👤 Meng-Shiou Lee

Using a genome-wide array-based comparative genomic hybridization (array-CGH), DNA copy number changes in uterine leiomyosarcoma were analyzed. We analyzed 4 cases of uterine leiomyoma and 7 cases of uterine leiomyosarcoma. The paraffin-fixed tissue samples were microdissected under microscope and DNA was extracted. Array-based CGH and fluorescence in situ hybridization (FISH) were carried out with Genome database (Gene Ontology). Uterine leiomyoma showed no genetic alterations, while all of 7 cases of uterine leiomyosarcoma showed specific gains and losses. The percentage of average gains and losses were 4.86% and 15.1%, respectively. The regions of high level of gain were 7q36.3, 7q33-q35, 12q13-12q15, and 12q23.3. And the regions of homozygous loss were 1p21.1, 2p22.2, 6p11.2, 9p21.1, 9p21.3, 9p22.1, 14q32.33, and 14q32.33 qter. There were no recurrent regions of gain, but recurrent regions of loss were 1p21.1-p21.2, 1p22.3-p31.1, 9p21.2-p22.2, 10q25-q25.2, 11q24.2-q25, 13q12-q12.13, 14q31.1-q31.3, 14q32.32-q32.33, 15q11-q12, 15q13-q14, 18q12.1-q12.2, 18q22.1-q22.3, 20p12.1, and 21q22.12-q22.13. In the high level of gain regions, BAC clones encoded HMGIC, SAS, MDM2, TIM1 genes. Frequently gained BAC clone-encoded genes were TIM1, PDGFR-beta, REC Q4, VAV2, FGF4, KLK2, PNUTL1, GDNF, FLG, EXT1, WISP1, HER-2, and SOX18. The genes encoded by frequently lost BAC clones were LEU1, ERCC5, THBS1, DCC, MBD2, SCCA1, FVT1, CYB5, and ETS2/E2. A subset of cellular processes from each gene was clustered by Gene Ontology database. Using array-CGH, chromosomal aberrations related to uterine leiomyosarcoma were identified. The high resolution of array-CGH combined with human genome database would give a chance to find out possible target genes present in the gained or lost clones. Show less

The Delta-Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for cell fate specification. Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. We now report that mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulated in the plasma membrane, while it was localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling. Show less

The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination. Show less

Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axonal regeneration following injury. The Nogo receptor complex, composed of the Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75), and LINGO-1, represses axon regeneration upon binding to these myelin components. The limited expression of p75 to certain types of neurons and its temporal expression during development prompted speculation that other receptors are involved in the NgR1 complex. Here, we show that an orphan receptor in the TNF family called TAJ, broadly expressed in postnatal and adult neurons, binds to NgR1 and can replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA in the presence of myelin inhibitors. In vitro exogenously added TAJ reversed neurite outgrowth caused by MAIFs. Neurons from Taj-deficient mice were more resistant to the suppressive action of the myelin inhibitors. Given the limited expression of p75, the discovery of TAJ function is an important step for understanding the regulation of axonal regeneration. Show less

Whole genome association studies of complex human diseases represent a new paradigm in the postgenomic era. In this study, we report application of the Affymetrix, Inc. (Santa Clara, CA) high-density single nucleotide polymorphism (SNP) array containing 11,555 SNPs in a pilot case-control study of esophageal squamous cell carcinoma (ESCC) that included the analysis of germ line samples from 50 ESCC patients and 50 matched controls. The average genotyping call rate for the 100 samples analyzed was 96%. Using the generalized linear model (GLM) with adjustment for potential confounders and multiple comparisons, we identified 37 SNPs associated with disease, assuming a recessive mode of transmission; similarly, 48 SNPs were identified assuming a dominant mode and 53 SNPs in a continuous mode. When the 37 SNPs identified from the GLM recessive mode were used in a principal components analysis, the first principal component correctly predicted 46 of 50 cases and 47 of 50 controls. Among all the SNPs selected from GLMs for the three modes of transmission, 39 could be mapped to 1 of 33 genes. Many of these genes are involved in various cancers, including GASC1, shown previously to be amplified in ESCCs, and EPHB1 and PIK3C3. In conclusion, we have shown the feasibility of the Affymetrix 10K SNP array in genome-wide association studies of common cancers and identified new candidate loci to study in ESCC. Show less

