epidermal-growth-factor and pervanadate

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2A

Topics: Amino Acid Sequence; Animals; Antibodies; Antibodies, Monoclonal; Antibody Specificity; Cross Reactions; Epidermal Growth Factor; HEK293 Cells; Humans; Leucine; Lymphocyte Activation; Methylation; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Peptides; Phosphorylation; Phosphotyrosine; Protein Phosphatase 2; Protein Processing, Post-Translational; src-Family Kinases; T-Lymphocytes; Vanadates

Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Amino Acids; Animals; Cell Membrane; Chlorocebus aethiops; COS Cells; Diglycerides; Epidermal Growth Factor; GTPase-Activating Proteins; HeLa Cells; Humans; Models, Molecular; Molecular Sequence Data; Neoplasm Proteins; Oncogene Proteins; Proline-Rich Protein Domains; Protein Binding; Protein Transport; rac GTP-Binding Proteins; src Homology Domains; Structure-Activity Relationship; Vanadates

Membrane compartments function as segregated signaling platforms for different cellular functions. It is not clear how Src is regulated at different membrane compartments. To visualize local Src activity in live cells, a FRET-based Src biosensor was targeted in or outside of lipid rafts at the plasma membrane, via acylation or prenylation modifications on targeting tags either directly fused to the biosensor or coupled to the biosensor through an inducible heterodimerization system. In response to growth factors and pervanadate, the induction of Src activity in rafts was slower and weaker, dependent on actin and possibly its mediated transportation of Src from perinuclear regions to the plasma membrane. In contrast, the induction of Src activity in nonrafts was faster and stronger, dependent on microtubules. Hence, Src activity is differentially regulated via cytoskeleton at different membrane compartments.

Topics: Actins; Biosensing Techniques; Cell Membrane; Cells, Cultured; Cytoskeleton; Epidermal Growth Factor; Fluorescence Resonance Energy Transfer; HeLa Cells; Humans; Microtubules; Platelet-Derived Growth Factor; Protein Transport; src-Family Kinases; Vanadates

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Line, Tumor; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Neoplasms; Peptides; Phenylmercuric Acetate; Protease Inhibitors; Protein Isoforms; Protein Kinase C; Protein Structure, Tertiary; Sequence Alignment; Spectrometry, Mass, Electrospray Ionization; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Vanadates

Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.

Topics: Animals; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Fibroblasts; Humans; Membrane Proteins; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Phosphorylation; Proteasome Endopeptidase Complex; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Serine; Tamoxifen; Tyrosine; Ubiquitin; Vanadates

Spred proteins modulate growth factor receptor signaling by inhibiting the Ras-MAPK cascade. Here, we show that Spred-1, Spred-2, and Spred-3 are ubiquitinated in HEK293T cells stimulated with epidermal growth factor (EGF) or pervanadate. Spred-2 tyrosines Y228 and/or Y231 in the Kit binding domain were identified as putative phosphorylation site(s) critical for Spred-2 ubiquitination. Depletion of Cbl and Cbl-b E3 ubiquitin ligases by RNA interference, or overexpression of a Cbl dominant inhibitory mutant (Cbl-N), inhibited Spred-2 ubiquitination, while conversely, wild type Cbl enhanced Spred-2 ubiquitination. Interaction of Spred-2 with Cbl-N was detectable by co-immunoprecipitation and required the Cbl SH2 domain and Spred-2 Y228 and Y231 residues. Studies on endogenous Spred-2 in ME4405 melanoma cells showed that pervanadate induced Spred-2 ubiquitination and a marked reduction in Spred-2 steady-state levels that was partially blocked by the proteasomal inhibitor, MG-132. These results suggest a role for Spred-2 tyrosine phosphorylation and ubiquitination in controlling Spred-2 expression levels.

