Activation of Raf-1 during Experimental Gastric Ulcer Healing Is Ras-Mediated and Protein Kinase C-Independent
1999; Elsevier BV; Volume: 155; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65491-0
ISSN1525-2191
AutoresRama Pai, Michael K. Jones, Morimasa Tomikawa, Andrzej S. Tarnawski,
Tópico(s)Phytochemistry and Bioactive Compounds
ResumoOur previous study demonstrated increases in epidermal growth factor receptor (EGF-R) phosphorylation and receptor tyrosine kinase and extracellular signal-regulated kinase (ERK1 and ERK2) activities in the ulcer margin of experimental gastric ulcer during healing. However, the intermediate steps linking activated receptor tyrosine kinase to ERKs during ulcer healing are as yet unknown. Raf-1 is upstream of mitogen-activated protein kinases (MAPK/ERK) and can be activated by Ras-dependent and/or Ras-independent mechanisms. Therefore, we studied Raf-1 activity, its potential activators protein kinase C (PKC) and Ras, and expression and associations of adapter proteins Shc, Grb2, and Sos during experimental gastric ulcer healing. To investigate if Raf-1–ERK activation is attributable to the epithelial component of ulcer margins, we studied the effect of EGF on PKC, Ras, and ERK activities in a rat gastric epithelial cell line (RGM1). Our results demonstrate that gastric ulceration significantly increases Raf-1 kinase activity, Grb2 and Ras protein, and Shc-Grb2 and Grb2-Sos complex levels. In contrast, PKC activity and protein level were significantly decreased in the ulcer margins. In RGM1 cells, EGF significantly increased Ras and ERK2 activities without affecting PKC activity. These findings indicate that Raf-1 activation during gastric ulcer healing is Ras mediated, involves Shc-Grb2-Sos, and is PKC-independent. Our previous study demonstrated increases in epidermal growth factor receptor (EGF-R) phosphorylation and receptor tyrosine kinase and extracellular signal-regulated kinase (ERK1 and ERK2) activities in the ulcer margin of experimental gastric ulcer during healing. However, the intermediate steps linking activated receptor tyrosine kinase to ERKs during ulcer healing are as yet unknown. Raf-1 is upstream of mitogen-activated protein kinases (MAPK/ERK) and can be activated by Ras-dependent and/or Ras-independent mechanisms. Therefore, we studied Raf-1 activity, its potential activators protein kinase C (PKC) and Ras, and expression and associations of adapter proteins Shc, Grb2, and Sos during experimental gastric ulcer healing. To investigate if Raf-1–ERK activation is attributable to the epithelial component of ulcer margins, we studied the effect of EGF on PKC, Ras, and ERK activities in a rat gastric epithelial cell line (RGM1). Our results demonstrate that gastric ulceration significantly increases Raf-1 kinase activity, Grb2 and Ras protein, and Shc-Grb2 and Grb2-Sos complex levels. In contrast, PKC activity and protein level were significantly decreased in the ulcer margins. In RGM1 cells, EGF significantly increased Ras and ERK2 activities without affecting PKC activity. These findings indicate that Raf-1 activation during gastric ulcer healing is Ras mediated, involves Shc-Grb2-Sos, and is PKC-independent. Ulcer healing requires interaction of various cellular and connective tissue components.1Tarnawski A Cellular mechanisms of gastric ulcer healing.in: Domschke W Konturek SJ The Stomach. Springer-Verlag, Berlin1993: 177-192Crossref Google Scholar, 2Tarnawski A Stachura J Krause WJ Douglass T Gergely H Quality of gastric ulcer healing: a new emerging concept.J Clin Gastroenterol. 1991; 13: S42-S47Crossref PubMed Scopus (114) Google Scholar It involves reconstruction of glandular structures, reepithelialization of the mucosal surface, and restoration of the connective tissue components.1Tarnawski A Cellular mechanisms of gastric ulcer healing.in: Domschke W Konturek SJ The Stomach. Springer-Verlag, Berlin1993: 177-192Crossref Google Scholar, 2Tarnawski A Stachura J Krause WJ Douglass T Gergely H Quality of gastric ulcer healing: a new emerging concept.J Clin Gastroenterol. 