2 and 6 (TRAF2 and TRAF6). An endogenous interaction between JNKBP1 and TRAF2 or TAK1 was confirmed by immunoprecipitation analysis. We also found that JNKBP1 could enhance the NF-kappaB activation induced by TAK1 and TRAF2, and could promote TRAF2 polyubiquitination. These results suggest a scaffolding role for JNKBP1 in the TRAF2-TAK1-NF-kappaB signaling pathway.

金沢大学がん研究所がん分子細胞制御, Cerebellar granule cell precursors (GCPs) proliferate in the outer part of the external granular layer (EGL). They begin their differentiation by exiting the cell cycle and migrating into the inner part of the EGL. Here we report that JSAP1, a scaffold protein for JNK signaling pathways, is expressed predominantly in the post-mitotic GCPs of the inner EGL. JSAP1 knockdown or treatment with a JNK inhibitor enhances the proliferation of cultured GCPs, but the overexpression of wild-type JSAP1 leads to increased proportions of p27Kip1- and NeuN-positive cells, even with saturating concentrations of Sonic hedgehog (Shh), a potent GCP mitogen. However, these differentiation-promoting effects on GCPs are attenuated significantly in cells overexpressing a mutant JSAP1 that lacks the JNK-binding domain. Together, these data suggest that JSAP1 antagonizes the mitogenic effect of Shh on GCPs and promotes their exit from the cell cycle and differentiation, by modulating JNK activity. © 2008 Elsevier Inc. All rights reserved.

金沢大学がん研究所がん分子細胞制御, The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. © 2008 Springer Science+Business Media B.V.

We previously identified c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffolding factor for JNK intracellular signaling pathways. Targeted gene-disruption studies have shown that JSAP1-null mice are unable to breathe and die shortly after birth. Although neural defects might be responsible for their death, there has been no convincing evidence for this. Here we first generated genetically engineered mice carrying a loxP-flanked (floxed) jsap1 gene. To evaluate the validity of this deletion as a jsap1 conditional knockout (KO), we created mice in which the same exon was deleted in all cell lineages, and compared their phenotypes with those of the jsap1 conventional KO mice reported previously. The two KO lines showed indistinguishable phenotypes, i.e., neonatal death and morphological defects in the telencephalon, indicating that the conditional deletion was a true null mutation. We then introduced the floxed jsap1 deletion mutant specifically into the neural lineage, and found that the jsap1 conditional KO mice showed essentially the same phenotypes as the JSAP1-null mice. These results strongly suggest that the neonatal death of jsap1-deficient mice is caused by defects in the nervous system.

JNK/stress-activated protein kinase-associated protein 1 (JSAP1) and JNK-associated leucine zipper protein (JLP) are structurally related scaffolding proteins that are highly expressed in the brain. Here, we found that JSAP1 and JLP play functionally redundant and essential roles in mouse cerebellar Purkinje cell (PC) survival. Mice containing PCs with deletions in both JSAP1 and JLP exhibited PC axonal dystrophy, followed by gradual, progressive neuronal loss. Kinesin-1 cargoes accumulated selectively in the swollen axons of Jsap1/Jlp-deficient PCs. In addition, autophagy inactivation in these mice markedly accelerated PC degeneration. These findings suggest that JSAP1 and JLP play critical roles in kinesin-1-dependent axonal transport, which prevents brain neuronal degeneration. © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., Embargo Period (12 mouths)

The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.