Topics: Adaptor Proteins, Signal Transducing; Cells, Cultured; Epidermal Growth Factor; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Phosphorylation; Protein Kinase Inhibitors; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-cbl; Repressor Proteins; RNA Interference; Tyrosine; Ubiquitin; Vanadates

Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Calcium; Cell Membrane; CHO Cells; Cloning, Molecular; Cricetinae; DNA, Complementary; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Epitopes; HeLa Cells; Humans; Ionophores; Kinetics; Metalloendopeptidases; Protein Kinase C; Protein Structure, Tertiary; Radioimmunoassay; Rats; Time Factors; Transfection; Tumor Necrosis Factor-alpha; Vanadates; Zinc

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.

Topics: Animals; Binding Sites; Blotting, Western; Catenins; Cell Adhesion Molecules; Cell Line; COS Cells; Cytoplasm; Delta Catenin; DNA Mutational Analysis; Electrophoresis, Gel, Two-Dimensional; Epidermal Growth Factor; Gene Deletion; Genes, Dominant; Glutathione Transferase; Humans; Mutagenesis, Site-Directed; Mutation; Phenylalanine; Phosphoproteins; Phosphorylation; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins pp60(c-src); Transfection; Tyrosine; Vanadates

Glandular epithelia express the keratin intermediate filament (IF) polypeptides 8, 18 and 19 (K8/18/19). These proteins undergo significant serine phosphorylation upon stimulation with growth factors and during mitosis, with subsequent modulation of their organization and interaction with associated proteins. Here we demonstrate reversible and dynamic tyrosine phosphorylation of K8 and K19, but not K18, upon exposure of intact mouse colon or cultured human cells to pervanadate. K8/19 tyrosine phosphorylation was confirmed by metabolic 32PO4-labeling followed by phosphoamino acid analysis, and by immunoblotting with anti-phosphotyrosine antibodies. Pervanadate treatment increases keratin solubility and also indirectly increases K8/18 serine phosphorylation at several known sites, some of which were previously shown to be associated with EGF stimulation, extracellular signal-regulated kinase (ERK), or p38 kinase activation. However, K8/19 tyrosine phosphorylation is independent of EGF signaling or ERK activation while inhibition of p38 kinase activity blocks pervanadate-induced K8/19 tyrosine phosphorylation. Our results demonstrate tyrosine phosphatase inhibitor-mediated in vivo tyrosine phosphorylation of K8/19, but not K18, and suggest that tyrosine phosphorylation may be a general modification of other IF proteins. K8/19 tyrosine phosphorylation involves a pathway that utilizes the p38 mitogen-activated protein kinase, but appears independent of EGF signaling or ERK kinase activation.

Topics: Animals; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Colon; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Keratins; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Serine; Signal Transduction; Solubility; Tyrosine; Vanadates

Epidermal growth factor (EGF)- and insulin-dependent mammary epithelial cell mitogenesis is mediated by specific tyrosine kinase receptors. Receptor tyrosine kinase activity is highly regulated in normal cells, whereas amplification of intracellular protein tyrosine phosphorylation is associated with abnormal growth and/or neoplastic transformation. Since protein tyrosine phosphatases (PTPs) are involved in regulating receptor tyrosine kinase signaling, studies were conducted to determine the effects of the PTP inhibitors, vanadate and pervanadate, on mitogen-receptor signal transduction and cell growth. Mammary epithelial cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 2-8 microM vanadate or pervanadate greatly increased intracellular protein tyrosine phosphorylation. However, in the presence of optimal mitogenic stimulation (10 ng/ml EGF and 10 microg/ml insulin), these treatments induced a slight, but significant decrease in cell growth. In contrast, these treatments significantly increased mammary epithelial cell growth, albeit less than optimally, under submitogenic culture conditions (500 pg/ml EGF and 10 microg/ml insulin). Neither vanadate nor pervanadate was found to mimic the mitogenic actions of EGF and/or insulin in these cells. The growth-stimulatory effects of PTP inhibitors in submitogenic conditions appear to result from enhanced receptor tyrosine kinase mitogenic signaling, whereas PTP inhibitor attenuation of optimal cell growth may be due to the suppression of PTP activity associated with cell cycle progression. In addition, treatment with PTP inhibitors was not found to stimulate anchorage-independent growth, as determined by the inability of single cells to form colonies in soft agarose. In conclusion, these data demonstrate that optimal mitogen-dependent mammary epithelial cell growth requires both receptor tyrosine kinase and PTP activity. Furthermore, PTP inhibitor-induced amplification of receptor tyrosine kinase mitogenic signaling is not in itself sufficient to induce enhanced cell growth or phenotypic expression of neoplastic transformation.

