Biomedical subjects

A Ullrich

Publications and source records attributed to A Ullrich.

At least 163 records · Page 9Linked to original sources

PubMed1994

Effects of type-I and -II interferons on 90K antigen expression in ovarian carcinoma cells.

Antigen 90K is produced by several tumor-cell lines and by patients with cancer. Its function has not yet been clarified, although recent reports suggest that it plays a role in the tumor-host relationship--for example by stimulation of natural killer and lymphokine-activated killer-cell activity. Previous studies have indicated that 90K expression may be under the influence of interferon-alpha. Here, we provide evidence that both interferon-alpha and -gamma can enhance the secretion of 90K and augment the level of specific mRNA expression in 3 ovarian carcinoma cell lines (OVCAR-3, HTB-77 and SKOV-6). However, interferon-gamma leads to depletion of cellular 90K whereas interferon-alpha increases both secreted and cellular 90K levels. In equimolar concentrations, Interferon-alpha was always superior to interferon-gamma in augmenting 90K protein or mRNA levels. Combinations of TNF with interferon-gamma were highly synergistic both in reducing cell proliferation and in increasing 90K secretion and mRNA expression. This synergism was seen to a lesser extent with interferon-alpha.

Antigens, Neoplasm↗

PubMed1994

Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2.

BACKGROUND: Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein with three Src homology (SH) domains in the order SH3-SH2-SH3. Both SH3 domains of GRB2 are necessary for interaction with the protein Son of sevenless (Sos), which acts as a Ras activator. Thus, GRB2 mediates signal transduction from growth factor receptors to Ras and is thought to be a key molecule in signal transduction. RESULTS: The three-dimensional structure of the carboxy-terminal SH3 domain of GRB2 (GRB2 C-SH3) was determined by NMR spectroscopy. The SH3 structure consists of six beta-strands arranged in two beta-sheets that are packed together perpendicularly with two additional beta-strands forming the third beta-sheet. GRB2 C-SH3 is very similar to SH3 domains from other proteins. The binding site of the ligand peptide (VPP-PVPPRRR) derived from the Sos protein was mapped on the GRB2 C-SH3 domain indirectly using 1H and 15N chemical shift changes, and directly using several intermolecular nuclear Overhauser effects. CONCLUSIONS: Despite the structural similarity among the known SH3 domains, the sequence alignment and the secondary structure assignments differ. We therefore propose a standard description of the SH3 structures to facilitate comparison of individual SH3 domains, based on their three-dimensional structures. The binding site of the ligand peptide on GRB2 C-SH3 is in good agreement with those found in other SH3 domains.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Inhibition of glioma cell growth by a truncated platelet-derived growth factor-beta receptor.

Abnormal expression of platelet-derived growth factor (PDGF) receptors has been observed in malignant glioma and other tumors such as osteosarcomas and malignant melanomas. However, their role in the development and maintenance of the tumors is not understood. Signaling through the PDGF receptors is activated by ligand-induced dimerization. Thus, introduction of mutant receptors that are kinase deficient but still dimerization competent is one strategy to study the importance of PDGF receptors in glioma cell growth. A truncated PDGF-beta receptor was introduced into C6 rat glioma cells and the PDGF-mediated signaling and subsequent cell growth studied. In clones expressing the mutant receptor, PDGF-BB-induced tyrosine phosphorylation of the endogenous receptor was significantly reduced. In addition, these cells grew to lower density in culture and formed smaller colonies in soft agar than the C6 parental cells. Furthermore, the ability of cells expressing the truncated receptor to grow as xenografts in nude mice was significantly impaired. These results support the important role for the PDGF-beta receptor in C6 glioma cell growth. They also demonstrate the usefulness of dominant-negative mutants of the PDGF receptor for the evaluation of the role of the receptor in tumorigenesis.

Animals↗

PubMed1994

Novel signaling pathway suggested by SH3 domain-mediated p95vav/heterogeneous ribonucleoprotein K interaction.

