Biomedical subjects

Yoshiki Murakumo

Publications and source records attributed to Yoshiki Murakumo.

At least 19 recordsLinked to original sources

PubMed2006

Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase.

During development of the central and peripheral nervous systems, neurite extension mediated via glial-cell-line-derived neurotrophic factor (GDNF) and its receptor RET is critical for neuronal differentiation. In the present study, we investigated the role of the RET substrate Dok-4 in neurite outgrowth induced by the GDNF/RET signaling pathway. In TGW neuroblastoma cells, which endogenously express both RET and Dok-4, depletion of Dok-4 through treatment with small interfering RNA resulted in a marked decrease in GDNF-stimulated neurite outgrowth. By contrast, exogenous expression of wild-type Dok-4 induced sustained p44/42 mitogen-activated protein kinase (ERK1/2) activation and enhanced neurite outgrowth. Expression of Dok-4 mutants in which the tyrosine residues at codons 187, 220 and 270, conserved between Dok-4, -5, and -6, were each replaced with a phenylalanine inhibited sustained ERK1/2 activation and neurite outgrowth. We also found that Dok-4 induced a significant activation of the small G protein Rap1 and that expression of a dominant active Rap1 mutant restored neurite outgrowth in Dok-4-depleted cells. By contrast, expression of a dominant negative Rap1 mutant impaired GDNF-stimulated neurite outgrowth from TGW cells. Finally, we found that neurite formation in cultured rat hippocampal neurons was enhanced by the expression of Dok-4. Together, our results suggest that Dok-4, through activation of the Rap1-ERK1/2 pathway, regulates GDNF-mediated neurite outgrowth during neuronal development.

Adaptor Proteins, Signal Transducing↗

PubMed2006

RET and neuroendocrine tumors.

The RET proto-oncogene encodes a receptor tyrosine kinase that is a main component of the signaling pathway activated by the glial cell line-derived neurotrophic factor family ligands. Gene targeting studies revealed that signaling through RET plays a crucial role in neuronal and renal organogenesis. It is well-known that germline mutations in RET lead to the human inherited diseases, multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung's disease, and that somatic rearrangements of RET cause papillary thyroid carcinoma. Due to marked advances in understanding of the molecular mechanisms of the development of MEN 2, a consensus on MEN 2 management associated with RET status is being reached and currently put into general use as a guideline. In this review, we summarize progress in the study of RET from bench to bedside, focusing on pathophysiology of neuroendocrine tumors.

Adrenal Gland Neoplasms↗

PubMed2006

Analyses of ultraviolet-induced focus formation of hREV1 protein.

Translesional DNA synthesis (TLS) is one of the DNA damage tolerance mechanisms that allow cells with DNA damage to continue DNA replication. Each of the mammalian Y-family DNA polymerases (Pol eta, Pol iota, Pol kappa, and REV1) has been shown to carry out TLS by itself or in combination with another enzyme in vitro. Recently, the C-terminal region of mammalian REV1 (the total 1251 residues in human) was found to interact with Pol eta, Pol iota, and Pol kappa, as well as with the REV7 subunit of another TLS enzyme, Pol zeta. Thus, it is proposed that REV1 plays a pivotal role in TLS in vivo. We here describe our study on the localization of human REV1 protein (hREV1) in nondamaged and ultraviolet (UV)-irradiated cells. Ectopically expressed hREV1 in mammalian cells was localized to the nucleus and exhibited dozens of tiny foci in approximately 3% of nondamaged cells. The percentage of focus-forming cells markedly increased after UV irradiation in a time- and dose-dependent manner. The focus formation was associated with UV-induced DNA damage. Interestingly, although the hREV1 foci in S-phase cells colocalized with PCNA foci, suggesting the association of hREV1 with the replication machinery, hREV1 focus formation was observed not only in the S phase but also outside S phase. Furthermore, it was found that the hREV1 focus formation after UV irradiation required a region near the C-terminal (826-1178).

Animals↗

PubMed2006

RET receptor signaling: dysfunction in thyroid cancer and Hirschsprung's disease.