1991; 13: S42-S47Crossref PubMed Scopus (114) Google Scholar, 3Wang JY Johnson LR Induction of gastric and duodenal mucosal ornithine decarboxylase during stress.Am J Physiol. 1989; 257: G259-G265PubMed Google Scholar A number of growth factors, including epidermal growth factor (EGF) and transforming growth factor α (TGF-α), have been shown to participate in the repair of tissue injury by stimulating the cell proliferation and migration necessary for reepithelialization and ulcer healing.4Johnson LR Regulation of gastrointestinal growth.in: Johnson LR ed 3. Physiology of The Gastrointestinal Tract. vol 1. Raven, New York1994: 611-641Google Scholar, 5Konturek SJ Dembinski A Warzecha A Brzozowski T Gregory H Role of epidermal growth factor in healing of chronic gastroduodenal ulcers in rats.Gastroenterology. 1988; 94: 1300-1307PubMed Google Scholar, 6Podolsky DK Peptide growth factors in the gastrointestinal tract.in: Johnson LR ed 3. Physiology of the Gastrointestinal Tract. vol 1. Raven, New York1994: 129-167Google Scholar, 7Basson MD Modlin IM Madri JA Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor.J Clin Invest. 1992; 90: 15-23Crossref PubMed Scopus (194) Google Scholar Immunohistochemical studies have shown overexpression of EGF and its receptor (EGF-R) in epithelial cells lining ulcer margins and regenerating glands,8Tarnawski A Stachura J Durbin T Sarfeh IJ Gergely H Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats.Gastroenterology. 1992; 102: 695-698PubMed Google Scholar, 9Wright NA Pike C Elia G Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells.Nature. 1990; 43: 82-85Crossref Scopus (426) Google Scholar, 10Lee H Hansson H-A Norstrom E Helander HF Immuno-reactivities for epidermal growth factor (EGF) and for EGF receptors in rats with gastric ulcer.Cell Tissue Res. 1991; 265: 211-218Crossref PubMed Scopus (51) Google Scholar indicating that these cells are major targets for the proliferation-stimulating action of EGF. Our recent study demonstrated that gastric ulceration triggers increased receptor tyrosine kinase activity, EGF-R phosphorylation, and extracellular signal regulated kinase 1 and 2 (ERK1 and ERK2) activity in epithelial cells of the ulcer margins.11Pai R Ohta M Itani RM Sarfeh IJ Tarnawski A Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 14: 706-713Abstract Full Text Full Text PDF Scopus (90) Google Scholar Biochemical and genetic studies in various cell systems (other than gastric mucosa) have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras, but upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK).12Kyriakis JM App H Zhang XF Banerjee P Brautigan DL Rapp UR Avruch J Raf-1 activates MAP kinase-kinase.Nature. 1992; 358: 417-421Crossref PubMed Scopus (980) Google Scholar, 13Dent P Haser W Haystead TAJ Vincent LA Roberts TM Sturgill TW Activation of mitogen-activated protein kinase by v-Raf in NIH 3T3 cells and in vitro.Science. 1992; 257: 1404-1407Crossref PubMed Scopus (501) Google Scholar Raf-1 activity can be modulated by both Ras-dependent and Ras-independent pathways.14Morrison DK Mechanisms regulating Raf-1 activity in signal transduction pathways.Mol Reprod Dev. 1995; 42: 507-514Crossref PubMed Scopus (62) Google Scholar In addition to Ras, Raf-1 activators may include protein kinase C (PKC), activated tyrosine kinases, or as yet unidentified serine threonine kinases and phosphatases.14Morrison DK Mechanisms regulating Raf-1 activity in signal transduction pathways.Mol Reprod Dev. 1995; 42: 507-514Crossref PubMed Scopus (62) Google Scholar The intermediate steps linking activated receptor tyrosine kinase to MAP kinases (ERK1 and ERK2) during gastric ulcer healing in vivo remain unknown. In some cellular sytems other than gastric cells (eg, Rat1, A431, and Her14 cells), EGF-R activation has been shown to cause binding of the SH2-containing adapter protein (Shc) to growth factor receptor-bound protein (Grb2), leading to recruitment of Son of sevenless (Sos) to the plasma membrane and Ras activation.15Buday L Downward J Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (936) Google Scholar, 16Lowenstein EJ Daly RJ Batzer AG Li W Margolis B Lammers R Ullrich A Skolnik EY Bar-Sagi D Schlessinger J The SH2 and SH3 domain-containing protein Grb2 links receptor tyrosine kinases to ras signaling.Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1348) Google Scholar, 17Pellici G Lanfrancone L Grignani G McGlace J Cavallo F Forni G Nicoletti I Grignani F Pawson T Pellici PG A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1140) Google Scholar, 18Pawson T Schlessinger J SH2 and SH3 domains.Curr Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (577) Google Scholar Phospholipase C-γ (PLC-γ) is another signaling protein that contains SH domains and is activated by tyrosine kinase.19Rhee SG Inositol phospholipids-specific phospholipase C: interaction of the γ 1 isoform with tyrosine kinase.Trends Biochem Sci. 1991; 16: 297-301Abstract Full Text PDF PubMed Scopus (193) Google Scholar PLC-γ1 can act either as an enzyme that generates diacylglycerol and IP3, leading to PKC activation,20Hernandez-Sotomayor SMT Carpenter G Non-catalytic activation of phospholipase C-γ in vitro by epidermal growth factor receptor.Biochem J. 1993; 293: 507-511Crossref PubMed Scopus (29) Google Scholar, 21Meisenhelder J Suh P Rhee SG Hunter T Phospholipase C-γ is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro.Cell. 1989; 57: 1109-1122Abstract Full Text PDF PubMed Scopus (608) Google Scholar or as an adapter protein by binding to activated growth factor receptors via its SH2 domains and a downstream molecule via its SH3 domains.22Wang Z Gluck S Zhang L Moran MF Requirement for phospholipase C-γ1 enzyme activity in growth factor-induced mitogenesis.Mol Cell Biol. 1998; 18: 590-597Crossref PubMed Google Scholar PKC can activate MAPK, which in turn activates the gene transcription involved in cell proliferation and differentiation.23Sylvester PW Birkenfeld HP Hosick HL Briski KP Fatty acid modulation of epidermal growth factor-induced mouse mammary epithelial cell proliferation in vitro.Exp Cell Res. 1994; 214: 145-153Crossref PubMed Scopus (63) Google Scholar, 24Kazlauskas A Cooper JA Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42.J Cell Biol. 1988; 106: 1395-1402Crossref PubMed Scopus (89) Google Scholar, 25Schonwasser DC Marias RM Marshall CJ Parker PJ Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes.Mol Cell Biol. 1988; 18: 790-798Google Scholar, 26Blenis J Signal transduction via the MAP kinases: proceed at your own RSK.Proc Natl Acad Sci USA. 1993; 90: 5889-5892Crossref PubMed Scopus (1158) Google Scholar, 27Davis RJ The mitogen-activated protein kinase signal transduction pathway.J Biol Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar Growth factor-mediated activation of the Raf-1–MAPK/ERK cascade may therefore involve either Ras, PKC, or both.13Dent P Haser W Haystead TAJ Vincent LA Roberts TM Sturgill TW Activation of mitogen-activated protein kinase by v-Raf in NIH 3T3 cells and in vitro.Science. 1992; 257: 1404-1407Crossref PubMed Scopus (501) Google Scholar, 23Sylvester PW Birkenfeld HP Hosick HL Briski KP Fatty acid modulation of epidermal growth factor-induced mouse mammary epithelial cell proliferation in vitro.Exp Cell Res. 1994; 214: 145-153Crossref PubMed Scopus (63) Google Scholar, 24Kazlauskas A Cooper JA Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42.J Cell Biol. 1988; 106: 1395-1402Crossref PubMed Scopus (89) Google Scholar, 25Schonwasser DC Marias RM Marshall CJ Parker PJ Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes.Mol Cell Biol. 1988; 18: 790-798Google Scholar, 26Blenis J Signal transduction via the MAP kinases: proceed at your own RSK.Proc Natl Acad Sci USA. 1993; 90: 5889-5892Crossref PubMed Scopus (1158) Google Scholar, 27Davis RJ The mitogen-activated protein kinase signal transduction pathway.J Biol Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar Previous studies have shown increased tyrosine kinase activity and PLC-γ1 phosphorylation during the early phase of acute injury repair in rat gastric mucosa.28Relan NK Fligiel SEG Dutta S Tureaud J Chauhan DP Majumdar APN Induction of EGF-R tyrosine kinase during early reparative phase of gastric mucosa and effects of aging.Lab Invest. 