The role of the protooncogene product p95vav in signal transduction was investigated by characterizing its interactions with proteins that may represent components of a novel signaling pathway. We demonstrate here stable association of p95vav with the heterogeneous ribonucleoprotein K (hnRNP-K), a protein that not only was found to be part of hnRNP particles but has also been implicated in transcriptional regulation of the c-myc gene. Through the PLPPPPPPRG sequence, hnRNP-K specifically interacts with the SH3 domain of p95vav and thus represents a novel SH3 binding protein that may be capable of translating cell surface receptor signals through p95vav activation into regulatory events on the level of gene expression.

Amino Acid Sequence↗

PubMed1994

Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain.

A 6.2-kb full-length clone encoding a distinct protein-tyrosine phosphatase (PTP; EC 3.1.3.48), PTPD1, was isolated from a human skeletal muscle cDNA library. The cDNA encodes a protein of 1174 amino acids with N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family, which also includes the two PTPs H1 and MEG1. The PTP domain is positioned in the extreme C-terminal part of PTPD1, and there is an intervening sequence of about 580 residues without any apparent homology to known proteins separating the ezrin-like and the PTP domains. Thus, PTPD1 and the closely related, partially characterized, PTPD2 belong to the same family as PTPH1 and PTPMEG1, but because of distinct features constitute a different PTP subfamily. Northern blot analyses indicate that PTPD1 and PTPD2 are expressed in a variety of tissues. In transient coexpression experiments PTPD1 was found to be efficiently phosphorylated by and associated with the src kinase pp60src.

Amino Acid Sequence↗

PubMed1994

EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor.

BACKGROUND: Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting with receptors that have protein tyrosine kinase activity. The stimulation of PC12 phaeochromocytoma cells with nerve growth factor leads to the sustained activation and nuclear translocation of the p42 and p44 isoforms of MAP kinase and induces the differentiation of these chromaffin cells to a sympathetic-neuron-like phenotype. In contrast, stimulation with epidermal growth factor induces a transient activation of p42 and p44 MAP kinases without pronounced nuclear translocation and does not trigger cell differentiation. We have examined whether the differential activation of MAP kinases forms the basis of the differential response of the cells to the two factors. RESULTS: By overexpressing either wild-type or mutant receptors for epidermal growth factor in PC12 cells, we found that p42 and p44 MAP kinase activity remains elevated for longer in cells that overexpress receptors than in untransfected cells. Epidermal growth factor promotes both a striking nuclear translocation of p42 MAP kinase and the differentiation of the overexpressing cells. CONCLUSIONS: Our results strongly suggest that the distinct effects of nerve growth factor and epidermal growth factor on PC12 cell differentiation can be explained by differences in the extent and duration of activation of p42 and p44 MAP kinases in response to the two factors, without invoking a signal transduction pathway specific to nerve growth factor.

Animals↗

PubMed1994

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B.

Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status. One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B. To obtain sufficient quantities for structural investigations, truncated forms of PTP1B encompassing the catalytic domain were over-expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography. The activity of these purified enzymes has been compared with the wild-type enzyme expressed in mammalian cells. By measuring the activities against p-nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTP1B retained the same characteristics as the full-length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C-terminus. Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme. In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.

Base Sequence↗

PubMed1994

Stable expression of truncated inositol 1,4,5-trisphosphate receptor subunits in 3T3 fibroblasts. Coordinate signaling changes and differential suppression of cell growth and transformation.