Topics: Animals; Cell Division; Cells, Cultured; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Female; Insulin; Mammary Glands, Animal; Mice; Mice, Inbred BALB C; Phosphotyrosine; Protein Tyrosine Phosphatases; Tetradecanoylphorbol Acetate; Vanadates

Activation of the phosphatidylinositol 3-kinase (PI3K) plays an important role in the mitogenic response of many cell types. Recently, two serine/threonine kinases Akt and p70(S6k) have been identified as physiological targets of PI3K. Observations that expression of activated forms of Akt led to the activation of p70(S6k) implied Akt might mediate mitogenic signaling through activation of p70(S6k). To clarify the relationship between signaling through these two kinases, we have examined their regulation by various mitogenic stimuli. In this study we have focused on the role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts. Depletion of intracellular calcium stores by EGTA pretreatment has no effect on growth factor-induced Akt activation but completely abolishes p70(S6k) stimulation. Increase of intracellular calcium induced by ionomycin or thapsigargin results in a full activation of p70(S6k), whereas little or no activation of Akt is observed. Furthermore, although PI3K in anti-phosphotyrosine immunoprecipitates is only very weakly activated by ionomycin, the calcium-induced stimulation of p70(S6k) is completely inhibited by the specific PI3K inhibitor wortmannin. We conclude Akt and p70(S6k) lie on separate signaling pathways. Activation of signaling to Akt is insufficient for the activation of p70(S6k), which can be achieved independently of Akt. p70(S6k) requires a separate calcium-dependent and wortmannin-sensitive process that is likely to be independent of type IA PI3K family members.

Topics: 3T3 Cells; Androstadienes; Animals; Calcium; Down-Regulation; Epidermal Growth Factor; Ionomycin; Mice; Mice, Inbred BALB C; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Signal Transduction; Tetradecanoylphorbol Acetate; Vanadates; Wortmannin

Previous studies have shown that the envelope protein of the mouse mammary tumor virus (MMTV) rapidly upregulates prolactin (PRL) receptors by shifting them from internal pools to the cell surface and downregulates epidermal growth factor (EGF) receptors by inducing their internalization and degradation. This study shows that the effect on PRL receptors is mediated by the nitric oxide (NO)/cGMP pathway, since it can be mimicked by an NO donor or 8-bromo-cGMP and can be blocked by an NO synthase inhibitor. In contrast, the effect on EGF receptors is mediated by tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K), since it can be blocked by either a tyrosine kinase inhibitor or by a PI3K inhibitor. Both of these pathways can be activated by a calcium ionophore and inhibited by calcium chelation. Therefore, it appears that the mouse mammary tumor virus envelope protein, like other retroviral envelope proteins, initially elevates cytoplasmic calcium, which can then stimulate both the NO/cGMP and the tyrosine phosphorylation/PI3K pathways, leading to PRL receptor upregulation and EGF receptor downregulation, respectively.