Apolipoprotein A5 plays an important role in modulating triacylglycerol metabolism in experimental animal models. The objective was to determine associations of the common apolipoprotein A5 gene (APOA5) -1131T-->C polymorphism with postprandial lipemic response and other cardiovascular disease risk factors in humans. Healthy, nonobese subjects [n = 158; mean (+/-SEM) age: 33.8 +/- 1.2 y; body mass index (in kg/m(2)): 23.3 +/- 0.3] were subdivided into 3 genotype groups: TT (n = 85), TC (n = 56), and CC (n = 17). We measured fasting and postprandial lipid concentrations, lipid peroxidation, C-reactive protein concentrations, and DNA damage. Fasting triacylglycerol concentrations in carriers of the C allele were higher (P < 0.05) than in carriers of the TT genotype. No other significant genotype-related differences were observed for any of the other baseline measures. After consumption of a mixed meal, carriers of the C allele had significantly greater increases in total chylomicron and VLDL triacylglycerol than did subjects with the TT genotype. Moreover, carriers of the C allele had higher dense LDL, serum C-reactive protein, and urinary 8-epi-prostaglandin F(2alpha) concentrations and more lymphocyte DNA damage. Conversely, we did not find significant genotype-related differences in postprandial glucose, insulin, or free fatty acid measures. Our data confirm the genetic modulation of serum fasting triacylglycerol concentrations by the APOA5 gene polymorphism and extend this observation to postprandial triacylglycerol concentrations and to markers of oxidation and inflammation. The presence of the C allele in the APOA5 promoter region at position 1131 could be a significant factor contributing to higher cardiovascular disease risk in Koreans independently of common environmental factors. Show less

Variation in the APOA5 gene has been shown to be associated with triglyceride levels in several independent population studies. It was our objective to determine if a relationship existed between selected genotypes or haplotypes of the APOA5 gene and findings on selective coronary angiography (SCA) in an independent cohort. The Vancouver SCA Cohort consists of individuals referred for angiography between 1993 and 1995. DNA was extracted from 537 patients and analyzed for the -1131T>C and the c.56C>G polymorphisms which define three common haplotypes of the APOA5 gene. Plasma triglycerides and the fractional esterification rate in apoB-depleted lipoproteins (FER(HDL)), an index of high-density lipoprotein (HDL) composition, were significantly higher (P = 0.01 and P = 0.001, respectively), and HDL cholesterol (HDL-C) was significantly lower (P = 0.03) in Caucasians with genotypes containing the minor allele of the -1131T>C polymorphism compared to the homozygotes for the major allele. However, there was no relationship between the c.56C>G polymorphism of the APOA5 gene and any of the measured lipid and lipoprotein parameters. Subjects homozygous for the common haplotype APOA5*1 had decreased triglyceride levels and FER(HDL) (P = 0.04 and P < 0.001, respectively) and increased HDL-C levels (P = 0.01) compared to subjects with all other haplogenotypes. Multivariate linear regression analysis indicated that the -1131T>C polymorphism remained an independent predictor of triglyceride, HDL-C, and FER(HDL) following adjustment of several variables including age, gender, body mass index, diabetes, lipid lowering and beta-blocker medication. The APOA5*1/*1 haplogenotype remained an independent predictor of HDL-C and FER(HDL) following adjustment of the same variables. The relationship between APOA5 genotype or haplogenotype and FER(HDL) remained significant even after the addition of both HDL-C and triglyceride to the model. However, there was no association between APOA5 gene polymorphisms or haplotypes and coronary artery disease as determined by angiography. Show less

Axon regeneration in the adult CNS is prevented by inhibitors in myelin. These inhibitors seem to modulate RhoA activity by binding to a receptor complex comprising a ligand-binding subunit (the Nogo-66 receptor NgR1) and a signal transducing subunit (the neurotrophin receptor p75). However, in reconstituted non-neuronal systems, NgR1 and p75 together are unable to activate RhoA, suggesting that additional components of the receptor may exist. Here we describe LINGO-1, a nervous system-specific transmembrane protein that binds NgR1 and p75 and that is an additional functional component of the NgR1/p75 signaling complex. In non-neuronal cells, coexpression of human NgR1, p75 and LINGO-1 conferred responsiveness to oligodendrocyte myelin glycoprotein, as measured by RhoA activation. A dominant-negative human LINGO-1 construct attenuated myelin inhibition in transfected primary neuronal cultures. This effect on neurons was mimicked using an exogenously added human LINGO-1-Fc fusion protein. Together these observations suggest that LINGO-1 has an important role in CNS biology. Show less