Stable clones of NIH 3T3 fibroblasts transfected with the cDNA of either the wild-type or deletion forms of the rat type I (or cerebellar) inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) were investigated. The delta form, missing the NH2-terminal sequence that includes the IP3-binding site, is expected to be still assembled with wild-type subunits to yield a tetrameric Ca2+ channel across the endoplasmic reticulum membrane; the s form, missing the membrane-spanning sequences, is expected to remain as a soluble monomer in the cytosol. With respect to control clones transfected with the vector only, the synthesis fo IP3Rs was markedly stimulated in the receptor-transfected clones. The mass accumulation, however, was increased only moderately (deletion forms = 15-30% of the endogenous IP3R), apparently because of a compensatory increase in receptor turnover. Coordinate changes in IP3 generation and Ca2+ release were revealed in the delta clones by experiments in both intact and permeabilized cells. In these clones, the IP3R was more sensitive to IP3, and IP3 generation at the ATP P2u surface receptor was decreased. This latter effect was due neither to a defect in G protein coupling nor to changes in phospholipase C expression, but to down-regulation of the P2u receptor. In the cells expressing the s- and delta-IP3R subunits, no differences with respect to the controls were observed in epidermal growth factor-induced DNA synthesis, whereas long-term growth stimulated by serum was reduced. Even more marked, especially in the delta clones (-90%), was the inhibition of cell transformation induced by autocrine stimulation with transforming growth factor alpha of the overexpressed epidermal growth factor receptors or by other growth factor receptors and oncogenes (platelet-derived growth factor/platelet-derived growth factor receptor beta, HER2/neu, and v-erbB). These effects appear not to be connected to the signaling processes mediated by tyrosine phosphorylation since the latter was unchanged in the delta clones. These results demonstrate for the first time (a) that the changes in cellular homeostasis directly induced by deleted IP3R subunits (increased receptor synthesis and increased IP3R sensitivity) are largely compensated by indirect coordinate changes apparently aimed to keep near normal the signaling properties of the cells; (b) that modulation of intracellular Ca2+ channels induces profound consequences that differentially affect growth and oncogenesis; and (c) that IP3Rs and the Ca2+ stores are important cross-roads of intracellular signaling pathways.

3T3 Cells↗

PubMed1994

The secreted tumor-associated antigen 90K is a potent immune stimulator.

Immunization of mice with conditioned media from human breast cancer cells yielded the monoclonal antibody SP-2, which recognized an antigen of approximately 90-95 kDa. This protein, designated 90K, was found to be present in the serum of healthy individuals and at elevated levels in the serum of subpopulations of patients with various types of cancer and AIDS. Here we report the primary structure of the SP-2 antigen and demonstrate its relationship to a family of proteins which carry a scavenger receptor cysteine-rich domain. Northern blot analysis of normal tissues, primary tumors, and tumor-derived cell lines indicates a broad expression spectrum of the 90K gene at widely varying levels. Functional characterization reveals stimulatory effects of 90K on host defense systems, such as natural killer cell and lymphokine-activated killer cell activity, and indicates that its immunostimulatory effects may be mediated through the induction of interleukin-2 and possibly other cytokines.

3T3 Cells↗

PubMed1994

Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2.

Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryonic fibroblasts that bind specifically to the SH3 domain of phospholipase C gamma, a primary substrate of receptor tyrosine kinases, and characterized the 110-kDa band as the microtubule-activated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaction does not require the dynamin GTPase function and involves a proline-rich target sequence between residues 812 and 820 of dynamin.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Prolactin-induced proliferation of Nb2 cells involves tyrosine phosphorylation of the prolactin receptor and its associated tyrosine kinase JAK2.

The interaction of prolactin with its receptor in the Nb2 cell line has been shown to induce the phosphorylation of cell-associated proteins and mitogenesis. It has been reported previously that one of these proteins, phosphorylated upon prolactin stimulation, was a tyrosine kinase. We have identified this kinase as JAK2, and demonstrate its association with the prolactin receptor. In addition, we show that the prolactin receptor itself becomes tyrosine phosphorylated upon ligand stimulation in Nb2 cells. These actions are time-dependent and occur rapidly after prolactin stimulation, with first the kinase being activated within 5 min and then the receptor being phosphorylated maximally at 20 min. Moreover, phosphorylation of both JAK2 and the receptor as well as Nb2 cell proliferation are dependent on the concentration of lactogenic hormone, resulting in a bell-shaped response curve similar to that observed in the two site model of hGH action. This indicates that early events in signal transduction as well as later events like mitogenesis and proliferation involve prolactin receptor dimerization. Together these data indicate that the prolactin receptor in Nb2 cells is associated to JAK2 and that upon ligand stimulation, and receptor dimerization, the kinase and the receptor are tyrosine-phosphorylated, which represents the first event in the process of prolactin receptor signal transduction in Nb2 cells.