Gain-of-function mutations within the receptor tyrosine kinase gene RET cause inherited and non-inherited thyroid cancer. Somatic gene rearrangements of RET have been found in papillary thyroid carcinoma and germline point mutations in multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC). Conversely, loss-of-function mutations are responsible for the development of Hirschsprung's disease, a congenital malformation of the enteric nervous system. Comparison between normal RET signaling activated by the RET ligand glial cell line-derived neurotrophic factor (GDNF) and abnormal RET signaling caused by various mutations has led to a deeper understanding of disease mechanisms. The focus of the present review is on recent progress in the study of RET signaling dysfunction in human diseases.

Animals↗

PubMed2005

GDNF-inducible zinc finger protein 1 is a sequence-specific transcriptional repressor that binds to the HOXA10 gene regulatory region.

The RET tyrosine kinase receptor and its ligand, glial cell line-derived neurotrophic factor (GDNF) are critical regulators of renal and neural development. It has been demonstrated that RET activates a variety of downstream signaling cascades, including the RAS/mitogen-activated protein kinase and phosphatidylinositol-3-kinase(PI3-K)/AKT pathways. However, nuclear targets specific to RET-triggered signaling still remain elusive. We have previously identified a novel zinc finger protein, GZF1, whose expression is induced during GDNF/RET signaling and may play a role in renal branching morphogenesis. Here, we report the DNA binding property of GZF1 and its potential target gene. Using the cyclic amplification and selection of targets technique, the consensus DNA sequence to which GZF1 binds was determined. This sequence was found in the 5' regulatory region of the HOXA10 gene. Electrophoretic mobility shift assay revealed that GZF1 specifically binds to the determined consensus sequence and suppresses transcription of the luciferase gene from the HOXA10 gene regulatory element. These findings thus suggest that GZF1 may regulate the spatial and temporal expression of the HOXA10 gene which plays a role in morphogenesis.

Animals↗

PubMed2005

Akt/PKB regulates actin organization and cell motility via Girdin/APE.

The serine/threonine kinase Akt (also called protein kinase B) is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. Here, we identify an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 was replaced with alanine, formed abnormal elongated shapes and exhibited limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility.

Actins↗

PubMed2005

The RET proto-oncogene: a molecular therapeutic target in thyroid cancer.

The RET proto-oncogene is responsible for the development of several human inherited and non-inherited diseases. Germline point mutations were identified in multiple endocrine neoplasia types 2A and 2B, and familial medullary thyroid carcinoma. More than 10 rearranged forms of RET, referred to as RET/PTC 1-9, ELKS/RET and RFP/RET, have been cloned from sporadic and radiation-associated papillary thyroid carcinomas. These mutations induced oncogenic activation of RET tyrosine kinase by different mechanisms. To date, various kinds of therapeutic approaches have been developed for the treatment of RET-associated cancers, including tyrosine kinase inhibitors, gene therapy with dominant negative RET mutants, and RNA interference to abrogate oncogenic mutant RET expression. RET and some signaling molecules that function downstream of RET could be potential targets for the development of selective cancer therapeutics.

Antineoplastic Agents↗

PubMed2005

CD109 expression in squamous cell carcinoma of the uterine cervix.

CD109 is a cell surface protein, a member of the alpha(2) macroglobulin/C3,C4,C5 family of thioester-containing proteins. The authors have recently reported that high expression of the CD109 gene was detected in approximately half of the examined lung and esophageal squamous cell carcinomas as well as in the testis, and that CD109 has the characteristics of a cancer-testis antigen. In the present study CD109 expression in cervical squamous cell carcinoma was compared with that in endometrial adenocarcinoma by reverse transcription polymerase chain reaction (RT-PCR). The result demonstrated that CD109 expression is significantly higher in cervical squamous cell carcinomas than in endometrial adenocarcinomas and normal cervix and endometrium. In contrast, when expression of RET finger protein (RFP) and bromodomain testis-specific (BRDT) genes, which are also known to be highly expressed in the testis, was examined, no significant difference in their expression levels was observed between squamous cell carcinomas and adenocarcinomas. These findings suggest that CD109 may become a molecular target for the development of new therapeutics for squamous cell carcinoma of various tissue origins.

