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Biomedical subjects

A Ullrich

Publications and source records attributed to A Ullrich.

At least 127 records · Page 7Linked to original sources
PubMed1996

Interaction of the receptor tyrosine kinase p145c-kit with the p210bcr/abl kinase in myeloid cells.

The chimaeric bcr/abl oncogene is detected in virtually all cases of chronic myelogenous leukaemia (CML). It encodes a constitutively active tyrosine kinase of 210 kDalton, p210bcr/abl, which stimulates a variety of cytosolic signalling intermediates. The effects of bcr/abl on the activity of growth factor receptors are less well known. In order to investigate interaction of p210bcr/abl with the receptor tyrosine kinase p145c-kit, we used two myeloid, factor-dependent cell lines, MO7 and 32D, to generate bcr/abl positive sublines, MO7p210 and 32Dp210, by transfection with the bcr/abl gene. Since 32D and 32Dp210 cells did not express p145c-kit, a c-kit retrovirus was used to generate c-kit positive cell lines (32Dkit, 32Dp210kit). In contrast to MO7 and 32Dkit cells, MO7p210 and 32Dp210kit cells were factor independent and did not respond to the growth-promoting effects of recombinant human Steel factor (rhSF). Preincubation with a monoclonal antibody (MAb) neutralizing the binding of SF to p145c-kit did not affect the growth of MO7p210 cells, thus eliminating the possibility of an autocrine SF secretion. 32Dkit cells transfected with bcr/abl containing an inactivating point mutation (Lys-->Arg271) in the Abl kinase domain (32Dp210(Arg271)kit) retained their responsiveness to the effects of rhSF. Immune complex kinase assays showed that the kinase activity of p145c-kit was several-fold higher in MO7p210 and 32Dp210kit cells than in MO7, 32Dkit and 32Dp210(Arg271)kit cells, suggesting that Abl kinase activity was necessary to activate p145c-kit. Co-immunoprecipitation experiments with anti-Kit and anti-Abl MAbs demonstrated that p145c-kit and p210bcr/abl were associated in an intracellular complex in human bcr/abl positive, c-kit positive cell lines (MO7p210; GM/SO). Finally, colony assays with bone marrow from bcr/abl positive CML patients showed that the haemopoietic progenitors of three of four patients did not respond to rhSF. Taken together, the results suggest that p145c-kit can be activated by p210bcr/abl via an Abl-kinase dependent mechanism involving the complex formation of both proteins. These findings could explain some clinical features (basophilia, increase of immature myeloid cells) of chronic-phase CML.

Cell Division↗
PubMed1996

90K (Mac-2 BP) in human milk.

Human 90K, also known as Mac-2 binding protein (Mac-2 BP), is a secreted glycoprotein which is widely expressed, binds to the human macrophage-associated lectin Mac-2, and may have a role in host defence. We have measured the concentrations of 90K in human breast milk from eight healthy mothers delivering mature healthy infants after full-term pregnancy using a specific immunoenzymatic assay. Maximal 90K concentrations were observed on days 2-3 post-partum, and ranged from 13.4 to 79.2 microg/ml. The concentrations of 90K in the milk had no correlation with those in the maternal blood.

Adult↗
PubMed1996

Motogenic and morphogenic activity of epithelial receptor tyrosine kinases.

Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c-ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c-met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial cells in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal.

Amino Acid Sequence↗
PubMed1996

Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression.

The proto-oncogene product Vav plays a critical role in hematopoietic signal transduction. By using the yeast two-hybrid system, we identified a novel human protein, ENX-1, which interacts specifically with Vav both in vitro and in vivo. ENX-1 represents the human homolog of the Drosophila Enhancer of zeste gene, a member of the Polycomb group of genes, which are transcriptional regulators of homeobox gene expression. Interaction with ENX-1 suggests that Vav functions as an upstream element in the transcriptional regulation of homeobox genes, known to be important effectors in the hematopoietic system.

Amino Acid Sequence↗
PubMed1996

The adhesion molecule E-cadherin and a surface antigen recognized by the antibody 9C4 are selectively expressed on erythroid cells of defined maturational stages.