Blotting, Western↗

PubMed1994

Autocrine stimulation of the Xmrk receptor tyrosine kinase in Xiphophorus melanoma cells and identification of a source for the physiological ligand.

The melanoma-inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. Using a highly specific antiserum produced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cell line revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cell supernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.

Animals↗

PubMed1994

Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma.

Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal-transducing proteins such as phospholipase C-gamma (PLC gamma), phosphatidylinositol-3'-kinase (PI3'-K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain-containing substrates within the Trk cytoplasmic domain suggests a non-essential function of PI3'-K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co-operative function of the PLC gamma-mediated pathway for neuronal differentiation of PC12 cells.

Animals↗

PubMed1994

Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an inter-receptor disulfide bond.

Populations of the epidermal growth factor receptor (EGFR) with both high and low affinity for EGF are found on the surface of cells. It has been hypothesized that the high-affinity state of the EGFR represents the receptor dimer and that this is also the kinase-active form. We describe here studies aimed at addressing this question directly. To stabilize homodimers of EGFR, we have generated a mutated form of the receptor by inserting a cysteine residue in the extracellular juxtamembranous region, in order to cross-link the extracellular domains of two receptors via disulfide bond formation. The mutation resulted in ligand-induced appearance of covalently linked EGF receptor dimers and, in parallel, increased the number of high-affinity receptors present on the surface of cells expressing the mutated EGFR. Comparison of the tyrosine kinase activity of the covalently linked dimeric and the monomeric forms of the EGF receptor, separated by sucrose density gradient centrifugation, showed that the dimer was significantly more active than monomer in the phosphorylation of exogenous substrate. We conclude that the dimeric form of the EGF-receptor represents the active form, and that dimer formation is associated with the appearance of high-affinity binding EGF receptors on the cell surface.

3T3 Cells↗

PubMed1994

Mutagenesis of Phe381 and Phe382 in the extracellular domain of the insulin receptor: effects on receptor biosynthesis, processing, and ligand-dependent internalization.

Mutations of the extracellular domain of the insulin receptor impair processing and transport of receptors to the plasma membrane. We have previously reported that a mutation substituting Val for Phe382 in the alpha-subunit of the insulin receptor impairs intracellular processing and insulin-induced autophosphorylation of the mutant receptor. In this investigation, we have generated two independent mutations of amino acids Phe381 and Phe382 of the insulin receptor: Val for Phe381 and Leu for Phe382. These substitutions cause a slight impairment of intracellular processing and transport of the mutant receptors. Furthermore, insulin-dependent internalization of the mutant receptors is unaffected by these mutations. Thus, of the three substitutions studied to date, Val for Phe382 is the only mutation of the Phe381-Phe382 sequence that causes a major defect in post-translational processing of the receptor.

3T3 Cells↗

PubMed1994

Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant.

Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.

Animals↗

PubMed1994

Structure of the N-terminal SH3 domain of GRB2 complexed with a peptide from the guanine nucleotide releasing factor Sos.

Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins. We have determined the high-resolution NMR structure of the complex between the amino-terminal SH3 domain of GRB2 and a ten amino acid peptide derived from the guanine nucleotide releasing factor Sos. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.

Adaptor Proteins, Signal Transducing↗

PubMed1994

One bead, one chemical compound: use of the selectide process for anticancer drug discovery.

A technology for chemical synthesis and testing of libraries of millions of chemical entities has been developed for rapid molecular and cellular screening for drug leads. Each individual compound in the library is on a separate resin bead. Screening for binding activity can be conducted directly on the beads. Biological activity is assessed in solution phase assay by cleaving a portion of the compound from each bead. The molecular structure of the compound of interest is obtained by automated peptide sequencing from the bead of origin. We have applied this technology to anticancer drug discovery as well as to other pharmaceutical targets. For anticancer drug development, current molecular targets include B-cell lymphoma, the EGF receptor, and the HER2-neu receptor. Solution phase screening with dual cleavable libraries is being used for growth inhibition of human tumor cell lines. Initial in vitro leads have been identified in each of these areas of anticancer drug discovery.

Antibodies, Monoclonal↗