1995; 73: 717-726PubMed Google Scholar, 29Majumdar APN Fligiel SEG Jaszewski R Tureaud J Dutta S Chelluderai B Inhibition of gastric mucosal regeneration by tyrphostin: evaluation of the role of epidermal growth factor receptor tyrosine kinase.J Lab Clin Med. 1996; 128: 173-180Abstract Full Text PDF PubMed Scopus (31) Google Scholar However, involvement of Ras or PKC in signaling pathways during chronic gastric ulcer healing is not known. The present study was aimed at determining whether during experimental gastric ulcer healing Raf-1 is activated and to identify potential activators of Raf-1 by examining the Ras and PKC protein levels, PKC activity, and Shc-Grb2, Grb2-Sos complex levels. To determine whether activation of the Raf-1–ERK cascade during gastric ulcer healing occurs predominantly in the epithelial component, we studied the effect of EGF on Ras activation, and PKC and ERK activities in an in vitro model using a rat gastric epithelial cell line (RGM1. derived from normal rat gastric mucosa. Kinase-inactive MEK, mouse monoclonal anti-PKC (MC5; recognizes α, β, and γ isoforms), anti-Ras, rabbit polyclonal anti-ERK2, and anti-Sos antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Shc antibody, mouse monoclonal anti-Grb2, and anti-c-Raf-1 antibodies were purchased from Transduction Laboratories (Lexington, KY). A Protein kinase C Assay System was purchased from Gibco BRL (Gaithersburg, MD). [γ-32P]ATP was purchased from Dupont NEN Research Products (Boston, MA), and all other molecular biology-grade chemicals were purchased from Sigma Chemical Company (St. Louis, MO). This study was approved by the Subcommittee for Animal Studies of the VA Medical Center (Long Beach, CA). Male Sprague-Dawley rats (Crl:CD(SD)BR; Charles River Laboratories; Wilmington, MA) were fasted for 12 hours and underwent laparotomy under Nembutal anesthesia (60 mg/kg body weight). One hundred percent acetic acid (50 μl) was applied to the serosa of lower gastric corpus at the posterior wall through a polyethylene tube (4.0 mm i.d.) for 90 seconds. The serosal area was then washed with isotonic saline and the abdomen closed. Sham-operated rats underwent a similar procedure without acetic acid administration. Rats with gastric ulcers (n = 54) and sham-operated rats (n = 36) were euthanized 3 and 7 days after operation. Ulcer margins were carefully dissected from granulation tissue, snap-frozen in liquid nitrogen, and stored at −80°C. Raf-1 kinase activity was determined using the method previously described.30Morrison DK Activation of Raf-1 by Ras in intact cells.Methods Enzymol. 1995; 255: 301-310Crossref PubMed Scopus (19) Google Scholar In brief, Raf-1 was immunoprecipitated from protein normalized tissue lysates (0.03 mg), using protein A Sepharose-antibody complex, and washed four times in lysis buffer. After the final wash, the immunoprecipitated Raf-1 complexes were resuspended and incubated for 20 minutes at 25°C in 40 μl kinase buffer containing 30 mmol/L HEPES (pH 7.4), 7 mmol/L manganese chloride, 5 mmol/L magnesium chloride, 1 mmol/L dithiothreitol, 15 μmol/L ATP, 10 μCi of [γ-32P]ATP (3000 Ci/mmol. Dupont NEN), and 4 units of kinase-inactive MEK. To terminate the assay, 15 μl of 4× Laemmli sample buffer was added and heated for 5 minutes at 100°C and analyzed by sodium dodecyl sulfate-polyacrylamide gel electophoresis (SDS-PAGE) and autoradiography. Quantification was performed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). To determine whether exogenous EGF could modulate Raf-1 activity during the healing process, ulcer