Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

A phosphoglycolate phosphatase/AUM-dependent link between triacylglycerol turnover and epidermal growth factor signaling

Mammalian phosphoglycolate phosphatase (PGP, also known as AUM or glycerol-3-phosphate phosphatase) is a small molecule-directed phosphatase important for metabolite repair and lipid metabolism. Although PGP was first characterized as an enzyme involved in epidermal growth factor (EGF) signaling, PGP protein substrates have remained elusive. Here we show that PGP depletion facilitates fatty acid flux through the intracellular triacylglycerol/fatty acid cycle, and that phosphatidylinositol-4,5-bisphosphate (PIP2), produced in a side branch of this cycle, is critical for the impact of PGP activity on EGF-induced signaling. Loss of endogenous PGP expression amplified both EGF-induced EGF receptor autophosphorylation and Src-dependent tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1). Furthermore, EGF enhanced the formation of circular dorsal ruffles in PGP-depleted cells via Src/PLCγ1/protein kinase C (PKC)-dependent signaling to the cytoskeleton. Inhibition of adipose triglyceride lipase normalized the increased PIP2 content, reduced EGF-dependent PLCγ1 hyperphosphorylation, and decreased the elevated dorsal ruffle formation of PGP-depleted cells. Our data explain how PGP exerts control over EGF-induced cellular protein tyrosine phosphorylation, and reveal an unexpected influence of triacylglycerol turnover on growth factor signaling.     

Sphingolipids are involved in N-methyl-N'-nitro-N-nitrosoguanidine-induced epidermal growth factor receptor clustering

Previously we have found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, can induce the clustering of cellular surface receptors including tumor necrosis factor receptor (TNFR) and epidermal growth factor receptor (EGFR). Since sphingolipids, especially ceramide, have been suggested as major players in ligand-induced receptor clustering, their involvement in this ligand-independent, chemical-induced receptor clustering was evaluated. It was shown that MNNG-induced EGFR clustering occurred primarily at lipid rafts, as nystatin, which can disrupt lipid raft structure, significantly decreasing MNNG-induced EGFR clustering. Lipidomic studies revealed that MNNG treatment induced profound changes in sphingolipids metabolism, which were not the same as those induced by EGF treatment. Acid sphingomyelinase (ASM) is responsible for hydrolyzing sphingomyelin to generate ceramide, and it was demonstrated that MNNG treatment caused ASM distribution changing from diffused state to concentrated area of cells, which colocalized with lipid rafts. Nystatin treatment also abolished the redistribution of ASM. In addition, blockage of ceramide production by ASM inhibitor imipramine interrupted MNNG-induced receptor clustering. Taken together, these data suggested that sphingolipids are involved in MNNG-induced receptor clustering; however, the specific species involved may be different from those involved in EGF-mediated receptor clustering.     

Nitric oxide and superoxide inhibit platelet-derived growth factor receptor phosphotyrosine phosphatases

Platelet derived growth factor receptor (PDGFR) became tyrosine autophosphorylated in rat mesangial cells shortly after platelet derived growth factor (PDGF) ligation in a tyrosine kinase inhibitor (tyrphostin AG 1296) sensitive manner. Ligand-independent, massive tyrosine PDGFR phosphorylation was achieved by diverse NO releasing compounds. Phosphorylation was slow compared to PDGF, revealed a concentration- and time-dependency, and was not mimicked by lipophilic cyclic-GMP analogues. Interleukin-1 beta/cAMP activated mesangial cells released NO and in turn showed PDGFR phosphorylation. A NO-synthase involvement was assured by L-NG-nitroarginine methyl ester inhibition. PDGFR phosphorylation was also achieved by the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. NO- and O2(.-)-evoked PGDFR phosphorylation was N-acetylcysteine reversible. Cell free dephosphorylation assays revealed PDGFR dephosphorylation by tyrosine phosphatases. Receptor dephosphorylation by cytosolic phosphatases was completed within 30 min and was sensitive to the readdition of NO donors or orthovanadate. In addition, phosphatase activity determined in a direct dephosphorylation assay using the substrate para-nitrophenyl phosphate was attenuated by NO or vanadate. We conclude that cytosolic protein tyrosine phosphatases are targeted by exogenously supplied or endogenously generated NO in mesangial cells. Radical (NO. or O2.-) formation shifts the phosphorylation--dephosphorylation equilibrium towards phosphorylation, thus integrating redox-mediated responses into established signal transducing pathways.     