Scaffold proteins of mitogen-activated protein kinase (MAPK) intracellular signal transduction pathways mediate the efficient and specific activation of the relevant MAPK signaling modules. Previously, our group and others have identified c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffold protein for JNK MAPK pathways. Although JSAP1 is expressed in the testis in adults, its expression during development has not been investigated. In addition, it is unknown which types of cells in the testis express the scaffold protein. Here, we examined the expression of JSAP1 in the testis of mice aged 14 days, 20 days, 6 weeks, and 12 weeks by immunohistochemistry and Western blotting. The specificity of the anti-JSAP1 antibody was evaluated from its reactivity to exogenously expressed JSAP1 and a structurally related protein, and by antigen-absorption experiments. The immunohistochemical analyses with the specific antibody showed that the JSAP1 protein was selectively expressed in the spermatogonia and spermatocytes, but not in other cell types, including spermatids and somatic cells, during development. However, not all spermatogonia and spermatocytes were immunopositive either, especially in the 12-week-old mouse testis. Furthermore, we found by Western blotting that the expression levels of JSAP1 protein vary during development; there is high expression until 6 weeks after birth, which approximately corresponds to the end of the first wave of spermatogenesis. Collectively, these results suggest that JSAP1 function may be important in spermatogenic cells during early postnatal development.

We previously reported that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, is important in embryonic stem (ES) cells during neurogenesis. In that study, we also observed the altered expression of mesodermal marker genes, which indicated that JSAP1 is involved in the differentiation of mesodermal lineages. Here, we investigated the function of JSAP1 in cardiomyocyte development using JSAP1-null ES cells, and found that cardiomyogenesis was impaired in the JSAP1-null mutant. The JSAP1 deficiency resulted in lower gene expression of the cardiac transcription factor Nkx2.5 and contractile proteins. In contrast, the mutant showed a significantly higher expression of mesoderm-related markers other than those of the cardiomyocyte lineage. Together, these results suggest that JSAP1 may be important for the differentiation of the mesodermal lineages, functioning as a positive factor for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into other lineages.

金沢大学がん研究所がん分子細胞制御, Receptor activator of NF-κB ligand (RANKL) plays an essential role in osteoclast formation and bone resorption. Although genetic and biochemical studies indicate that RANKL regulates osteoclast differentiation by activating receptor activator of NF-κB and associated signaling molecules, the molecular mechanisms of RANKL-regulated osteoclast differentiation have not yet been fully established. We investigated the role of the transcription factor c-Jun, which is activated by RANKL, in osteoclastogenesis using transgenic mice expressing dominant-negative c-Jun specifically in the osteoclast lineage. We found that the transgenic mice manifested severe osteopetrosis due to impaired osteoclastogenesis. Blockade of c-Jun signaling also markedly inhibited soluble RANKL-induced osteoclast differentiation in vitro. Overexpression of nuclear factor of activated T cells 1 (NFAT1) (NFATc2/ NFATp) or NFAT2 (NFATc1/NFATc) promoted differentiation of osteoclast precursor cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated osteoclast-like cells even in the absence of RANKL. Overexpression of NFAT1 also markedly transactivated the TRAP gene promoter. These osteoclastogenic activities of NFAT were abrogated by overexpression of dominant-negative c-Jun. Importantly, osteoclast differentiation and induction of NFAT2 expression by NFAT1 overexpression or soluble RANKL treatment were profoundly diminished in spleen cells of the transgenic mice. Collectively, these results indicate that c-Jun signaling in cooperation with NFAT is crucial for RANKL-regulated osteoclast differentiation.

P19 embryonic carcinoma cells, a model system for studying early development and differentiation, can differentiate into neurons and primitive endoderm-like cells depending on the culture conditions. We have previously reported that the activation of c-Jun amino-terminal kinase (JNK) is required for the retinoic acid-induced neural differentiation of P19 cells. However, the signaling pathway(s) responsible for the activation of JNK has not been known. In this study, we demonstrated that activities of MAPK kinase 4 (MKK4) and TAK1, one of the upstream kinases of MKK4, were enhanced in the neurally differentiating cells. Inhibition of the neural differentiation by an overexpression of protein phosphatase 2Cepsilon, an inactivator of TAK1, suggested a critical role of the TAK1 signaling pathway during the differentiation. Confocal microscopic analysis indicated that TAK1, phospho-MKK4, and phospho-JNK were colocalized with tubulin in the neurites and localized also in the nuclei of the differentiating cells. In contrast, two TAK1-binding proteins, TAB1 and TAB2, which are involved in the activation of TAK1, were localized in the neurites and the nuclei of the differentiating cells, respectively. These results suggest that two distinct TAK1-MKK4-JNK signaling pathways are independently activated at the different intracellular locations and may participate in the regulation of the neural differentiation of P19 cells.

