Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases
2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês
10.1074/jbc.m111357200
ISSN1083-351X
AutoresMichael Haas, Haojie Wang, Jiang Tian, Zijian Xie,
Tópico(s)Cellular transport and secretion
ResumoBinding of ouabain to Na+/K+-ATPase activates tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), Src, and p42/44 mitogen-activated protein kinases (MAPKs) in both cardiac myocytes and A7r5 cells. Here, we explored the roles of Src and the EGFR in the ouabain-invoked pathways that lead to the activation of MAPKs. Exposure of A7r5 and LLC-PK1 cells to ouabain caused a dose-dependent inhibition of Na+/K+-ATPase activity, which correlated well with ouabain-induced activation of Src and MAPKs in these cells. Immunoprecipitation experiments showed that ouabain stimulated Src binding to Na+/K+-ATPase in a dose- and time-dependent manner and increased phosphorylation of Src at Tyr418 but had no effect on Tyr529phosphorylation. Ouabain failed to activate MAPKs in A7r5 cells that were pretreated with the Src inhibitor PP2 and in SYF cells in which Src family kinases are knocked out. Preincubation with AG1478, but not AG1295, also blocked the effects of ouabain on p42/44 MAPKs in A7r5 cells. Significantly, both herbimycin A and PP2 abrogated ouabain-induced but not epidermal growth factor-induced Src binding to the EGFR and the subsequent EGFR tyrosine phosphorylation. Ouabain also failed to affect tyrosine phosphorylation of the EGFR in SYF cells. In addition, unlike epidermal growth factor, ouabain did not increase EGFR autophosphorylation at Tyr1173. These findings clearly indicate that ouabain transactivates the EGFR by activation of Src and stimulation of Src binding to the EGFR. Furthermore, we found that the transactivated EGFR was capable of recruiting and phosphorylating the adaptor protein Shc. This resulted in increased binding of another adaptor protein Grb2 to the Src-EGFR complex and the subsequent activation of Ras and MAPKs. Taken together, these new findings suggest that Src mediates the inter-receptor cross-talk between Na+/K+-ATPase and the EGFR to transduce the signals from ouabain to the Ras/MAPK cascade. Binding of ouabain to Na+/K+-ATPase activates tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), Src, and p42/44 mitogen-activated protein kinases (MAPKs) in both cardiac myocytes and A7r5 cells. Here, we explored the roles of Src and the EGFR in the ouabain-invoked pathways that lead to the activation of MAPKs. Exposure of A7r5 and LLC-PK1 cells to ouabain caused a dose-dependent inhibition of Na+/K+-ATPase activity, which correlated well with ouabain-induced activation of Src and MAPKs in these cells. Immunoprecipitation experiments showed that ouabain stimulated Src binding to Na+/K+-ATPase in a dose- and time-dependent manner and increased phosphorylation of Src at Tyr418 but had no effect on Tyr529phosphorylation. Ouabain failed to activate MAPKs in A7r5 cells that were pretreated with the Src inhibitor PP2 and in SYF cells in which Src family kinases are knocked out. Preincubation with AG1478, but not AG1295, also blocked the effects of ouabain on p42/44 MAPKs in A7r5 cells. Significantly, both herbimycin A and PP2 abrogated ouabain-induced but not epidermal growth factor-induced Src binding to the EGFR and the subsequent EGFR tyrosine phosphorylation. Ouabain also failed to affect tyrosine phosphorylation of the EGFR in SYF cells. In addition, unlike epidermal growth factor, ouabain did not increase EGFR autophosphorylation at Tyr1173. These findings clearly indicate that ouabain transactivates the EGFR by activation of Src and stimulation of Src binding to the EGFR. Furthermore, we found that the transactivated EGFR was capable of recruiting and phosphorylating the adaptor protein Shc. This resulted in increased binding of another adaptor protein Grb2 to the Src-EGFR complex and the subsequent activation of Ras and MAPKs. Taken together, these new findings suggest that Src mediates the inter-receptor cross-talk between Na+/K+-ATPase and the EGFR to transduce the signals from ouabain to the Ras/MAPK cascade. Na+/K+-ATPase, or the sodium pump, is a ubiquitous transmembrane enzyme that has long been ascribed the function of transporting Na+ and K+ across the plasma membrane (1Skou J.C. Methods Enzymol. 1988; 156: 1-25Crossref PubMed Scopus (108) Google Scholar, 2Lingrel J.B. Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar, 3Kelly R.A. Smith T.W. J. Am. Coll. Cardiol. 1993; 22: 107A-112ACrossref PubMed Scopus (44) Google Scholar). By specific binding to the extracellular surface of the α-subunit of Na+/K+-ATPase, cardiotonic steroids such as ouabain inhibit the ion pumping function of the enzyme. In recent years, we have demonstrated that the binding of ouabain to cardiac Na+/K+-ATPase can also activate multiple signal transduction pathways including activation of Ras and p42/44 MAPKs 1The abbreviations used are: MAPKmitogen-activated protein kinaseEGFepidermal growth factorEGFREGF receptorPDGFplatelet-derived growth factorPDGFRPDGF receptorRIPAradioimmune precipitation bufferSHSrc homology 1The abbreviations used are: MAPKmitogen-activated protein kinaseEGFepidermal growth factorEGFREGF receptorPDGFplatelet-derived growth factorPDGFRPDGF receptorRIPAradioimmune precipitation bufferSHSrc homology and increased production of reactive oxygen species (4Xie Z. Cell. Mol. Biol. 2001; 47: 383-390PubMed Google Scholar, 5Peng M. Huang L. Xie Z. Huang W.