Measurement of downstream kinase activity modulation as indicator of epidermal growth factor receptor inhibitor efficacy

The acoustic membrane micro particle (AMMP) technology has been used to quantify single analytes out of multiple sample types. In this study the technology is used to reveal molecular interactions of components of kinase pathways. Specifically, the downstream kinase activity of the EGFR receptor in the presence or absence of EGFR inhibitors is investigated. These experiments substantiate that EGFR stimulation predominantly activates the MEK/ERK pathway. The EGFR inhibitors tested had varying effectiveness at preventing phosphorylation at the EGFR or downstream kinase activity. These experiments reveal the use of the AMMP technology for observing multiple signaling pathways in a single experiment.     

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC₅₀ values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.     

Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Cao2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Cao2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Cao2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Cao2+. This is consistent with the known expression of TGFalpha by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Cao2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation

We have previously developed Epitheliome, a software agent representation of the growth and repair characteristics of epithelial cell populations, where cell behaviour is governed by a number of simple rules. In this paper, we describe how this model has been extended to incorporate an example of a molecular 'mechanism' behind a rule-in this case, how signalling by both endogenous and exogenous ligands of the epidermal growth factor receptor (EGFR) can impact on the proliferation of cell agents. We have developed a mathematical model representing release of endogenous ligand by cells, three-dimensional diffusion of the secreted molecules through a volume of cell culture medium, ligand-receptor binding, and bound receptor internalization and trafficking. Information relating to quantities of molecular species associated with each cell agent is frequently exchanged between the agent and signalling models, and the ratio of bound to free receptors determines cell cycle progression and hence the proliferative behaviour of the cell agents. We have applied this integrated model to examine the effect of plating density on tissue growth via autocrine/paracrine signalling. This predicts that cell growth is dependent on the concentration of exogenous ligand, but where this is limited, then growth becomes dependent on cell density and the availability of endogenous ligand. We have further modified the calcium concentration of the medium to modulate the formation of intercellular bonds between cells and shown that the increased propensity for cells to form colonies in physiological calcium does not result in significantly different patterns of receptor occupancy. In conclusion, our approach demonstrates that by combining agent-based and mathematical modelling paradigms, it is possible to probe the complex feedback relationship between the behaviour of individual cells and their interaction with one another and their environment.     

N-methyl-N'-nitro-N-nitrosoguanidine interferes with the epidermal growth factor receptor-mediated signaling pathway

Many environmental factors, such as ultraviolet (UV) and arsenic, can induce the clustering of cell surface receptors, including epidermal growth factor receptor (EGFR). This is accompanied by the phosphorylation of the receptors and the activation of ensuing cellular signal transduction pathways, which are implicated in the various cellular responses caused by the exposure to these factors. In this study, we have shown that N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, also induced the clustering of EGFR in human amnion FL cells, which was similar in morphology to that of epidermal growth factor treatment. However, MNNG treatment did not activate Ras, the downstream mediator in EGFR signaling pathway, as compared to EGF treatment. The autophosphorylation of tyrosine residues Y1068 and Y1173 at the intracellular domain of EGFR, which is related to Ras activation under EGF treatment, was also not observed by MNNG exposure. Interestingly, although MNNG did not affect the binding of EGF to EGFR, MNNG can interfere with EGF function. For instance, pre-incubating FL cells with MNNG inhibited the autophosphorylation of EGFR by EGF treatment, as well as the activation of Ras. In addition, the phosphorylation of Y845 on EGFR by EGF, which is mediated through c-Src or related kinases but not autophosphorylation, was also affected by MNNG. Therefore, MNNG may influence the tyrosine kinase activity as well as the phosphorylation of EGFR through its interaction with EGFR.     

Proteolytic domains of the epidermal growth factor receptor of human placenta

Microsomal membranes from human placenta, which bind 5–20 pmol of ¹²⁵I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with ¹²⁵I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR₁), and by PAR₁ inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR₁-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.