The Jsap1 gene encodes a scaffold protein for c-Jun N-terminal kinase cascades. We established c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1)-null mouse embryonic stem cell lines by homologous recombination. The JSAP1-null embryonic stem cells were viable, however, exhibited hyperplasia of the ectoderm during embryoid body formation, and spontaneously differentiated into neurons more efficiently than did wild type. The expression of components of c-Jun N-terminal kinase cascades and a subset of marker mRNAs during early embryogenesis was altered in the JSAP1-null mutants. Retinoic acid dramatically increased the expression of JSAP1 and JNK3, which were co-precipitated with anti-JNK3 in the neuroectoderm of wild type but not JSAP1-null embryoid bodies. In the neurons differentiated from the wild type embryoid bodies, JSAP1 was localized in the soma, neurites, and growth cone-like structure of the neurites, and neurite outgrowth from the JSAP1-null embryoid bodies was apparently less efficient than from wild type. JSAP1 and c-Jun N-terminal kinase 3 were coexpressed in the embryonic ectoderm of E7.5 mouse embryo, whereas Wnt1 and Pax2 were coexpressed with JSAP1 at the midbrain-hindbrain junction in E12.5 mouse embryo, thus suggesting that JSAP1 is required for early embryonic neurogenesis.

The mitogen-activated protein kinase (MAPK) cascades consist of MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). The specificity of activation of MAPK cascades may be determined, in part, by scaffold proteins that organize multi-enzyme complexes. We have earlier reported a scaffold protein JSAP1 (also known as JIP3) in the JNK MAPK cascade. We also showed that, of the adult mouse tissues tested, JSAP1 mRNA was predominantly expressed in brain. Here we report the localization of JSAP1 protein in mouse embryos and adult brain by immunohistochemical analysis. In embryos (E11-16), JSAP1 immunoreactivity was mainly found in the central and peripheral nervous systems, where it was localized to the cell bodies and/or axons of developing neurons, but not neural precursor cells. In the adult brain, immunoreactive JSAP1 was localized mostly to cell bodies in almost all neurons. We also showed that the expression of JSAP1 transcripts and proteins gradually increased during the neural differentiation of mouse P19 embryonal carcinoma (EC) cells. Furthermore, we showed that overexpressed JSAP1 facilitated the efficient activation of JNK by MEKK1 in P19 cells. These results suggest that JSAP1 may function as a scaffold protein for the JNK signaling module in neuronal cells.

We have identified four isoforms of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein that participates in JNK mitogen-activated protein kinase cascades, termed JSAP1a, JSAP1b, JSAP1c, and JSAP1d. The previously identified JSAP1 was renamed JSAP1a to avoid confusion. Analyses of the exon–intron structure of the jsap1 gene indicated that the isoforms are generated through alternative splicing involving exons 5 and 6. The mRNA expression levels of the JSAP1 isoforms differed among the mouse tissues examined. We also investigated the region of JSAP1 responsible for its interaction with JNK, and found that the JNK-binding domain is located between aa residues 201 and 217 in JSAP1a, which is encoded by part of exon 6. As all the JSAP1 isoforms contain this binding domain, we examined the binding affinity of the JSAP1 isoforms for JNK1, JNK2, and JNK3. JSAP1c and JSAP1d, which contain a 31-aa sequence not present in JSAP1a or JSAP1b, had a lower binding affinity for the JNKs, especially JNK3. These results suggest that JSAP1c and JSAP1d may attenuate the scaffolding activity of JSAP1a and/or JSAP1b in JNK cascades, especially the JNK3 cascades.

We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF-β-activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.