The antigen expression of immature erythroid bone marrow cells was studied using two recently generated monoclonal antibodies (mAb), mAb 67A4 and 9C4, with specificities for the epithelial cell adhesion molecule E-cadherin (E-cad; mAb 67A4), and a novel 110 kDa differentiation antigen (mAb 9C4) with unknown molecular structure. Pappenheim staining of FACS-purified cells labeled with mAb 9C4 and anti-glycophorin A (GA) revealed that the majority of the 9C4+GA- and 9C4+GA+ cells consisted of erythroblasts. In contrast, the E-cad-positive population comprised normoblasts and erythroblasts. While the E-cad+GA- fraction contained mainly erythroblasts and basophilic normoblasts, the E-Cad+GA+ population was enriched in orthochromatic and polychromatophilic normoblasts. By colony assays of affinity column-purified cells it could be shown that erythroid colony forming units (CFU-E) were enriched and erythroid burst forming units (BFU-E) were depleted in the 9C4- and E-cad-positive fractions. Flow cytometric analysis of bone marrow cells double-labeled with mAb 67A4 and anti-CD71, anti-CD117, anti-CD34, or anti-GA revealed that about 90% of the E-cad-positive cells coexpressed CD71, about 70% were positive for CD117, about 50% for GA, and only about 5% coexpressed CD34. The expression pattern of 9C4 antigen was similar to that of E-Cad with the exception that only a minority of the 9C4-positive cells coexpressed GA. Lymphoid and myeloid markers were negative on both the E-Cad- and 9C4-positive populations. In these studies we describe the identification of a new mAb-defined antigen which is specifically expressed on erythroblasts and CFU-E(9C4) and demonstrate that E-Cad is not only expressed on epithelial cells but also on erythropoietic cells of defined maturational stages.

Antibodies, Monoclonal↗
PubMed1996

Multiple in vivo phosphorylated tyrosine phosphatase SHP-2 engages binding to Grb2 via tyrosine 584.

SHP-2 (also named PTP1D, syp, or SH-PTP2) has been identified as a phosphotyrosine phosphatase comprising two src-homology-2 (SH2) domains. Upon growth factor stimulation, SHP-2 becomes tyrosine phosphorylated, thereby increasing its catalytic activity. Here, we identified SHP-2 to be phosphorylated on multiple tyrosine residues in response to different stimuli and unmasked the carboxyl-terminal tyrosine 584 as a major phosphorylation site in human cell lines. Tyrosine 584 shares, together with tyrosine 546, the consensus sequence pY-X-N-X, a characteristic of potential binding sites for the SH2 domain of growth factor receptor-bound protein 2 (Grb2). We show here that mutation of tyrosine 584, but not tyrosine 546, to phenylalanine totally abolished the binding of Grb2 to SHP-2. By using a systematic mutagenesis approach, phosphorylation of additional tyrosines in each of the SH2 domains of SHP-2 was detected after coexpression of epidermal growth factor receptor, but not after coexpression of platelet-derived growth factor receptor, whereas tyrosine 263 located in the interspace between SH2 and catalytic domain appears to be exclusively recognized by platelet-derived growth factor receptor. Immunoprecipitation of SHP-2 from a panel of mammary carcinoma cell lines copurifies several tyrosine phosphorylated proteins; the most prominent band has an apparent molecular weight of M(r) 115,000.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Adapter function of protein-tyrosine phosphatase 1D in insulin receptor/insulin receptor substrate-1 interaction.

Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.

Amino Acid Sequence↗
PubMed1995

The 90K tumor-associated antigen and clinical progression in human immunodeficiency virus infection.