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3. Show less

Chromosomal rearrangements resulting in gene fusions are frequently involved in carcinogenesis. Here, we describe a semiautomatic procedure for identifying fusion gene transcripts by using publicly available mRNA and EST databases. With this procedure, we have identified 96 transcript sequences that are derived from 60 known fusion genes. Also, 47 or more additional sequences appear to be derived from 20 or more previously unknown putative fusion genes. We have experimentally verified the presence of a previously unknown IRA1/RGS17 fusion in the breast cancer cell line MCF7. The fusion gene encodes the full-length RGS17 protein, a regulator of G protein-coupled signaling, under the control of the IRA1 gene promoter. This study demonstrates that databases of ESTs can be used to discover fusion genes resulting from structural rearrangement of chromosomes. Show less

Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator beta-catenin. Recent studies have demonstrated that axin, which coordinates beta-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. We have developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3beta, APC, axin, beta-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein-protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. We initially defined a reference state of kinetic, thermodynamic, and flux data from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which we analyzed the effects of prolonged and transient Wnt stimulation on beta-catenin and axin turnover. We predict several unusual features of the Wnt pathway, some of which we tested experimentally. An insight from our model, which we confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. We can also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and we show that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of beta-catenin at decreased APC concentrations. By applying control analysis to our mathematical model, we demonstrate the modular design, sensitivity, and robustness of the Wnt pathway and derive an explicit expression for tumor suppression and oncogenicity. Show less

The components of the Wnt-signaling pathway are mutated in tumors, but the relationship between these components and cervical cancer has not been elucidated. In this study, we used immunohistochemistry, single strand confirmation polymorphism (SSCP) and direct sequencing methods to analyze the mutation and protein expressions of both CTNNB1 and AXIN1 in cervical cancer. Among the 30 tested cervical cancers, no mutation of CTNNB1 but 3 polymorphisms were found. Mutation analysis of AXIN1 revealed that one specimen had a heterozygous mutation at codon 740 (GCC right curved arrow ACC) and six polymorphisms were also found. Immunohistochemistry showed no relationship between the protein expression patterns and mutation of AXIN1 and CTNNB1. Mutations of CTNNB1 may not be a factor, whereas mutations of AXIN1 may play a limited role in tumorigenesis of cervical cancer. In addition, aberrant expression patterns are not mutation related, so that other factors may be responsible for these changes. Show less

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1. Show less

Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene (CPS1) encoding a protein with two AMP-binding domains. CPS1 orthologs were detected in all Cochliobolus spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketidepeptide hybrids. Disruption of CPS1 caused reduced virulence of both race T and race O of C. heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat. These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes. Show less

In human atherosclerotic lesions, degranulated mast cells are found in the vicinity of macrophage foam cells. Mast cell granules contain tryptase, a tetrameric serine protease requiring glycosaminoglycans for stabilization. No endogenous inhibitors have been described for tryptase, and the physiological functions of the enzyme are poorly understood. Here, we investigated the effects of human tryptase on the integrity of high density lipoprotein (HDL)3 and on its ability to release cholesterol from cultured mouse macrophage foam cells. Incubation of HDL3 with tryptase led to degradation of its apolipoproteins. Tryptase predominantly degraded a quantitatively minor subfraction of HDL3 that is lipid poor, exhibits electrophoretic pre-beta mobility, and contains either apolipoprotein A-I or apolipoprotein A-IV as its sole apolipoprotein. Moreover, tryptase caused functional changes in HDL3 by destroying its ability to promote high-affinity efflux of cholesterol from macrophage foam cells, ie, the pre-beta-HDL-dependent component of the process. Human aortic proteoglycans increased the ability of tryptase to proteolyze HDL3, suggesting that the proteoglycan-rich extracellular matrix of the arterial intima provides an appropriate environment for the extracellular actions of tryptase. By depleting pre-beta-HDL, mast cell tryptase may impair the initial step of reverse cholesterol transport and will then favor cellular accumulation of cholesterol during atherogenesis. Show less

