Takahiro Kamimoto

Intracellular Inclusions Containing Mutant α1-Antitrypsin Z Are Propagated in the Absence of Autophagic Activity

Mutant α1-antitrypsin Z (α1-ATZ) protein, which has a tendency to form aggregated polymers as it accumulates within the endoplasmic reticulum of the liver cells, is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary α1-antitrypsin (α1-AT) deficiency. Previous studies have suggested that efficient intracellular degradation of α1-ATZ is correlated with protection from liver disease in α1-AT deficiency and that the ubiquitin-proteasome system accounts for a major route, but not the sole route, of α1-ATZ disposal. Yet another intracellular degradation system, autophagy, has also been implicated in the pathophysiology of α1-AT deficiency. To provide genetic evidence for autophagy-mediated disposal of α1-ATZ, here we used cell lines deleted for the Atg5 gene that is necessary for initiation of autophagy. In the absence of autophagy, the degradation of α1-ATZ was retarded, and the characteristic cellular inclusions of α1-ATZ accumulated. In wild-type cells, colocalization of the autophagosomal membrane marker GFP-LC3 and α1-ATZ was observed, and this colocalization was enhanced when clearance of autophagosomes was prevented by inhibiting fusion between autophagosome and lysosome. By using a transgenic mouse with liver-specific inducible expression of α1-ATZ mated to the GFP-LC3 mouse, we also found that expression of α1-ATZ in the liver in vivo is sufficient to induce autophagy. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for α1-ATZ and suggest that autophagic degradation plays a fundamental role in preventing toxic accumulation of α1-ATZ.

TNFalpha-induced ATF3 expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.

ATF3 (Activating transcription factor 3), a member of the CREB/ATF family, can be induced by stress and growth factors in mammalian cells, and is thought to play an important role in the cardiovascular system. However, little is currently known about how the induction of ATF3 is regulated, except that the JNK pathway is involved. Here, we investigated the differential roles of the MAPK pathways involved in TNFα (tumour necrosis factor α)‐induced ATF3 expression in vascular endothelial cells. In human umbilical vein endothelial cells, the expression of constitutively active MKK7 (MAPK kinase 7) increased the number of ATF3‐positive cells, and dominant negative MKK7 suppressed the TNFα‐induced expression of ATF3, indicating a requirement for the JNK pathway. In contrast, the expression of constitutively active or dominant negative MEK1/2 (MAPK/ERK kinase 1/2) suppressed or enhanced TNFα‐mediated induction of ATF3, respectively. In support of this, the MEK1/2 specific inhibitor U0126 enhanced the expression of ATF3 induced by TNFα. Furthermore, the ERK pathway inhibits the TNFα‐mediated induction of ATF3 mRNA, but not its stability, suggesting the involvement of ERK activity in the transcriptional regulation of the ATF3 gene. Our results suggest that TNFα‐induced ATF3 gene expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.

Dymple, a Novel Dynamin-like High Molecular Weight GTPase Lacking a Proline-rich Carboxyl-terminal Domain in Mammalian Cells

We have cloned human dymple, a novel dynamin family member. The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa. This amino acid sequence most resembles yeast DNM1P and VPS1P. Dymple lacks a proline-rich carboxyl-terminal domain through which dynamin binds to SH3 domains to be activated. Northern blot analysis revealed two transcript sizes of 2.5 and 4.2 kilobases with alternative polyadenylation at the highest levels in brain, skeletal muscle, and testis. It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner. When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, whereas an amino-terminal deletion mutant formed large aggregates bounded by a trans-Golgi network marker. Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecule in each specific vesicle transport pathway has been predicted. Our findings suggest that dymple may be the first example of such a subfamily in mammalian cells other than dynamin itself, although its precise role and membrane localization remain to be resolved.

Scaffold role of a mitogen-activated protein kinase phosphatase, SKRP1, for the JNK signaling pathway.

Stress-activated protein kinase (SAPK) pathway-regulating phosphatase 1 (SKRP1) has been identified as a member of the mitogen-activated protein kinase (MAPK) phosphatase (MKP) family that interacts physically with the MAPK kinase (MAPKK) MKK7, a c-Jun N-terminal kinase (JNK) activator, and inactivates the MAPK JNK pathway. Although these findings indicated that SKRP1 contributes to the precise regulation of JNK signaling, it remains to be elucidated how SKRP1 is integrated into this pathway. We report that SKRP1 also plays a scaffold role for the JNK signaling, judged by the following observations. SKRP1 selectively formed the stable complexes with MKK7 but not with MKK4 and biphasically regulated the MKK7 activity and MKK7-induced gene transcription in vivo. Co-precipitation analysis between SKRP1 and MKK7-activating MAPKK kinases (MAPKKKs) revealed that SKRP1 also interacted with the MAPKKK, apoptosis signal-regulating kinase 1 (ASK1), but not with MAP kinase kinase kinase 1 (MEKK1). Consistent with these findings, SKRP1 expression increased the ASK1-MKK7 complexes in a dose-dependent manner and specifically enhanced the activation of MKK7 by ASK1. Thus, our findings are, to our knowledge, the first evidence to show that an MKP also functions as a scaffold protein for the particular MAPK signaling.

