Showing papers by "Axel Ullrich published in 1996"

Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors

Abstract: Transduction of a mitogenic signal from the cell membrane to the nucleus involves the adapter proteins SHC and Grb2, which mediate activation of the Ras/mitogen-activated protein (MAP) kinase pathway. In contrast to receptor tyrosine kinases (RTKs), the signalling steps leading to Ras/MAP kinase activation by G-protein-coupled receptors (GPCRs) are still poorly characterized but appear to include beta gamma subunits of heterotrimeric G-proteins and as-yet unidentified tyrosine kinases. We report here that the epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the GPCR agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppressed MAP kinase activation and strongly inhibited induction of fos gene expression and DNA synthesis. Our results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk.

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation

Abstract: ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

Tumor necrosis factor-alpha- and hyperglycemia-induced insulin resistance. Evidence for different mechanisms and different effects on insulin signaling.

Abstract: Inhibition of insulin receptor signaling by high glucose levels and by TNF-alpha was recently observed in different cell systems. The aim of the present study was to characterize the mechanism of TNF-alpha-induced insulin receptor inhibition and to compare the consequences of TNF-alpha- and hyperglycemia-induced insulin receptor inhibition for signal transduction downstream from the IR. TNF-alpha (0.5-10 nM) and high glucose (25 mM) showed similar rapid kinetics of inhibition (5-10 min, > 50%) of insulin receptor autophosphorylation in NIH3T3 cells overexpressing the human insulin receptor. TNF-alpha effects were completely prevented by the phosphotyrosine phosphatase (PTPase) inhibitors orthovanadate (40 microM) and phenylarsenoxide (35 microM), but they were unaffected by the protein kinase C (PKC) inhibitor H7 (0.1 mM), the phosphatidylinositol-3 kinase inhibitor wortmannin (5 microM), and the thiazolidindione troglitazone (CS045) (2 microgram/ml). In contrast, glucose effects were prevented by PKC inhibitors and CS045 but unaffected by PTPase inhibitors and wortmannin. To assess effects on downstream signaling, tyrosine phosphorylation of the following substrate proteins of the insulin receptor was determined: insulin receptor substrate-1, the coupling protein Shc, focal adhesion kinase (FAK125), and unidentified proteins of 130 kD, 60 kD. Hyperglycemia (25 mM glucose) and TNF-alpha showed analogous (> 50% inhibition) effects on tyrosine phosphorylation of insulin receptor substrate-1, Shc, p60, and p44, whereas opposite effects were observed for tyrosine phosphorylation of FAK125, which is dephosphorylated after insulin stimulation. Whereas TNF-alpha did not prevent insulin-induced dephosphorylation of FAK125, 25 mM glucose blocked this insulin effect completely. In summary, the data suggest that TNF-alpha and high glucose modulate insulin receptor-signaling through different mechanisms: (a) TNF-alpha modulates insulin receptor signals by PTPase activation, whereas glucose acts through activation of PKC. (b) Differences in modulation of the insulin receptor signaling cascade are found with TNF-alpha and high glucose: Hyperglycemia-induced insulin receptor inhibition blocks both insulin receptor-dependent tyrosine phosphorylation and dephosphorylation of insulin receptor substrate proteins. In contrast, TNF-alpha blocks only substrate phosphorylation, and it does not block insulin-induced substrate dephosphorylation. The different effects on FAK125 regulation allow the speculation that long-term cell effects related to FAK125 activity might develop in a different way in hyperglycemia- and TNF-alpha-dependent insulin resistance.

PTP1D is a positive regulator of the prolactin signal leading to beta-casein promoter activation.

Abstract: Stimulation of the prolactin receptor (PRLR), a member of the cytokine/growth hormone receptor family, results in activation of the associated Jak2 tyrosine kinase and downstream signaling pathways. We report that PTP1D, a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 (SH2) domains, physically associates with the PRLR-Jak2 complex and is tyrosine-phosphorylated upon stimulation with prolactin. The formation of the trimeric PRLR-Jak2-PTP1D complex is critical for transmission of a lactogenic signal, while PTP1D phosphorylation is necessary, but not sufficient. The dominant negative inhibitory effect of a phosphatase-deficient mutant on expression of a beta-casein promoter-controlled reporter gene is evidence for an essential role of fully functional PTP1D in the regulation of milk protein gene transcription.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity

Abstract: We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

SH3 domain-dependent interaction of the proto-oncogene product Vav with the focal contact protein zyxin.