Juvenile-onset neuronal ceroid lipofuscinosis (JNCL; Batten disease) features hallmark membrane deposits and loss of central nervous system (CNS) neurons. Most cases of the disease are due to recessive inheritance of an approximately 1 kb deletion in the CLN3 gene, encoding battenin. To investigate the common JNCL mutation, we have introduced an identical genomic DNA deletion into the murine CLN3 homologue (Cln3) to create Cln3( Deltaex7/8) knock-in mice. The Cln3( Deltaex7/8) allele produced alternatively spliced mRNAs, including a variant predicting non-truncated protein, as well as mutant battenin that was detected in the cytoplasm of cells in the periphery and CNS. Moreover, Cln3( Deltaex7/8) homozygotes exhibited accrual of JNCL-like membrane deposits from before birth, in proportion to battenin levels, which were high in liver and select neuronal populations. However, liver enzymes and CNS development were normal. Instead, Cln3( Deltaex7/8) mice displayed recessively inherited degenerative changes in retina, cerebral cortex and cerebellum, as well as neurological deficits and premature death. Thus, the harmful impact of the common JNCL mutation on the CNS was not well correlated with membrane deposition per se, suggesting instead a specific battenin activity that is essential for the survival of CNS neurons. Show less

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells. Show less

A partial C-terminal cDNA sequence of a novel Drosophila mitogen-activated protein kinase phosphatase (MKP), designated DMKP-3, was identified from an epitope expressed sequence tag database, and the missing N-terminal cDNA fragment was cloned from a Drosophila cDNA library. DMKP-3 is a protein of 411 amino acids, with a calculated molecular mass of 45.8 kDa; the deduced amino acid sequence is most similar to that of mammalian MKP-3. Recombinant DMKP-3 produced in Escherichia coli retained intrinsic tyrosine phosphatase activity. In addition, DMKP-3 specifically inhibited extracellular-signal-regulated kinase (ERK) activity, but was without a significant affect on c-Jun N-terminal kinase (JNK) and p38 activities, when it was overexpressed in Schneider cells. DMKP-3 interacted specifically with Drosophila ERK (DERK) via its N-terminal domain. In addition, DMKP-3 specifically inhibited Elk-1-dependent trans-reporter gene expression in mammalian CV1 cells, and dephosphorylated activated mammalian ERK in vitro. DMKP-3 is uniquely localized in the cytoplasm within Schneider cells, and gene expression is tightly regulated during development. Thus DMKP-3 is a Drosophila homologue of mammalian MKP-3, and may play important roles in the regulation of various developmental processes. Show less

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by growth of benign bone tumors. This genetically heterozygous disease comprises three chromosomal loci: the EXT1 gene on chromosome 8q23-q24, EXT2 on 11p11-p13, and EXT3 on 19p. Both EXT1 and EXT2 have been cloned and defined as a new family of potential tumor suppressor genes in previous work. However, no studies have been conducted in the Taiwanese population. To determine if previous results can also be applied to the Taiwanese, we analyzed 5 Taiwanese probands with clinical features of HME: 1 of them is a sporadic case, and the others are familial cases. Linkage studies were performed in the familial cases before the mutation analysis to determine to which of the three EXT chromosomes these cases could be assigned. Our results showed that one proband is linked to the EXT1 locus and three are linked to the EXT2 locus; the sporadic case was subsequently found to involve EXT1. We then identified four new mutations that have not been found in other races: two in EXT1--frameshift (K218fsX247) and nonsense (Y468X) mutations and two in EXT2-missense (R223P) and nonsense (Y394X) mutations. Our results indicate that in familial cases, linkage analysis can prove useful for preimplantation genetic diagnosis. Show less

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity. Show less

The wnt pathway regulates the steady state level of beta-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit beta-catenin turnover via its competition with axin and adenomatous polyposis for beta-catenin binding. A mutant of beta-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of beta-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between beta-catenin and Tcf3 stimulate beta-catenin degradation, indicating this interaction normally plays an important role in regulating beta-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1epsilon stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces. Show less

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation. Show less

The proto-oncogene Raf is a major regulator of growth and differentiation. Previous studies from a number of laboratories indicate that Raf activates a signaling pathway that is independent of the classic MEK1,2-ERK1,2 cascade. However, no other signaling cascade downstream of Raf has been identified. We describe a new member of the mitogen-activated protein kinase family, p97, an ERK5-related kinase that is activated and Raf associated when cells are stimulated by Raf. Furthermore, p97 is selectively responsive to different growth factors, providing a mechanism for specificity in cellular signaling. Thus, p97 is activated by the neurogenic factor fibroblast growth factor (FGF) but not the mitogenic factor epidermal growth factor (EGF) in neuronal cells. Conversely, the related kinase ERK5 is activated by EGF but not FGF. p97 phosphorylates transcription factors such as Elk-1 and Ets-2 but not MEF2C at transactivating sites, whereas ERK5 phosphorylates MEF2C but not Elk-1 or Ets-2. Finally, p97 is expressed in a number of cell types including primary neural and NIH 3T3 cells. Taken together, these results identify a new signaling pathway that is distinct from the classic Raf-MEK1,2-ERK1,2 kinase cascade and can be selectively stimulated by growth factors that produce discrete biological outcomes. Show less