金沢大学がん研究所がん分子細胞制御, FK506 is a powerful immunosuppressive drug currently in use that inhibits the activation of several transcription factors (nuclear factor (NF)-AT and NF-κB) critical for T cell activation. We show here that, contrary to the situation in T cells, FK506 activates transcription factor NF-κB in non-lymphoid cells such as fibroblasts and renal mesangial cells. We further show that FK506 induces NF-κB-regulated IL-6 production in vitro and in vivo, in particular in kidney. IL-6 has been shown previously to produce renal abnormalities in vivo, such as mesangioproliferative glomerulonephritis. Similar renal abnormalities were also observed in FK506-treated animals. These results thus suggest a causal relationship between FK506-induced NF-κB activation/IL-6 production and some of FK506-induced renal abnormalities.

Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCFβTrCP-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH2-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCFβTrCP E3 ligase upon phosphorylation at two Ser residues in the βTrCP-binding-motif-like sequence D94AGLCMDSPSP104. Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCFβTrCP, and point mutation of DAG to AAG or DAA eradicated both βTrCP binding and ubiquitylation. Further analysis of the mode of βTrCP binding to this region revealed that the PEST-like sequence from E82SS to D94AG is crucially involved in both the βTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E82SS to SPSP104 region with Asp resulted in βTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCFβTrCP requires the phosphorylation of S101PS103P in the βTrCP-binding-motif-like and adjacent PEST-like sequences. © 2011. Published by The Company of Biologists Ltd.

MafB is a basic leucine zipper transcription factor that plays important roles in development and differentiation processes. During osteoclastogenesis, its expression is downregulated at the transcriptional level via the JNK and p38 MAP kinase pathways. In the present study, we demonstrated that MafB protein stability is regulated by JNK and identified a phosphorylation site, Thr62. The expression of a constitutively active form of JNK (a fusion protein MKK7α1-JNK1β1) promoted the degradation of MafB in COS7 cells, and a T62A substitution significantly reduced the instability of MafB. The introduction of a four-fold (T58A/T62A/S70A/S74A) substitution in an acidic transcription-activating domain almost protected the instability resulting from the activation of JNK. Furthermore, treatment with proteasome inhibitors increased the MafB level, and a high-molecular-weight smear, characteristic of polyubiquitination, was observed in lysates from cells in which MafB, ubiquitin, and MKK7α1-JNK1β1 were co-expressed. These results suggest that phosphorylation of MafB by JNK confers susceptibility to proteasomal degradation., Article, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. 494(1):94-100 (2010)

金沢大学がん研究所がん分子細胞制御, Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein Gα13, showed little or no effect on the cell migration defect. Furthermore, although a constitutively active Gα13 enhanced the migration of control HeLa cells, the Gα13-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with Gα13. © The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

最近のいくつかの研究から, アルツハイマー病における痴呆の直接的原因となる選択的神経細胞死は, 細胞内情報伝達分子JNK3によって仲介されていると考えられる. 本研究では, JNK3と相互作用する蛋白質因子の同定およびその解析を試みた. 酵母 two-hybrid 法により, 新規JNK3結合蛋白 (BP#2と命名) のcDNAクローニングに成功した. BP#2は, 脊椎動物MAPキナーゼの中ではJNK3と特異的に結合し, さらにJNK活性化キナーゼSEK1との結合能も有することから, JNK3情報伝達系におけるスキャフォールド蛋白として機能していると考えられる. また, 繊維芽細胞内でBP#2を強制的に過剰発現させることによりJNK3の活性化が抑制されることを見出した. 神経細胞においても同様の現象が認められるならば, BP#2の過剰発現によりアルツハイマー病における神経細胞死を特異的に阻害することが可能となるかもしれない.

