Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N‐terminal domain of the ost proto‐oncogene and was highly tumorigenic in nude mouse assays. The proto‐ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP‐binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP‐bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha‐tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.
The Eph family of receptors is the largest family of RTKs. Eph receptors are stimulated by a family of membrane-linked ligands designated ephrins (6, 7). Both biochemical and genetic studies have established the central role that ephrins have in the control of cell contact repulsion, boundary formation, cell migration, and repulsive axon guidance (6). Repulsive axon guidance appears to be caused by modulation of cytoskeletal organization leading to regulation of neural growth-cone development (8). Eph-receptors also regulate cell-matrix interaction and cell proliferation by affecting signaling by integrins (9-11) and by modulation of MAPK response (12)(13)(14).In this article, we demonstrate that EphA4 binds directly and specifically via the N-terminal portion of its protein tyrosine kinase core to the juxtamembrane (JM) region of FGFRs. In cells that express EphA4 and FGFRs, the interactions between the cytoplasmic domains of EphA4 and FGFRs can lead to transreceptor activation, resulting in tyrosine phosphorylation of FRS2␣ and MAPK activation. The synergistic effect of ephrin-A1 stimulation on FGF2-induced cellular responses may influence the biological outcome of the activation of these two families of RTKs. Materials and MethodsYeast Two-Hybrid Experiments. The yeast two-hybrid system was used as described (15). The bait used for screening was 81 aa (amino acids 398-478), derived from the JM domain of human FGFR3. A human brain cDNA library in a pJG4-5 vector consisting of 3.5 ϫ 10 6 primary transformants (Clontech) was used for screening for proteins that interact with the JM domain of FGFR3. Fig. 1A shows the constructs used to detect interactions between the cytoplasmic domain of EphA4 and the JM domain of FGFR3.Cells. HEK293 cells were maintained in DMEM supplemented with 10% calf serum. For neural differentiation, P19 cells were maintained in ␣-MEM supplemented with 10% FBS containing 0.5 M retinoic acid for 3 days. Rat L6 myoblasts were maintained in DMEM supplemented with 10% FBS.Preparation of Ephrin-A1. Ephrin-A1 fused to human IgG-Fc was purchased from Sigma-Aldrich. Before application to the cells, 5 g of ephrin-A1-Fc was oligomerized by mixing with 12 g of rabbit anti-human IgG-Fc (Jackson ImmunoResearch) in 1 ml of PBS at 4°C for at least 1 h. As a control, a human IgG-Fc fragment (Jackson ImmunoResearch) was also applied after oligomerization.Expression Plasmids. Full-length cDNA of human EphA4 was prepared by RT-PCR using total RNA from a human brain extract (Clontech) as the template. The cDNA of human FGFR4 was prepared by RT-PCR using K562 cell-derived RNA as the template. The cDNAs for FGFR1 and FGFR2 were provided by W. McKeehan (Texas A&M University, College Station, TX). The cDNA for FGFR3 was provided by D. E. Johnson (University of Pittsburgh, Pittsburgh). Receptor mutants were prepared by applyConflict of interest statement: No conflicts declared.
Familial multiple endocrine neoplasia type 1 (MEN-1) is characterized by tumors of the parathyroids, endocrine pancreas, and anterior pituitary. Since the gene associated with MEN-1, located on chromosome 11 (11q13), may normally inhibit tumor proliferation, tumors could arise from inactivation of one or both of the alleles. However, parathyroid tumors in patients with MEN-1 have been considered to result from polyclonal hyperplasia. Using genetic probes, we tested parathyroid tumors for a monoclonal component, represented by a loss of alleles at any of eight loci along chromosome 11. Ten of 16 tumors from 14 patients with familial MEN-1 had losses of alleles from chromosome 11. Tumors with losses were larger than those without (1.6 vs. 0.2 g; P less than 0.002), suggesting that a monoclonal adenoma may develop after a phase of polyclonal hyperplasia. In 7 of 10 tumors, the subregion of loss was less than the full length of chromosome 11 but always included one copy of the MEN-1 locus. Of 34 sporadic adenomas from patients without MEN-1, 9 showed similar allelic losses in chromosome 11; in 7 the losses included the apparent MEN-1 locus. We conclude that many "hyperplastic" parathyroid tumors in familial MEN-1 are in fact monoclonal and may progress or even begin to develop by inactivation of the MEN-1 gene (at 11q13) in a precursor cell. Some sporadic adenomas have allelic losses on chromosome 11, which may also involve the MEN-1 gene.
Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF consists of a series of structural units, including an amino-terminal segment with a hairpin loop, four kringle domains, and a serine protease-like region. In this study, we demonstrate that the amino-terminal (
A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.
Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.
A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAGJ). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAGI sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAGJ is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRA4G).The fibroblast growth factors (FGFs) are a family of related proteins with roles in mitogenesis, differentiation, wound healing, and organogenesis (reviewed in ref. 1). Nine FGFs, acidic FGF (aFGF)/FGF-1, basic FGF/FGF-2, INT-2/ FGF-3, HST/FGF-4, FGF-5, HST-2/FGF-6, keratinocyte growth factor (KGF)/FGF-7, androgen-induced growth factor/FGF-8, and glia-activating factor/FGF-9, have been identified and cloned. While most of the members can stimulate the growth of fibroblasts, KGF/FGF-7 and glia-activating factor/ FGF-9 are involved in epithelial and glial cell proliferation, respectively (2, 3). The biological responses of the FGFs are mediated through specific high affinity receptor tyrosine kinases (reviewed in ref. 4). These receptors, FGFR1/Flg, FGFR2/Bek, FGFR3, and FGFR4, encode structurally related proteins (5-8). The diversity of FGFRs are increased by alternative RNA splicing, which generates additional isoforms of FGFRs (9-11). In particular, the FGFR2/BEK gene encodes two receptor species, FGFR2 and KGF receptor (KGFR), which differ with respect to ligand-binding specificity; FGFR2 binds aFGF and basic FGF, whereas KGFR binds aFGF and KGF (9). These receptors differ only in 49 amino acids in their third Ig-like domains.We have developed an efficient expression cloning system which we have used to isolate several unique transforming cDNAs (12, 13). Here we report the isolation of a constitutively active FGFR2 with an altered C terminus by expression cloning with a rat osteosarcoma (ROS) cDNA library. This FGFR2 isoform (FGFR2-ROS) resulted from a chromosomal rearrangement of the FGFR2 gene with a novel gene, designated FGFR activating gene 1 (FRAG1), in ROS cells. Activation of FGFR2-ROS through C-terminal fusion with FRAG1 suggests a third activation mechanism of receptor tyrosine kin...
This is the first report that universally examined mismatch repair immunohistochemical screening for upper urinary tract urothelial cancers. The prevalence (5%) of putative Lynch syndrome-associated upper urinary tract urothelial cancers is much higher than we had expected. We ascertained that putative Lynch syndrome-associated upper urinary tract urothelial cancers were clinically in the early stage and histologically classified into low-grade malignancy with its characteristic pathological features. The clinicopathological characteristics that we found in the present study could become additional possible markers in the diagnosis of Lynch syndrome-associated upper urinary tract urothelial cancers.
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