Abstract: Scr homology 3 (SH3) domain-mediated protein-protein interactions have been implicated in the localization of proteins to specific sites within the cell. We present evidence that the product of the vav proto-oncogene, p95vav, interacts specifically with the focal adhesion protein zyxin both in vitro and in yeast two hybrid system. Solution binding and two-hybrid system experiments demonstrate that association of Vav with the LIM domain protein zyxin is mediated by the C-terminal SH3 domain of the Vav and involves the proline-rich N-terminus of zyxin. The interaction appears to be selective, since no binding of the proline-rich N-terminus of zyxin with other SH3 domain-containing proteins such as GRB-2, phospholipase C gamma, GTPase-activating protein, or p85 was detected.

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

Abstract: The chimaeric bcr/abl oncogene is detected in virtually all cases of chronic myelogenous leukaemia (CML). It encodes a constitutively active tyrosine kinase of 210kDalton, 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.

Characterization of PCP-2, a novel receptor protein tyrosine phosphatase of the MAM domain family.

Abstract: DNA sequences encoding a novel member of the receptor protein tyrosine phosphatase (R-PTP) family, termed PCP-2, were identified in a human pancreatic adenocarcinoma cDNA library. Human PCP-2 cDNA predicts a protein of 1430 amino acids with a calculated Mr of 160 kDa. The predicted PCP-2 enzyme consists of a 740 amino acid extracellular region, a single transmembrane domain, and a 666 amino acid intracellular portion. The extracellular sequence contains a MAM (meprin/A5/PTPmu) domain, an immunoglobulin-like domain and four fibronectin type III-like repeats, suggesting that it is a member of the PTPkappa and PTPmu subfamily. The intracellular region contains two tandemly-repeated protein tyrosine phosphatase domains. Northern blot analyses revealed a single transcript of 5.5 kilobases, which is expressed at different levels in many human tissues except spleen and placenta. Upon transfection of PCP-2 cDNA into human embryonic kidney fibroblast 293 cells, a protein with an apparent Mr of 180 000 was detected by immunoblot analysis. This size was reduced to the predicted Mr upon treatment with endoglycosidase F, indicating that PCP-2 is glycosylated and, hence, expressed at the cell surface. A potential role of PCP-2 in cell-cell recognition and adhesion is supported by its co-localization with cell adhesion molecules, such as catenin and E-cadherin, at sites of cell-cell contact.

Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D.

Abstract: Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.

Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation.

Abstract: The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites resulted in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhanced its inositol trisphosphate (IP3) and calcium signals and resulted in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibited a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impaired the IP3/calcium signal and attenuated the MAP kinase activation and receptor dephosphorylation, but resulted in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. Our findings establish the significance of PLC gamma affinity for signal definition, the role of this receptor tyrosine kinase substrate as a negative feedback regulator and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations.

90K (Mac‐2 BP) in human milk

Abstract: 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.

Characterization of the pest family protein tyrosine phosphatase bdp1

Abstract: Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues.

Cck-4, a receptor tyrosine kinase, and methods for diagnosis and treatment of cck-4 signal transduction disorders

Abstract: The present invention relates to CCK-4 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Methods for treatment, diagnosis, and screening are provided for diseases or conditions characterized by an abnormality in a signal transduction disorder. The signal transduction pathway involves an interaction between a CCK-4 receptor tyrosine kinase and a receptor for the kinase.

Cck-4, une tyrosine kinase recepteur, techniques diagnostiques et therapeutiques de troubles de la transduction du signal cck-4

Abstract: Cette invention porte sur des polypeptides de CCK-4, des acides nucleiques codant ces polypeptides, des cellules, des tissus ainsi que sur des animaux possedant ces acides nucleiques, sur des anticorps de ces polypeptides, des analyses les utilisant ainsi que sur des procedes en rapport avec tout ce qui precede. L'invention offre des techniques de traitement, de diagnostic et de depistage de maladies ou d'etats pathologiques caracterises par une anomalie dans un trouble de la transduction de signal. La trajectoire de transduction de signal fait intervenir une interaction entre une tyrosine-kinase recepteur de CCK-4 et un recepteur de la kinase.

Tyrosine kinase associated polypeptides

Abstract: The present invention relates to TKA-1 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Methods for treatment, diagnosis, and screening are provided for TKA-1 related diseases or conditions characterized by an abnormal interaction between a TKA-1 polypeptide and a TKA-1 binding partner.