We investigated the possibility that a secreted glycoprotein of approximately 90,000 daltons, termed 90K and identified as a member of the protein superfamily characterized by the scavenger receptor cysteine-rich (SRCR) domain, might have value as a predictor of progression to acquired immunodeficiency syndrome (AIDS) in subjects infected with the human immunodeficiency virus (HIV). Among 488 HIV-seropositive intravenous drug users with a median follow-up of 32.5 months, high levels of serum 90K at baseline proved to be a significant predictor of faster progression to AIDS, either as a continuous variable (log 90K; p < 0.0001) or as a dichotomous variable with an optimized cutoff point of 30 U/ml (p < 0.00001). Analysis of 90K in relation to known prognostic factors found an association with CD4 count, beta 2-microglobulin, and p24 antigen but none with neopterin. In multivariate analysis, the baseline 90K level was an independent predictor of AIDS. As compared with subjects with low levels of 90K, the relative risk of developing AIDS was 3.5 (95% CI 1.9-6.5) among those with high levels of 90K. The predictive value of 90K was maintained after stratification by baseline CD4 count: among subjects with > or = 500 x 10(6)/L CD4 cells, the proportion in whom AIDS developed was 10.5% for those with 90K levels < or = 30 U/ml as compared with 20% for those with 90K above the cutoff point (p = 0.006). Serum 90K is an independent predictor of the risk for progression to AIDS in HIV-infected subjects, including those whose CD4 counts have not fallen.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Cloning, characterization, and differential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of the eck/eph family.

Using a polymerase chain reaction-based strategy for the cloning of developmentally regulated receptor tyrosine kinases, we identified two novel members of the eck/eph-related subfamily which, in analogy with the recently identified mouse developmental kinase 1 (MDK1), were designated MDK2 and MDK5. MDK2 is highly homologous to the mouse kinase Myk-1 and the human kinase Htk, whereas MDK5 represents the mouse homologue of human Hek2. Northern blot analyses of adult mouse tissues revealed a 4.7 kb transcript of MDK2 and a 4.8 kb transcript of MDK5 in various organ systems, including lung, liver, kidney, intestine, muscle, heart, and, in the case of MDK5, also the brain. In addition to the full-length transcripts, smaller fragments were identified that probably represent truncated receptors. Northern blot analysis and in situ hybridization of mouse embryos indicated abundant expression during embryonic development, with preferential involvement of tissues of epithelial and endothelial origin for both kinases and of the spinal cord gray matter for MDK5. Unlike most other members of the eck/eph-related subfamily, the expression of MDK2 and MDK5 is not primarily restricted to neuronal structures, and their abundant presence in various organ systems during embryonic development suggests an important role in gestational growth and differentiation.

Amino Acid Sequence↗
PubMed1995

Colon carcinoma kinase-4 defines a new subclass of the receptor tyrosine kinase family.

Complementary DNA (cDNA) encoding a novel member of the receptor tyrosine kinase (RTK) family has been isolated from colon carcinoma tissue. Colon carcinoma kinase 4 (CCK-4) mRNA is highly expressed in human lung tissue and at lower levels in the thyroid gland and ovary. While no mRNA was found in human adult colon tissues, expression varied remarkably in colon carcinoma-derived cell lines. CCK-4 cDNA encodes a chicken KLG-related, 1071 amino acid-long transmembrane glycoprotein containing several genetic alterations within the RTK consensus sequences. These define CCK-4 as a catalytically inactive member of the RTK family of proteins and, in analogy to HER3, suggest a potentially tumor-characteristic role as a signal amplifier or modulator for an as yet unidentified kinase-competent partner.

Adult↗
PubMed1995

Phosphotyrosine residues in the nerve-growth-factor receptor (Trk-A). Their role in the activation of inositolphospholipid metabolism and protein kinase cascades in phaeochromocytoma (PC12) cells.