Although most somatic cells cannot proliferate, immature cells proliferate continuously to produce mature cells. Recently, we cloned mouse PSF1 from a hematopoietic stem cell specific cDNA library and reported that PSF1 is indispensable for the proliferation of immature cells. To identify the PSF1-binding protein, we used the yeast two-hybrid system with PSF1 as bait, and identified and cloned SLD5. SLD5 interacted with a central region of PSF1. Tissue distribution of SLD5 was quite similar to that of PSF1. When overexpressed, SLD5 protein was co-localized with PSF1. These data suggest that PSF1 and SLD5 may cooperate in the proliferation of immature cell populations.

p53 and NF-kappaB RelA are activated by various genotoxic agents and mutually suppress each other's ability to activate transcription, most likely through competition for transcriptional coactivators such as CBP or p300. However, we found that the inhibition by RelA of p53 transcriptional activity is not completely restored by CBP/p300 overexpression and that a p53 mutant can not suppress RelA activity despite of its ability to bind CBP/p300. In the present study, we further present evidence that these two transcriptional factors directly interact both in vivo and in vitro. These results therefore indicate that the cross transcriptional interference between p53 and RelA is partly caused by the direct interaction between these two transcription factors which is mediated by their dimerization/tetramerization domains and results in inhibition of each other's transcriptional activity. Finally, cells derived from RelA knockout mice showed enhanced p53 transcriptional activity, suggesting that this cross transcriptional interference is physiologically important in cellular response to genotoxic stress.

We have generated a mouse model for hepatic ischemia in which surgical subcutaneous transposition of the spleen allows hepatic ischemia to be applied without affecting other tissues. Using this mouse model we investigated the relationship between the length of ischemic periods in the liver and subsequent liver function; furthermore, we assayed the activation of c-Jun N-terminal kinase (JNK) during ischemia and reperfusion. Although prior to this study only the activated form of JNK was known to be translocated to the nucleus, we found that JNK translocates to the nucleus during ischemia without activation and is then activated during reperfusion. These results suggest a novel mechanism of JNK activation.

Compared with the knowledge of maternal care, much less is known about the factors required for paternal parental care. Here we report that new sires of laboratory mice, though not spontaneously parental, can be induced to show maternal-like parental care (pup retrieval) using signals from dams separated from their pups. During this interaction, the maternal mates emit 38-kHz ultrasonic vocalizations to their male partners, which are equivalent to vocalizations that occur following pheromone stimulation. Without these signals or in the absence of maternal mates, the sires do not retrieve their pups within 5 min. These results show that, in mice, the maternal parent communicates to the paternal parent to encourage pup care. This new paradigm may be useful in the analysis of the parental brain during paternal care induced by interactive communication., Parental responsibilities in mice are usually carried out by the mother of the pups. In this study, the authors show that when mothers are separated from their mouse pups, they emit ultrasonic vocalizations to their male partners, who respond by administering paternal care to the pups.

Induction of phase 2 detoxification enzymes by phenolic antioxidants can account for prevention of tumor initiation but cannot explain why these compounds inhibit tumor promotion. Phase 2 genes are induced through an antioxidant response element (ARE). Although the ARE resembles an AP-1 binding site, we show that the major ARE binding and activating protein is not AP-1. Interestingly, AP-1 DNA binding activity was induced by the phenolic antioxidant tert-butylhydroquinone (BHQ), but the induction of AP-1 transcriptional activity by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by this compound. BHQ induced expression of c-jun, junB, fra-1, and fra-2, which encode AP-1 components, but was a poor inducer of c-fos and had no effect on fosB. Like c-Fos and FosB, the Fra proteins heterodimerize with Jun proteins to form stable AP-1 complexes. However, Fra-containing AP-1 complexes have low transactivation potential. Furthermore, Fra-1 repressed AP-1 activity induced by either TPA or expression of c-Jun and c-Fos. We therefore conclude that inhibitory AP-1 complexes composed of Jun-Fra heterodimers, induced by BHQ, antagonize the transcriptional effects of the tumor promoter TPA, which are mediated by Jun-Fos heterodimers. Since AP-1 is an important mediator of tumor promoter action, these findings may explain the anti-tumor-promoting activity of phenolic antioxidants.

The assignment of the human prealbumin (PALB) gene to chromosome region 18q11-q12.1 has been achieved using a human genomic probe in the study of human-mouse somatic cell hybrids and by in situ hybridization. Because familial amyloidotic polyneuropathy was reported previously to be due to a mutation in prealbumin, it can be inferred that the gene for this disorder also maps to 18q11.2-q12.1.

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.