Hiroyuki Misawa

A new protein containing an SH2 domain that inhibits JAK kinases

The proliferation and differentiation of cells of many lineages are regulated by secreted proteins known as cytokines. Cytokines exert their biological effect through binding to cell-surface receptors that are associated with one or more members of the JAK family of cytoplasmic tyrosine kinases. Cytokine-induced receptor dimerization leads to the activation of JAKs, rapid tyrosine-phosphorylation of the cytoplasmic domains, and subsequent recruitment of various signalling proteins, including members of the STAT family of transcription factors, to the receptor complex. Using the yeast two-hybrid system, we have now isolated a new SH2-domain-containing protein, JAB, which is a JAK-binding protein that interacts with the Jak2 tyrosine-kinase JH1 domain. JAB is structurally related to CIS, a cytokine-inducible SH2 protein,. Interaction of JAB with Jak1, Jak2 or Jak3 markedly reduces their tyrosine-kinase activity and suppresses the tyrosine-phosphorylation and activation of STATs. JAB and CIS appear to function as negative regulators in the JAK signalling pathway.

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK‐binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N‐terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB‐related proteins. An additional N‐terminal 12‐amino‐acid region (kinase inhibitory region) of JAB also contributes to high‐affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.

STAT5 Activation Correlates with Erythropoietin Receptor-mediated Erythroid Differentiation of an Erythroleukemia Cell Line

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.

Physiology and function of the erythropoietin receptor.

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. Targeted disruption of the EPO receptor (EPOR) gene have clearly demonstrated the importance of the EPO/EPOR system for definitive erythroid cell survival and proliferation; however, in vitro rescue experiments have revealed that it is not essential for differentiation. The three-dimensional structure of the EPOR has been determined, and a biologically active 20 amino acid peptide has been shown to cause dimerization of the extracellular domain of EPOR. EPO activates the JAK2-STAT5 pathway, and two tyrosine residues (Y343, Y401) in the cytoplasmic domain of EPOR are important for STAT5 activation. However, the physiologic role of STAT5 in erythroid cell proliferation and differentiation is still controversial. Mutations that result in C-terminal deletion of EPOR are frequently found in familial erythrocytosis. Hematopoietic cell phosphatase, which binds to the C-terminal region, could be involved in negative regulation of EPOR function.

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4‐II‐E cells overexpressing RC stably. H4‐II‐E cells were transfected with RC/pCXN2 vector and the multiple neomycin‐resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2‐transfected cells used in this study was 19.7‐fold as compared with that of the parental wild type H4‐II‐E cells. Wild type H4‐II‐E cells, pCXN2 vector‐transfected cells (mock type), and RC/pCXN2‐transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4‐II‐E cells was significantly suppressed in transfectants with culture for 12–48 h. The presence of anti‐RC monoclonal antibody (10–50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti‐RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 μM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4‐II‐E overexpressing RC stably. J. Cell. Biochem. 84: 143–149, 2002.

Identification of transcription factor in the promoter region of rat regucalcin gene: Binding of nuclear factor I‐A1 to TTGGC motif

Hepatic nuclear protein has been reported to bind specifically to the TTGGC sequence of the rat regucalcin gene promoter region in stimulating the promoter activity (Misawa and Yamaguchi [ 2000 ] Biochem. Biophys. Res. Commun. 279: 275–281). The present study was undertaken to identify transcription factor, which binds to TTGGC motif in the rat regucalcin gene promoter, using the yeast one‐hybrid system. The sequence between −525 and −504, which has been defined as a functional promoter element II‐b, was used as bait to screen a rat liver cDNA library. Two cDNA clones were identified as a nuclear factor I‐A1 (NF1‐A1). The results of gel mobility shift assay and mutation analysis using recombinant NF1‐A1 protein showed that this protein could specifically bind to TTGGC motif of the II‐b oligonucleotide in promoter region. The expression of NF1‐A1 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. This study demonstrates that NF1‐A1 is a transcription factor in stimulating the rat regucalcin gene promoter activity. J. Cell. Biochem. 84: 795–802, 2002.

Role of endogenous regucalcin in transgenic rats: Suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+‐ATPase activity

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5‐week‐old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild‐type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+‐ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+‐ATPase activity was significantly enhanced in TG rats. The changes in their enzymes activities in TG rats were completely abolished in the presence of anti‐regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo. J. Cell. Biochem. 86: 520–529, 2002.

Potential role of regucalcin as a specific biochemical marker of chronic liver injury with carbon tetrachloride administration in rats

The potential sensitivity of liver specific protein regucalcin as a biochemical marker of chronic liver injury with carbon tetrachloride (CCl4) administration in rats was investigated. CCl4 (10%; 1.0 ml/100 g body wt) was orally given 5 times at 3-day intervals to rats, and the animals were killed by bleeding at 3, 6, 18, and 30 days after the first administration of CCl4. The body weight of rats was significantly lowered 3 and 6 days after CCl4 administration as compared with that of control rats administered with corn oil, and then the weight was restored at 18 and 30 days. Serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities were significantly increased 3 days after the administration, while a significant increase in serum γ-glutamyltranspeptidase (γ-GTP) activity was seen at 3 and 6 days after the administration. Serum GOT, GPT, and γ-GTP activities were restored to control levels at 18 and 30 days after CCl4 administration. Serum albumin, α-fetoprotein, and ammonium levels were not changed by CCl4 administration. Meanwhile, serum regucalcin concentration was markedly increased 3 and 6 days after CCl4 administration, and a significant increase in serum regucalcin concentration was observed 18 and 30 days after the administration. Liver regucalcin mRNA and liver cytosolic regucalcin levels were significantly decreased 18 and 30 days after CCl4 administration. Liver content of calcium, which intracellular calcium homeostasis is maintained, was significantly increased between 3 and 30 days after CCl4 administration. Hepatic mitochondrial succinate dehydrogenase activity was significantly increased 30 days after the administration. The present study demonstrates that serum regucalcin has a potential sensitivity as a specific biochemical marker of chronic liver injury with CCl4 administration in rats.

Expression of Ca2+-binding protein regucalcin in rat brain neurons: inhibitory effect on protein phosphatase activity

The expression of Ca(2+)-binding protein regucalcin and its role in the regulation of protein phosphatase activity in rat brain neuronal cells obtained with primary culture was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain neuronal cells using rat regucalcin-specific primers. Moreover, regucalcin protein in brain neuronal cells was detected by Western blot analysis using a polyclonal rabbit anti-regucalcin antibody. The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward phosphotyrosine, phosphoserine and phosphothreonine in the reaction mixture containing the cytosol of neuronal cell homogenates. This increase was completely prevented by the addition of regucalcin (10(-8) M). Protein phosphatase activity toward three phosphoaminoacids was significantly elevated by the addition of Ca(2+) (100 microM) and calmodulin (5 microg/ml). This elevation was completely blocked by the addition of regucalcin (10(-8) M). The present study demonstrates that regucalcin is expressed in rat brain neuronal cells, and that it has an inhibitory effect on protein phosphatase activity in the cells.

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral‐diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [ 2002 ] Int. J. Mol. Med. 9:503–508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral‐diaphyseal tissues with fracture healing were cultured for 48 h in a serum‐free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N‐terminal sequencing of 66 kDa protein indicated that its N‐terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral‐diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum‐free medium containing either vehicle, parathyroid hormone (1–34) (10−7 M), insulin‐like growth factor‐I (10−8 M) or zinc acexamate (10−4 M), medium albumin was significantly increased in the presence of those bone‐stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral‐diaphyseal and ‐metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral‐diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components. J. Cell. Biochem. 89: 356–363, 2003.