A Novel Dual Specificity Phosphatase SKRP1 Interacts with the MAPK Kinase MKK7 and Inactivates the JNK MAPK Pathway IMPLICATION FOR THE PRECISE REGULATION OF THE PARTICULAR MAPK PATHWAY

Mitogen-activated protein kinases (MAPKs) are activated in response to various extracellular stimuli, and their activities are regulated by upstream activating kinases and protein phosphatases such as MAPK phosphatases (MKPs). We report the identification and characterization of a novel MKP termed SKRP1 (SAPK pathway-regulatingphosphatase 1). It contains an extended active site sequence motif conserved in all MKPs but lacks a Cdc25 homology domain. Immunoblotting analysis revealed that SKRP1 is constitutively expressed, and its transcripts of 4.0 and 1.0 kb were detected in almost tissues examined. SKRP1 was highly specific for c-Jun N-terminal kinase (JNK) in vitro and effectively suppressed the JNK activation in response to tumor necrosis factor α or thapsigargin. Endogenous SKRP1 was present predominantly in the cytoplasm and co-localized with JNK. However, SKRP1 does not bind directly to its target JNK, but co-precipitation of SKRP1 with the MAPK kinase MKK7, a JNK activator, was found in vitro and in vivo. Furthermore, we found that SKRP1 did not interfere with the co-precipitation of MKK7 with JNK. Together, our findings indicate that SKRP1 interacts with its physiological substrate JNK through MKK7, thereby leading to the precise regulation of JNK activity in vivo.

EXPRESSION CLONING AND INTRACELLULAR LOCALIZATION OF A HUMAN ZF5 HOMOLOGUE

We isolated a cDNA encoding a human homologue of ZF5 (hZF5), which has five Kruppel-like C2H2 type zinc fingers at carboxyl terminus and the BTB/POZ (poxvirus and zinc finger) at the amino terminus, using autoimmune sera from a patient with overlap syndrome (dermatomyositis and scleroderma). Sequencing of the entire cDNA revealed an open reading frame (ORF) of 1349 bp with a deduced protein sequence of 449 amino acid residues and a calculated molecular weight of 51.3 kDa. The deduced amino acid sequence of hZF5 is highly homologous to mouse ZF5 (99.3% identity). Immunofluorescence studies revealed that HA-tagged hZF5 transiently expressed in COS-7 cells showed the nuclear dot pattern in the BTB/POZ domain-dependent manner.

Anti-mitosin antibodies in a patient with chronic graft-versus-host disease after allogeneic bone marrow transplantation.

In a patient undergoing allogeneic BMT for ALL, chronic GVHD (cGVHD) with skin changes developed within 110 days after transplantation. One year post-BMT, anti-nuclear antibodies were detected. The patient’s serum was used for immunoscreening of a HeLa cDNA library. Ten different overlapping positive clones were found to be partial clones of mitosin, a 350-kDa nuclear phosphoprotein which shows a speckled nuclear distribution in S phase and which relocates to the centromere and mitotic apparatus in M phase. Although autoantibodies against centromere protein-F, which is very similar to mitosin, have been reported in patients with cancer, this is the first report of autoantibodies against mitosin in a patient with cGVHD.

Spectrum of autoantibodies against a dynamin-related protein, dymple.

OBJECTIVE To characterize the clinical features of patients with autoantibodies against dymple, a dynamin-related protein. METHODS Serum samples from 281 patients with rheumatic diseases were examined. The characteristics of antidymple and antibody-reactive determinants were investigated by immunoblotting with the recombinant dymple antigen, including its deletion mutants, and by immunofluorescence studies with affinity-purified serum. RESULTS Five serum samples (2%) were found to have antidymple. All of these patients were male, and 4 of them had interstitial pneumonitis. Their sera were considered to mainly recognize the N-terminus of dymple, which contains GTP-binding motifs. CONCLUSION Dymple, which joins the cytoplasmic autoantigens, could be a marker for a newly recognized subset of connective tissue diseases.