margins from 3-day ulcers were carefully dissected and incubated with 10 ng/ml of EGF in Dulbecco's minimum essential medium (DMEM)/F12 serum-free medium for 15 and 30 minutes in a humidified chamber with 5% CO2. After incubations, tissue samples were snap-frozen in liquid nitrogen and homogenized in lysis buffer, and Raf-1 activity was determined, following the procedure outlined above. PKC activity was determined using an assay system described in our previous study.31Jones MK Sarfeh IJ Tarnawski A Induction of in vitro angiogenesis in the endothelial-derived cell line, EA hy926, by ethanol is mediated through PKC and MAPK.Biochem Biophys Res Commun. 1998; 249: 118-123Crossref PubMed Scopus (52) Google Scholar In brief, frozen tissue samples were lysed in 0.5 ml PKC homogenization buffer (20 mmol/L Tris-HCl, pH 7.4; 2 mmol/L EGTA; 2 mmol/L EDTA; 1% NP-40; 0.33 mol/L sucrose; 0.2 mmol/L sodium orthovanadate; 100 mmol/L sodium fluoride; 10 mmol/L sodium pyrophosphate; 10 μg/ml aprotinin; 10 μg/ml leupeptin; 1 mmol/L phenylmethylsulfonyl fluoride). The protein-normalized lysates were diluted in ice-cold dilution buffer (20 mmol/L Tris-HCl, pH 7.4; 0.2 mol/L NaCl; 0.5 mmol/L EGTA; 0.5 mmol/L EDTA; 10 mmol/L β-mercaptoethanol), and 25 μl (1 μg of protein) was assayed using the PKC assay system according to the manufacturer's instructions (Gibco BRL, Gaithersburg, MD). Frozen tissues were homogenized in ice-cold lysis buffer (20 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 50 mmol/L sodium fluoride, 30 mmol/L sodium pyrophosphate, 5 mol/L EGTA, 10% glycerol, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, and 5 μg/ml aprotinin) and clarified by centrifugation at 14,000 rpm for 10 minutes. The protein concentration of the lysate was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins were incubated with specific primary antibody immobilized onto protein A Sepharose for 2 hours at 4°C under gentle rotation. Beads were washed extensively with lysis buffer, and immune complexes were eluted by heating for 5 minutes at 95°C in Laemmli buffer and microcentrifuged. The supernatant was subjected to SDS-PAGE, followed by immunoblotting with specific antibodies (listed in Materials and Methods). Western blot analysis was performed following the method previously described.11Pai R Ohta M Itani RM Sarfeh IJ Tarnawski A Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 14: 706-713Abstract Full Text Full Text PDF Scopus (90) Google Scholar Tissue lysates containing equal amounts of proteins (0.15 mg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were stained with Ponceau Red to ensure equal loading and complete transfer of proteins. The membrane containing the transferred proteins was incubated with blocking buffer, subsequently washed, and incubated with specific primary antibodies (listed in Materials and Methods) for 1 hour at room temperature. Blots were washed and incubated with specific peroxidase-conjugated secondary antibodies for 1 hour at room temperature. After washing, bound antibody was visualized with the ECL detection system (Amersham Corp, IL) according to the manufacturer's instructions. The density of the protein bands was analyzed using a laser densitometer (UltroScan XL Laser Densitometer; Pharmacia LKB Biotechnology, Uppsala, Sweden). Because there is no currently available method allowing determination of Ras activation in vivo, we examined Ras activation in rat gastric epithelial cells. RGM1 cells were plated in 100-mm tissue culture dishes and grown until ∼80% confluent in DMEM/F12 supplemented with 20% fetal bovine serum. The cells were serum-starved for 16 hours and metabolically labeled for an additional 8 hours in serum-free, phosphate-free DMEM containing 200 μCi/ml32Downward J Graves JD Warne PH Rayter S Cantrell DA Stimulation of p21ras upon T-cell activation.Nature. 