PC12 cells, which lack platelet derived-growth-factor (PDGF) receptors, have been stably transfected with a chimaera consisting of the extracellular domain of the beta-PDGF receptor and the intracellular and transmembrane domains of the nerve-growth-factor receptor Trk-A (termed PT-R). Mutation of the Trk-A residue Tyr490 to phenylalanine prevents the association with Shc, while similar mutations at Tyr751 or Tyr785 are reported to prevent interaction of Trk-A with the p85 subunit of inositol phospholipid 3-kinase and phospholipase C-gamma 1, respectively. The strong and sustained activation of p42 and p44 mitogen-activated-protein kinases induced by PDGF-B/B in PC12/PT-R cells was unaffected by mutation of Tyr785 or Tyr751 to phenylalanine, but was smaller and transient after mutation of Tyr490, and almost abolished by the double mutation of Tyr490 and Tyr785. Mutation of Tyr490 reduced by 70% the PDGF-induced increase in inositol phospholipid 3-kinase activity immunoprecipitated from cell extracts with antiphosphotyrosine monoclonal antibodies and greatly suppressed the PDGF-induced increase in the intracellular products of inositol phospholipid 3-kinase, while mutation of Tyr751 or Tyr785 had no effect. Mutation of Tyr785 (but not mutation of Tyr490 or Tyr751) abolished PDGF-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. Mutation of Tyr490, alone or in combination with mutation of Tyr751 and Tyr785, had no effect on the PDGF-induced activation of p70 S6 kinase (p70S6K). However, the activation of p70S6K by PDGF (or nerve growth factor), but not the activation of mitogen-activated-protein kinase, was prevented by two structurally unrelated inhibitors of inositol phospholipid 3-kinase, wortmannin or LY294002. Our results demonstrate the following: (1) the phosphorylation of Tyr490 plays a major role in the activation of inositol phospholipid 3-kinase and formation of 3-phosphorylated inositol lipids and confirm that the phosphorylation of Tyr 785 triggers the activation of phospholipase C-gamma 1 in vivo. (2) Tyr490 phosphorylation (but not inositol phospholipid 3-kinase activation) is also required for strong and sustained activation of mitogen-activated-protein kinase and neuronal differentiation, while the smaller and more transient activation of mitogen-activated-protein kinase, produced by the activation of phospholipase C-gamma 1 is insufficient to trigger the neuronal differentiation of PT-R cells. (3) Inositol phospholipid 3-kinase is required for the activation of p70S6K, but only a small increase in inositol phospholipid 3-kinase activity and the level of 3-phosphorylated inositol lipids is required for maximal p70S6K activation.

Amino Acid Sequence↗
PubMed1995

Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure.

Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation.

Animals↗
PubMed1995

Protein-tyrosine phosphatase 1D modulates its own state of tyrosine phosphorylation.

The insulin receptor-mediated signal transduction pathway involves insulin receptor substrate 1 and a variety of proteins containing Src homology-2 (SH2) domains, such as phosphatidylinositol 3-kinase, Grb2, and protein-tyrosine phosphatase 1D (PTP1D). Upon insulin stimulation of baby hamster kidney cells overexpressing the IR, the catalytically inactive mutant of PTP1D, C463A, becomes tyrosine-phosphorylated and coprecipitates with Grb2. Tyrosine phosphorylation of this mutant is significantly reduced when wild type PTP1D is coexpressed. Substitution of tyrosine residues 546 and 584 with phenylalanine abrogates tyrosine phosphorylation of the catalytically inactive mutant and abolishes its interaction with Grb2.

Animals↗
PubMed1995

Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases alpha and epsilon.

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase.

Animals↗
PubMed1995

Association of SH2 domain protein tyrosine phosphatases with the epidermal growth factor receptor in human tumor cells. Phosphatidic acid activates receptor dephosphorylation by PTP1C.

The SH2 domain protein tyrosine phosphatases (PTPases) PTP1C and PTP1D were found associated with epidermal growth factor (EGF) receptor which was purified from A431 cell membranes by several steps of chromatography. Both PTPases also associated with the EGF receptor upon exposure of immunoprecipitated receptor to lysates of MCF7 mammary carcinoma cells. The associated PTPases had little activity toward the bound receptor when it was autophosphorylated in vitro. Receptor dephosphorylation could, however, be initiated by treatment of the receptor-PTPase complex with phosphatidic acid (PA). When autophosphorylated EGF receptor was exposed to lysates of PTP1C or PTP1D overexpressing 293 cells, the association of PTP1C but not of PTP1D was enhanced in the presence of PA. In intact A431 cells, an association of PTP1C and PTP1D with the EGF receptor was detectable by coimmunoprecipitation experiments. PA treatment reduced the phosphorylation state of ligand activated EGF receptors in A431 cells and in 293 cells overexpressing EGF receptors together with PTP1C but not in 293 cells overexpressing EGF receptors alone or together with PTP1D. We conclude that PTP1C but not PTP1D participates in dephosphorylation of activated EGF receptors. A possible role of PA for physiological modulation of EGF receptor signaling is discussed.

ErbB Receptors↗
PubMed1995

Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3.

Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor-derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG-induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3-kinase in transfected 293 and mammary carcinoma-derived MCF-7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG-dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG-induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.

3T3 Cells↗