Topics: Androstadienes; Animals; Calcimycin; Cell Membrane; Cyclic GMP; Down-Regulation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Products, env; Genistein; Mammary Glands, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred C3H; Models, Biological; Nitric Oxide; Nitroprusside; omega-N-Methylarginine; Organ Culture Techniques; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Receptors, Prolactin; Signal Transduction; Up-Regulation; Vanadates; Wortmannin

The intraperitoneal injection of a vanadate/H2O2 mixture (peroxovanadate) into mice resulted within minutes in the appearance of numerous tyrosine-phosphorylated proteins in the liver and kidney. These effects are presumably due to the inhibition of phosphotyrosine phosphatase activity. Three of the tyrosine-phosphorylated proteins have been identified as the receptors for epidermal growth factor, insulin, and hepatocyte growth factor. The injection of peroxovanadate also enhanced the tyrosine phosphorylation of many of the proteins known to function downstream of these receptors, including SHC, signal transducer and activator of transcription (Stat) 1alpha,beta, Stat 3, Stat 5, phospholipase C-gamma, insulin receptor substrate 1, GTPase-activating protein, beta-catenin, gamma-catenin, p120cas, SHP-1, and SHP-2. The administration of peroxovanadate also induced nuclear translocation of a number of tyrosine-phosphorylated Stat proteins. In addition, the global effects on tyrosine phosphorylation permitted the detection of a number of novel intracellular protein interactions, including an association of Tyk2 with beta-catenin. The in situ administration of peroxovanadate may prove useful in the search for novel tyrosine-phosphorylated proteins and the identification of new interactions between previously identified tyrosine-phosphorylated substrates.

Topics: Animals; DNA-Binding Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Injections, Intraperitoneal; Kidney; Liver; Mice; Molecular Weight; Phosphorylation; Protein Sorting Signals; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; STAT1 Transcription Factor; Trans-Activators; Tyrosine; Vanadates

In this study we have investigated the relationships between the stimulation of tyrosine-specific protein phosphorylation and the state of assembly of cell-cell and cell-matrix adherens-type junctions. Bovine aortic endothelial (BAE) cells were treated with either the phosphotyrosine phosphatase inhibitor pervanadate or with epidermal growth factor (EGF), and the effect of the treatment on the organization of cell contacts and the actin cytoskeleton was evaluated by digital immunomicroscopy. We show here that pervanadate induced a dramatic (about 40-fold) increase in the level of phosphotyrosine labeling of cell-cell junctions, which reached maximal values following 20 minutes of incubation. Concomitantly, the junctional levels of vinculin, actin and plakoglobin increased, followed by a slower recruitment of cadherins to these sites. Upon longer incubation cell-cell junctions deteriorated and stress fibers and focal adhesions were formed. EGF stimulation of serum-starved BAE cells induced a rapid 'wave' of junctional tyrosine phosphorylation, followed by cyclic changes in the local levels of phosphotyrosine labeling. Periodic changes were also found in the intensity of labeling of junctional actin, vinculin and cadherins. These results suggest that tyrosine phosphorylation and the assembly of cell-cell adherens junctions are interdependent processes, and raise the possibility that the cross-talk between the two is responsible both for the regulation of junction formation and for adhesion-mediated signaling.

Topics: Animals; Aorta; Cattle; Cell Adhesion; Cells, Cultured; Endothelium, Vascular; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; Intercellular Junctions; Microscopy, Fluorescence; Phosphorylation; Tyrosine; Vanadates

In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Using several experimental approaches we demonstrate that Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Animals; Cadherins; Cell Adhesion; Cell Size; Enzyme Inhibitors; Epidermal Growth Factor; Genetic Techniques; Mice; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Tyrosine Phosphatases; Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; src Homology Domains; Tyrosine; Vanadates

In numerous studies on mammary epithelial cell lines multiple factors, added to the medium or contained in the serum, were required for casein gene expression. It has been shown in these systems that the mammary gland factor (MGF) is implicated in the activation of the beta-casein gene promoter. In the present study, we determined the relationship between known agents that affect casein gene expression and MGF activity using the properties of rabbit primary mammary epithelial cells to respond to PRL alone, when cultured in chemically defined medium. We demonstrate that MGF is rapidly activated by PRL alone or by human growth hormone, a natural ligand of many PRL receptors (PRL-Rs), in the cytoplasm and accumulated in the nucleus. The MGF activation by PRL occurred in the absence of endogenous extracellular matrix, a condition where casein synthesis is known to be markedly reduced. Different inhibitors of protein-tyrosine kinases, which have been shown to reduce casein mRNA synthesis, but not of protein kinase C, decrease the MGF activity. A tyrosine phosphatase inhibitor, sodium pervanadate, induced two GAS-binding complexes related to MGF and STAT1. Our data show that MGF is a latent cytoplasmic factor rapidly activated in mammary epithelial cells, by a mechanism involving a tyrosine kinase and a tyrosine phosphatase.