Adenocarcinoma↗

PubMed2005

Components of DNA damage checkpoint pathway regulate UV exposure-dependent alterations of gene expression of FHIT and WWOX at chromosome fragile sites.

Common chromosome fragile sites are highly recombinogenic and susceptible to deletions during the development of environmental carcinogen-induced epithelial tumors. Previous studies showed that not only genetic but also epigenetic alterations in cancerous cells are involved in inactivation of the genes FHIT and WWOX at chromosome fragile sites, reported to be potential tumor suppressor genes. Here we investigated the effect of UV light on the gene expression. After exposure to UV, the mRNA and protein of the two genes in murine embryonic fibroblasts (MEF) were unstable, apparently at the G1-S phase of the cell cycle, which was consistent with nuclear run-on assay. A study of MEFs synchronized via a double thymidine block indicated that, after the exposure, the expression of Fhit and Wwox was reduced in E2f-1-deficient cells and markedly in wild-type cells, whereas the reduction was partially inhibited in Trp53-deficient cells; cells at the S phase seemed to be sensitive to exogenous FHIT, suggesting a role of the checkpoint at the G1-S phase in the stability of gene expression and a possible involvement of FHIT function at the S phase. The transfection experiment showed that the UV-induced decrease in expression was partially inhibited by transfection of kinase-dead Atr (ataxia telangiectasia mutated and Rad3 related), which is a sensor of UV-induced damage. Taken together, the present study showed that UV-induced alterations of the fragile site gene expression are involved at least partially in the checkpoint function, suggesting the role in the process of carcinogenesis after exposure to UV.

Acid Anhydride Hydrolases↗

PubMed2004

Induction of CRMP-2 by GDNF and analysis of the CRMP-2 promoter region.

Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33 of Caenorhabditis elegans. Mutations of CRMP-2 result in abnormal axon termination. Recently, it was demonstrated that CRMP-2 binds to tubulin heterodimers to promote microtubule assembly that is critical for axonal differentiation and growth during development. Here we show that glial cell line-derived neurotrophic factor (GDNF) enhances CRMP-2 expression in TGW human neuroblastoma cells via activation of RET receptor tyrosine kinase. GDNF-mediated CRMP-2 expression was regulated mainly by the extracellular regulated kinase (ERK) pathway, but was independent of activation of phosphatidylinositol 3-kinase and Src family kinases. Analysis of the promoter region of the CRMP-2 gene revealed that the region 214-48 bp upstream of the transcriptional start site is important for CRMP-2 expression. The SP1, E2F, and GATA1/2 binding sites appeared to play some roles in regulation of CRMP-2 expression. As expected, the CRMP-2 protein accumulated in extended neurites of TGW cells treated with GDNF. However, neuritogenesis of TGW cells was mostly dependent on Src family kinase activity and not ERK activity, indicating that the increased expression of CRMP-2 alone was not sufficient for neuritogenesis.

Cell Line, Tumor↗

PubMed2004

Expression of CD109 in human cancer.

It was recently reported that the human CD109 gene encodes a glycosyl-phosphatidylinositol-anchored glycoprotein that is a member of the alpha(2)-macroglobulin/C3, C4, C5 family of thioester-containing proteins. In this study, we found that the expression of mouse CD109 gene was upregulated in NIH3T3 cells expressing RET tyrosine kinase with a multiple endocrine neoplasia 2B mutation. Northern blot analysis showed a high level of expression of the CD109 gene only in the testis in normal human and mouse tissues. In addition, its expression was high in some human tumor cell lines, which included squamous cell carcinoma and glioblastoma cell lines, whereas it was undetectable in neuroblastoma and small-cell lung carcinoma cell lines. When CD109 expression was examined in 33 cases of human lung cell carcinomas by quantitative RT-PCR, a significant high expression of CD109 was detected in about half of squamous cell carcinomas examined, but not in adenocarcinoma, large-cell carcinoma and small-cell carcinoma. Similarly, upregulation of CD109 was observed in nine out of 17 esophageal squamous cell carcinomas. Thus, these results suggested that CD109 might be a useful molecular target for the development of new therapeutics for malignant tumors, such as squamous cell carcinoma.

Amino Acid Sequence↗

PubMed2004

RET and neuroendocrine tumors.