1990; 346: 719-723Crossref PubMed Scopus (687) Google Scholar PO4. To optimize the dose and time of EGF treatment on Ras activation, cells were treated with various concentrations of EGF (1–100 ng/ml) for 5–60 minutes. Ras activation was determined according to a previously described method.32Downward J Graves JD Warne PH Rayter S Cantrell DA Stimulation of p21ras upon T-cell activation.Nature. 1990; 346: 719-723Crossref PubMed Scopus (687) Google Scholar Briefly, Ras proteins contained in the cell lysates were immunoprecipitated with rat monoclonal anti-Ras antibody (Y13–259; Santa Cruz Biotechnology, Santa Cruz, CA). The guanine nucleotides bound to the Ras proteins were eluted in 16 μl of 2 mmol/L EDTA, 5 mmol/L dithiothreitol, 1 mmol/L GTP, 1 mmol/L GDP, 0.2% SDS at 68°C for 20 minutes and fractionated by thin-layer chromatography. Quantification was performed with a phosphorimager (Molecular Dynamics). The percentage of GTP bound to Ras (as an indicator of Ras activation) was calculated as cpm in GTP/(cpm in GTP + cpm in GDP) normalized for moles of phosphate in each nucleotide. ERK activity in RGM1 cells was determined as in our previous study.11Pai R Ohta M Itani RM Sarfeh IJ Tarnawski A Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 14: 706-713Abstract Full Text Full Text PDF Scopus (90) Google Scholar In brief, serum-starved cells were treated with various concentrations of EGF (1–100 ng/ml) for 5–60 minutes. After incubation, cells were washed in cold phosphate-buffered saline and lysed in lysis buffer (100 mmol/L NaCl, 50 mmol/L HEPES (pH 7.5), 1 mmol/L EDTA, 1% NP-40, 1 μmol/L pepstatin, 0.2 mmol/L phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 0.2 mmol/L sodium orthovanadate, 40 mmol/L paranitrophenyl phosphate). ERK2 was immunoprecipitated from protein-normalized cell lysates (0.03 mg), using protein A Sepharose-antibody complex. Beads were washed twice with lysis buffer and twice with wash buffer (10 mmol/L HEPES, 10 mmol/L magnesium acetate, pH 7.5). Kinase reactions were performed in 30 μl of tracer buffer (4.0 μCi/tube [γ-32P]ATP, 50 μmol/L ATP, 10 mmol/L magnesium acetate, 7.5 mmol/L HEPES, pH 7.5) and 10 μl of myelin basic protein (MBP) (3.0 mg/ml) for 30 minutes at 30°C. Then 20 μl of 2× Laemmli sample buffer was added to each tube, and the samples were boiled for 5 minutes and subjected to 10% SDS-PAGE. The gel was stained with Coomassie blue R250, dried, and autoradiographed. After this, individual bands were cut out and counted for isotope labeling by liquid scintillation spectrometry. All data are presented as mean ± SD. Student's t-test was used to determine the statistical significance between ulcers and gastric tissues of sham-operated rats. One-way analysis of variance followed by Bonferroni correction was used for multiple comparisons. A P value of <0.05 was considered to be statistically significant. Because our previous study showed induction of the ERK cascade during gastric ulcer healing,11Pai R Ohta M Itani RM Sarfeh IJ Tarnawski A Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 14: 706-713Abstract Full Text Full Text PDF Scopus (90) Google Scholar in the present study we attempted to determine the activators of this cascade. We determined the Raf-1 kinase activity by in vitro phosphorylation of inactive MEK, using immunoprecipitated Raf-1 kinase from normal (sham operated) and ulcerated gastric mucosa (Figure 1). In ulcerated gastric mucosa there was about a twofold increase in Raf-1 activity versus controls (P < 0.01). Treatment of dissected ulcer margins with exogenous EGF for 15 minutes caused a 1.1-fold (statistically nonsignificant) increase in Raf-1 activity versus ulcer margins treated with medium alone. Treatment of dissected ulcer margins with exogenous EGF for 30 minutes did not cause any significant change compared with ulcer margins treated with medium alone. PKC has been shown to modulate ERK activity in several cell systems.23Sylvester PW Birkenfeld HP Hosick HL Briski KP Fatty acid modulation of epidermal growth factor-induced mouse mammary epithelial cell proliferation in vitro.Exp Cell Res. 1994; 214: 