Regulation of beta-catenin degradation by intracellular components of the wnt pathway was reconstituted in cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent beta-catenin degradation in extracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while dishevelled inhibits beta-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the APC-axin-GSK3 complex. Our results confirm and extend models in which inhibition of GSK3 has two synergistic effects: (1) reduction of APC phosphorylation and loss of affinity for beta-catenin and (2) reduction of beta-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. Dishevelled thus stabilizes beta-catenin, which can dissociate from the APC/axin complex and participate in transcriptional activation. Show less

We have cloned a cardiovascular-restricted basic helix-loop-helix factor that interacts with arylhydrocarbon receptor nuclear translocator (ARNT) in a yeast two-hybrid screen. Cardiovascular helix-loop-helix factor 1 (CHF1) is distantly related to the hairy family of transcriptional repressors. We analyzed its expression pattern during mouse embryo development. At day 8.5, the expression of CHF1 is first detected in the primitive ventricle of the primordial heart tube and persists throughout gestation. In rat hearts, this expression is down-regulated after birth, concurrent with terminal differentiation of cardiomyocytes. In the developing vasculature, CHF1 first appears in the dorsal aorta at day 9.0, which precedes the reported expression of smooth muscle cell markers, and persists into adulthood. In an in vitro system of smooth muscle cell differentiation, CHF1 mRNA was barely detectable in undifferentiated cells but was induced highly in differentiated smooth muscle cells. To determine whether CHF1 might affect the function of ARNT, we performed transfection studies. Co-transfection of CHF1 inhibited ARNT/EPAS1-dependent transcription by 85%, and this inhibition is dose-dependent. In electrophoretic mobility studies, CHF1 inhibited the binding of the ARNT/EPAS1 heterodimer to its target site. Our data suggest that CHF1 functions as a transcriptional repressor and may play an important role in cardiovascular development. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominantly inherited disease characterized by the formation of cartilage-capped prominences (exostoses) that develop from the juxtaepiphyseal regions of the long bones. Recently, EXT1 and EXT2 genes were cloned and germline mutations of EXT1 and EXT2 were identified in EXT families. In this study, we performed a mutational analysis of EXT1 and EXT2 genes in eight unrelated Korean EXT families by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. As a result, we were able to identify one family (SNU-OC3) with the EXT1 mutation and another family (SNU-OC15) with the EXT2 mutation. The EXT1 mutation was a 10-bp deletion at the 3' end of exon 5 (CTAATTTAGg) including the splice site of this exon. The EXT2 mutation identified in the SNU-OC15 family was a missense mutation at codon 85 of exon 2 (TGC-->CGC), resulting in an amino acid change from cysteine to arginine. This missense mutation cosegregated with the disease phenotype in this family, suggesting that it is the disease-causing mutation. These two mutations identified in EXT1 and EXT2 are novel ones. Show less

Xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs) cross-interfere to various extents in non-mouse species and in wild Asian mice, suggesting that they might use a common receptor for infection. Consistent with this hypothesis, the susceptibility of some wild mice to X-MLVs has been mapped to the P-MLV receptor locus at the distal end of mouse chromosome 1. In this study, we report the isolation and characterization of a cDNA for the human X-MLV cell surface receptor (X-receptor) by using a human T lymphocyte cDNA library in a retroviral vector. The predicted X-receptor contains 696 amino acids with multiple hydrophobic potential membrane-spanning sequences and with weak homologies to the yeast proteins SYG1, of unknown function, and PHO81, which has been implicated in a system that regulates transport of inorganic phosphate. Expression of the X-receptor in Chinese hamster ovary cells, which are substantially resistant to P-MLVs and to X-MLVs, made them susceptible to both of these virus groups. The mouse homologue of the X-receptor was mapped by hybridization to the distal end of chromosome 1 at the same position as the P-MLV receptor gene Rmc1. These results strongly support the hypothesis that a common gene encodes the receptors for X-MLVs and P-MLVs, with the human X-receptor preferentially mediating X-MLV infections and the homologous protein of inbred mice mediating only P-MLV infections. We propose that X-MLVs and P-MLVs comprise a single family of retroviruses that have coevolved in response to diversification in X-receptor genes of the host. Show less