Glial cell line-derived neurotrophic factor (GDNF), a ligand of RET tyrosine kinase, and its family ligands promote the survival and differentiation of a variety of neurons. Gene ablation studies have revealed that the GDNF-RET receptor system is essential for the development of kidney and peripheral neurons, including sympathetic, parasympathetic and enteric neurons. RET can activate various signaling pathways such as RAS/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/AKT, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. These signaling pathways are activated via binding of adaptor proteins to intracellular tyrosine residues of RET phosphorylated by its own kinase activity. The RET is profoundly involved in the development of several human neuroendocrine diseases. The constitutive activation of the RET by somatic rearrangement with other partner genes or germ-line mutations causes a considerable population of human papillary thyroid carcinomas or multiple endocrine neoplasia (MEN) type 2A and 2B, respectively, whereas the dysfunction of RET by germ-line missense and/or nonsense mutations causes Hirschsprung's disease. Biological properties of mutant RET protein determine the disease phenotype. For example, the MEN 2B mutation alters the substrate specificity of RET tyrosine kinase and RET carrying the MEN 2B mutation hereby induces the different set of genes from that carrying the MEN 2A mutation. In this review, we describe the current knowledge about the molecular mechanism of RET activation in human neuroendocrine tumors as well as the physiological roles and signal transduction of RET tyrosine kinase.

Animals↗

PubMed2004

Identification of a mouse cytoskeleton-associated protein, CKAP2, with microtubule-stabilizing properties.

Microtubule dynamics is an important factor in cell proliferation and one of the main targets of cancer chemotherapy. Since microtubule-associated proteins (MAPs) are known to influence microtubule stability, study of MAPs may contribute both to knowledge of cancer cell biology and to the production of new anti-cancer drugs. In this study, we identified a new mouse gene which is a homolog of human cytoskeleton-associated protein, CKAP2 gene, by differential display analysis. The level of expression of mouse CKAP2 (mCKAP2) was significantly higher in NIH3T3 cells expressing RET with a multiple endocrine neoplasia (MEN) 2A or MEN2B mutation than in parental NIH3T3 cells. Immunocytochemical analysis showed that mCKAP2 protein is localized in cytoplasm with a fibrillar appearance, and is co-localized with microtubules throughout the cell cycle. Furthermore, overexpression of mCKAP2 in cells appeared to stabilize microtubules against treatment with nocodazole, a microtubule-depolymerizing agent. In addition, levels of human CKAP2 were increased in some human tumor cell lines examined. These findings suggest that CKAP2 is a new MAP with microtubule-stabilizing properties and may represent a new molecular target for cancer chemotherapy.

Amino Acid Sequence↗

PubMed2004

Interaction of hREV1 with three human Y-family DNA polymerases.

Polkappa is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y-family of polymerases along with Poleta, Poliota and hREV1. Unlike Poleta encoded by the xeroderma pigmentosum variant (XPV) gene, Polkappa is unable to bypass UV-induced DNA damage in vitro, but it is able to bypass benzo[a]pyrene (B[a]P)-adducted guanines accurately and efficiently. In an attempt to identify factor(s) targeting Polkappa to its cognate DNA lesion(s), we searched for Polkappa-interacting proteins by using the yeast two-hybrid assay. We found that Polkappa interacts with a C-terminal region of hREV1. Poleta and Poliota were also found to interact with the same region of hREV1. The interaction between Polkappa and hREV1 was confirmed by pull-down and co-immunoprecipitation assays. The C-terminal region of hREV1 is known to interact with hREV7, a non-catalytic subunit of Polzeta that is another structurally unrelated TLS enzyme, and we show that Polkappa and hREV7 bind to the same C-terminal region of hREV1. Thus, our results suggest that hREV1 plays a pivotal role in the multi-enzyme, multi-step process of translesion DNA synthesis.

Amino Acid Sequence↗

PubMed2004

A targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia.

The Ret receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in Ret represents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, phosphatidylinositol 3-kinase/AKT, and Jun-associated N-terminal kinase pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice in which tyrosine 1062 in Ret was replaced with phenylalanine were generated. Although homozygous knock-in mice were born normally, they died by day 27 after birth and showed growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract, as observed in Ret-deficient mice. The rest of the mutant mice developed enteric neurons in the intestine to various extents, although the size and number of ganglion cells were significantly reduced. Unlike Ret-deficient mice, a small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to a decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.