145-153Crossref PubMed Scopus (63) Google Scholar, 24Kazlauskas A Cooper JA Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42.J Cell Biol. 1988; 106: 1395-1402Crossref PubMed Scopus (89) Google Scholar, 25Schonwasser DC Marias RM Marshall CJ Parker PJ Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes.Mol Cell Biol. 1988; 18: 790-798Google Scholar, 26Blenis J Signal transduction via the MAP kinases: proceed at your own RSK.Proc Natl Acad Sci USA. 1993; 90: 5889-5892Crossref PubMed Scopus (1158) Google Scholar, 27Davis RJ The mitogen-activated protein kinase signal transduction pathway.J Biol Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar To investigate whether PKC is involved in ERK activation during gastric ulcer healing, we determined PKC activity and protein levels in normal and ulcerated gastric mucosa. In ulcerated gastric mucosa, PKC activity was significantly reduced at 3 days (2.1-fold decrease; P < 0.002) and 7 days (1.4-fold decrease) compared to controls (sham operated) (Figure 2). PKC protein levels determined by Western blot analysis (Figure 3) showed a significant decrease at 3 days (1.9-fold decrease; P < 0.0001).Figure 3Gastric ulceration down-regulates PKC protein levels. Detergent-soluble lysates containing equal amounts of protein (0.15 mg) were subjected to Western blot analysis, using rabbit polyclonal anti-PKC antibody. A: Representative Western blot shows reduced PKC protein expression in ulcerated gastric mucosa when compared with normal (sham operated) controls. B: Quantitative analysis of PKC protein levels during ulcer healing. Data are expressed as the mean ± SD (n = 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because in some cell systems in vitro, growth factors such as EGF can induce activation of Ras through the recruitment of adapter proteins containing SH2 and SH3 domains such as Shc and Grb2,15Buday L Downward J Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (936) Google Scholar, 16Lowenstein EJ Daly RJ Batzer AG Li W Margolis B Lammers R Ullrich A Skolnik EY Bar-Sagi D Schlessinger J The SH2 and SH3 domain-containing protein Grb2 links receptor tyrosine kinases to ras signaling.Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1348) Google Scholar, 17Pellici G Lanfrancone L Grignani G McGlace J Cavallo F Forni G Nicoletti I Grignani F Pawson T Pellici PG A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1140) Google Scholar in the present study we examined whether activation of Raf-1 during ulcer healing in vivo involves these adapter proteins. Immunoprecipitation of Shc protein followed by immunoblotting with anti-Grb2 antibody revealed that gastric ulceration significantly increases Shc binding to Grb2 at both 3 and 7 days (Figure 4). At 3 days there was a 1.4-fold increase (P < 0.0001) and at 7 days there was a 1.2-fold increase (P < 0.0002) in Grb2 bound to Shc protein versus controls (sham operated). Because we found increased binding of Grb2 to Shc during ulcer healing, and because this association has been shown to result in recruitment of Sos and Ras activation,15Buday L Downward J Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (936) Google Scholar, 16Lowenstein EJ Daly RJ Batzer AG Li W Margolis B Lammers R Ullrich A Skolnik EY Bar-Sagi D Schlessinger J The SH2 and SH3 domain-containing protein Grb2 links receptor tyrosine kinases to ras signaling.Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1348) Google Scholar, 17Pellici G Lanfrancone L Grignani G McGlace J Cavallo F Forni G Nicoletti I Grignani F Pawson T Pellici PG A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1140) Google Scholar, 18Pawson T Schlessinger J SH2 and SH3 domains.Curr Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (577) Google Scholar we next examined whether the guanidine exchange factor Sos was bound to Grb2. This was analyzed by immunoprecipitation of Sos followed by immunoblotting with Grb2 antibody. In ulcerat
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