Yoshiki Murakumo

Interaction of hREV1 with three human Y-family DNA polymerases

Polκ is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y‐family of polymerases along with Polη, Polι and hREV1. Unlike Polη encoded by the xeroderma pigmentosum variant (XPV) gene, Polκ 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 Polκ to its cognate DNA lesion(s), we searched for Polκ‐interacting proteins by using the yeast two‐hybrid assay. We found that Polκ interacts with a C‐terminal region of hREV1. Polη and Polι were also found to interact with the same region of hREV1. The interaction between Polκ 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 Polζ that is another structurally unrelated TLS enzyme, and we show that Polκ 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.

Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor.

Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor α (GFRα). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-κB in GDNF-treated cells, respectively.

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.

Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus.

Disrupted-In-Schizophrenia 1 (DISC1), a susceptibility gene for major psychiatric disorders, regulates neuronal migration and differentiation during mammalian brain development. Although roles for DISC1 in postnatal neurogenesis in the dentate gyrus (DG) have recently emerged, it is not known how DISC1 and its interacting proteins govern the migration, positioning, and differentiation of dentate granule cells (DGCs). Here, we report that DISC1 interacts with the actin-binding protein girdin to regulate axonal development. DGCs in girdin-deficient neonatal mice exhibit deficits in axonal sprouting in the cornu ammonis 3 region of the hippocampus. Girdin deficiency, RNA interference-mediated knockdown, and inhibition of the DISC1/girdin interaction lead to overextended migration and mispositioning of the DGCs resulting in profound cytoarchitectural disorganization of the DG. These findings identify girdin as an intrinsic factor in postnatal development of the DG and provide insights into the critical role of the DISC1/girdin interaction in postnatal neurogenesis in the DG.

An Actin-Binding Protein Girdin Regulates the Motility of Breast Cancer Cells

Girdin (girders of actin filaments) is a novel actin-binding Akt substrate that plays an important role in actin organization and Akt-dependent cell motility in fibroblasts. Here, we find that Girdin is expressed in a variety of cancer cell lines, including the breast cancer cell line MDA-MB-231, and is phosphorylated by the stimulation of insulin-like growth factor (IGF-I). In vitro migration and invasion assays showed that Girdin is required for the IGF-I-dependent cell movement of MDA-MB-231 cells. Short hairpin interfering RNA directed against Girdin markedly inhibited the metastasis of s.c. transplanted MDA-MB-231 cells in nude mice. In addition, Girdin is highly expressed in a variety of human malignant tissues, including breast, colon, lung, and uterine cervical carcinomas. These findings highlight the important role of Girdin in tumor progression in which the Akt signaling pathway is aberrantly activated.

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. (Cancer Sci 2005; 96: 143–148)

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

ABSTRACT 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.

FEZ1/LZTS1 gene at 8p22 suppresses cancer cell growth and regulates mitosis

The FEZ1/LZTS1 gene maps to chromosome 8p22, a region that is frequently deleted in human tumors. Alterations in FEZ1/LZTS1 expression have been observed in esophageal, breast, and prostate cancers. Here, we show that introduction of FEZ1/LZTS1 into Fez1/Lzts1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S–G2/M stage of the cell cycle. Fez1/Lzts1 protein is hyperphosphorylated by cAMP-dependent kinase during cell-cycle progression. We found that Fez1/Lzts1 is associated with microtubule components and interacts with p34cdc2 at late S–G2/M stage in vivo. Present data show that FEZ1/LZTS1 inhibits cancer cell growth through regulation of mitosis, and that its alterations result in abnormal cell growth.

Characterization of Gene Expression Induced by RET with MEN2A or MEN2B Mutation

Germ-line point mutations of the RET gene are responsible for multiple endocrine neoplasia (MEN) type 2A and 2B that develop medullary thyroid carcinoma and pheochromocytoma. We performed a differential display analysis of gene expression using NIH 3T3 cells expressing the RET-MEN2A or RET-MEN2B mutant proteins. As a consequence, we identified 10 genes induced by both mutant proteins and eight genes repressed by them. The inducible genes include cyclin D1, cathepsins B and L, and cofilin genes that are known to be involved in cell growth, tumor progression, and invasion. In contrast, the repressed genes include type I collagen, lysyl oxidase, annexin I, and tissue inhibitor of matrix metalloproteinase 3 (TIMP3) genes that have been implicated in tumor suppression. In addition, six RET-MEN2A- and five RET-MEN2B-inducible genes were identified. Among 21 genes induced by RET-MEN2A and/or RET-MEN2B, six genes including cyclin D1, cathepsin B, cofilin, ring finger protein 11 (RNF11), integrin-alpha6, and stanniocalcin 1 (STC1) genes were also induced in TGW human neuroblastoma cells in response to glial cell line-derived neurotrophic factor stimulation. Because the STC1 gene was found to be highly induced by both RET-MEN2B and glial cell line-derived neurotrophic factor stimulation, and the expression of its product was detected in medullary thyroid carcinoma with the MEN2B mutation by immunohistochemistry, this may suggest a possible role for STC1 in the development of MEN 2B phenotype.

Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities

We developed a simple algorithm, i-Score (inhibitory-Score), to predict active siRNAs by applying a linear regression model to 2431 siRNAs. Our algorithm is exclusively comprised of nucleotide (nt) preferences at each position, and no other parameters are taken into account. Using a validation dataset comprised of 419 siRNAs, we found that the prediction accuracy of i-Score is as good as those of s-Biopredsi, ThermoComposition21 and DSIR, which employ a neural network model or more parameters in a linear regression model. Reynolds and Katoh also predict active siRNAs efficiently, but the numbers of siRNAs predicted to be active are less than one-eighth of that of i-Score. We additionally found that exclusion of thermostable siRNAs, whose whole stacking energy (ΔG) is less than −34.6 kcal/mol, improves the prediction accuracy in i-Score, s-Biopredsi, ThermoComposition21 and DSIR. We also developed a universal target vector, pSELL, with which we can assay an siRNA activity of any sequence in either the sense or antisense direction. We assayed 86 siRNAs in HEK293 cells using pSELL, and validated applicability of i-Score and the whole ΔG value in designing siRNAs.