Abnormalities, Multiple↗

PubMed2003

Identification of a novel glial cell line-derived neurotrophic factor-inducible gene required for renal branching morphogenesis.

In the developing kidney, activation of the rearrangement during transfection tyrosine kinase by glial cell line-derived neurotrophic factor (GDNF) is required for normal branching of the ureteric bud epithelium [corrected]. By differential display analysis we identified a novel GDNF-inducible gene (named GZF1) with a BTB/POZ (broad complex, tramtrack, and bric-a-brac)/(poxvirus and zinc finger) domain and 10 tandemly repeated zinc finger motifs. The up-regulation of the GZF1 gene showed two peaks at 1 h and 24-48 h after GDNF stimulation by Northern blotting. The late induction was also found at protein levels by Western blotting with anti-GZF1 antibody. As observed for other proteins with the BTB/POZ domain, the GZF1 protein had strong transcriptional repressive activity. Intriguingly, its expression was detected at high levels in branching ureteric buds and collecting ducts of mouse metanephric kidney in which RET was also expressed. Antisense phosphorothioated oligodeoxynucleotides of the GZF1 gene markedly impaired the ureteric bud branching in the metanephric organ culture, suggesting that the induction of GZF1 expression via the GDNF/RET signaling system is required for renal branching morphogenesis.

Amino Acid Sequence↗

PubMed2003

Cys611Ser mutation in RET proto-oncogene in a kindred with medullary thyroid carcinoma and Hirschsprung's disease.

Germline mutations in the RET proto-oncogene are responsible for the development of human hereditary diseases, including multiple endocrine neoplasia (MEN) type 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease (HSCR). It has been reported that some families developed both MEN 2A/FMTC and HSCR, in which a mutation in a cysteine residue at codon 609, 618, or 620 in the RET gene was present. Here we report a novel RET mutation detected in a Japanese family with medullary thyroid carcinoma and HSCR. A germline mutation in cysteine 611 of the RET gene was identified in this family, which introduced an amino-acid change from cysteine to serine. By biological and biochemical analyses of mutant RET proteins, we previously predicted the potentiality that amino-acid substitution for cysteine 611 as well as cysteines 609, 618, and 620 would promote the development of MEN 2A/FMTC and HSCR. This clinical case substantiates our suggestion for the mechanism of the development of both the diseases.

Amino Acid Substitution↗

PubMed2003

Establishment and characterization of mouse mammary carcinoma cell lines expressing RET with a multiple endocrine neoplasia 2A mutation.

We recently generated transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation (RET-MEN2A). Mammary tumors with frequent lung metastasis were developed in 22% of female transgenic mice in a stochastic fashion. In the current study, we established two cell lines (named MKK-f and MKK-s) from mammary tumors developed in RET-MEN2A transgenic mice. MKK-f and MKK-s were derived from well-differentiated ductal carcinoma and sarcomatous spindle cell carcinoma, respectively. MKK-f cells show epithelial-like morphology with a doubling time of 19 h, and MKK-s cells show spindle-shaped morphology with a doubling time of 15 h. When inoculated in immunodeficient mice, both cell lines were tumorigenic, metastasized to the lung and displayed histological features similar to those of the primary tumors. They maintained a high level of RET expression and activation of signaling molecules downstream of RET. Consistent with the histological phenotype, expression of E-cadherin was almost undetectable in MKK-s cells, whereas its expression was very high in MKK-f cells. When the difference of gene expression between the two cell lines was analyzed using cDNA microarrays including approximately 900 genes/ESTs, a total of 21 up- or down-regulated (> 2.0-fold) genes were identified. Differentially regulated genes included thymosin beta-10, fibroblast growth factor receptor 4, aldo-keto reductase and caspase 6 genes, which are known to be associated with tumor development and progression. These results may reflect the profiles of the transcriptional changes associated with dedifferentiation or progression of mammary carcinomas developed in genetically engineered mice.

Alcohol Oxidoreductases↗