Topics: Animals; Base Sequence; Biological Transport; Caseins; Cells, Cultured; DNA; DNA-Binding Proteins; Down-Regulation; Epidermal Growth Factor; Extracellular Matrix; Female; Glucocorticoids; Growth Hormone; Immune Sera; Interferon-gamma; Mammary Glands, Animal; Milk Proteins; Molecular Sequence Data; Pregnancy; Prolactin; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rabbits; RNA, Messenger; STAT1 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Vanadates

1. The contractile actions of vanadate (VO4) and pervanadate (PV, peroxide(s) of vanadate) were studied in rat gastric longitudinal muscle strips and in aortic rings. The roles of extracellular sodium and calcium were evaluated and the potential effects of nerve-released agonists were considered. The possibility that these responses were due to the potentiation of tyrosine kinase activity, as a result of PV-mediated tyrosine phosphatase inhibition was explored with the use of tyrosine kinase inhibitors (genistein, tyrphostin) and by Western blot analysis of phosphotyrosyl proteins in PV-treated tissues. The ability of PV to mimic the action of the tyrosine kinase receptor-associated agonist, epidermal growth factor-urogastrone (EGF-Uro), in the gastric preparation was also studied. 2. PV caused concentration-dependent contractions in both gastric and aorta-derived tissues, with a potency that was 1 to 2 orders of magnitude greater than that of VO4. 3. Although repeated exposure of gastric and aortic tissues to a fixed concentration of VO4 caused reproducible contractions in both tissues, repeated exposure of gastric tissue to PV caused an increased contractile response plateauing after 3 exposures. In contrast, a single exposure of aortic tissue to PV (20 microM) caused a prolonged desensitization of the tissue to the subsequent contractile actions of PV or other agonists. 4. The contractile responses to PV were unaffected in both preparations by tetrodotoxin, atropine, yohimbine and phenoxybenzamine; and in the aortic preparation, the responses to VO4 and PV were the same in the presence or absence of a functional endothelium. 5. PV-induced contractions in both tissues were observed in the absence of extracellular sodium but required extracellular calcium and were attenuated by 1 micro M nifedipine.6. In the gastric preparation, the characteristics of the contractile actions of PV paralleled those of EGF-Uro in terms of (1) inhibition by genistein, (2) inhibition by indomethacin and (3) a requirement for extracellular calcium. These response characteristics differed from those of other contractile agonists such as carbachol.7. In both the gastric and aortic preparations genistein was able to inhibit PV-induced contractions selectively without causing comparable inhibition of KCI-induced contractions. Tyrphostin (AG18) also selectively blocked PV-induced contractions in the gastric, but not in the aortic preparation.8. In both the gastric and aortic tissu

Topics: Angiotensin II; Animals; Aorta, Thoracic; Blotting, Western; Calcium; Epidermal Growth Factor; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Muscle, Smooth, Vascular; Phosphotyrosine; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Sodium; Stomach; Tyrosine; Vanadates

A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.

Topics: Aluminum Compounds; Animals; Cell Communication; Cell Line; Connexin 43; Cricetinae; Cricetulus; Embryo, Mammalian; Epidermal Growth Factor; Ethers, Cyclic; Fibroblasts; Fluorides; Intercellular Junctions; Iodine Radioisotopes; Kinetics; Lung; Marine Toxins; Mesocricetus; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Tetradecanoylphorbol Acetate; Vanadates; Vanadium Compounds