-H. Askari A. J. Biol. Chem. 1996; 271: 10372-10378Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 6Huang L., Li, H. Xie Z. J. Mol. Cell. Cardiol. 1997; 29: 429-437Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 7Kometiani P., Li, J. Gnudi L. Kahn B.B. Askari A. Xie Z. J. Biol. Chem. 1998; 273: 15249-15256Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 8Xie Z. Kometiani P. Liu J., Li, J. Shapiro J.I. Askari A. J. Biol. Chem. 1999; 274: 19323-19328Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Interestingly, the activation of some of the signal transduction pathways by ouabain is independent of the ouabain-induced changes in intracellular ion concentrations as well as the contractility of cardiac myocytes (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar,10Liu J. Tian J. Haas M. Shapiro J.I. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27838-27844Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Significantly, we have recently shown that the classic effects of ouabain on intracellular calcium also depend on the signal transducing function of Na+/K+-ATPase (11Tian J. Gong X. Xie Z. Am. J. Physiol. 2001; 281: H1899-H1907Crossref PubMed Google Scholar). Inhibition of either protein tyrosine phosphorylation or Ras/p42/44 MAPKs diminishes ouabain-induced increases in intracellular calcium. Furthermore, interplays among different pathways (e.g. MAPKs, calcium, and reactive oxygen species) in cardiac myocytes eventually lead to hypertrophic growth and changes in the expression of multiple growth-related genes (4Xie Z. Cell. Mol. Biol. 2001; 47: 383-390PubMed Google Scholar). mitogen-activated protein kinase epidermal growth factor EGF receptor platelet-derived growth factor PDGF receptor radioimmune precipitation buffer Src homology mitogen-activated protein kinase epidermal growth factor EGF receptor platelet-derived growth factor PDGF receptor radioimmune precipitation buffer Src homology Because there is sufficient evidence that activation of the Ras/MAPKs is important for regulating cell function and growth, we have recently initiated studies to explore the upstream signaling events that emanate from the binding of ouabain to Na+/K+-ATPase. Traditionally, binding of the growth factor to its receptor results in receptor dimerization and autophosphorylation (12Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4609) Google Scholar, 13Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2164) Google Scholar). The tyrosine-phosphorylated receptor can then serve as a scaffold to recruit Src and adaptor proteins, resulting in activation of the Ras/MAPK cascade. More recently, the EGFR and Src have been identified as critical elements that relay the signals from G protein-coupled receptors, cytokines, and cellular stress to a variety of cellular responses, which include the activation of MAPKs (14Prenzel N. Fischer O.M. Streit S. Hart S. Ullrich Endocr. Relat. Cancer. 2001; 8: 11-31Crossref PubMed Scopus (560) Google Scholar, 15Abram C.L. Courtneidge S.A. Exp. Cell Res. 2000; 254: 1-13Crossref PubMed Scopus (341) Google Scholar, 16Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar, 17Ma Y. Huang J. Ali S. Lowry W. Huang X. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 18Chen K. Vita J.A. Berk B.C. Keaney J.F., Jr. J. Biol. Chem. 2001; 276: 16045-16050Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 19Andreev J. Galisteo M.L. Kranenburg O. Logan S.K. Chiu E.S. Okigaki M. Cary L.A. Moolenaar W.H. Schlessinger J. J. Biol. Chem. 2001; 276: 20130-20135Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Our previous work demonstrated that ouabain caused a rapid activation of Src and stimulated tyrosine phosphorylation of the EGFR in both cardiac myocytes and A7r5 cells (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Furthermore, inhibition of tyrosine kinases by either genistein or herbimycin A antagonized the effects of ouabain on MAPKs. These findings led us to postulate that the activation of Src by ouabain was sufficient to transactivate the EGFR, resulting in the recruitment of Shc and Grb2 to the receptor and subsequent activation of the Ras/MAPK cascade (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). The present studies were performed to test this hypothesis and delineate the molecular mechanisms of ouabain-induced activation of Src and transactivation of the EGFR. Chemicals of the highest purity were used. Herbimycin A, PP2, tyrphostin AG1295, and tyrphostin AG1478 were obtained from Calbiochem. Sodium binding benzofuran isophthalate acetoxymethyl ester was from Molecular Probes (Eugene, OR). [γ-32P]ATP was obtained from PerkinElmer Life Sciences. The following antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): monoclonal anti-phosphotyrosine antibody (PY99), polyclonal anti-MAPK antibody, monoclonal anti-phospho-MAPK antibody, and goat anti-rabbit and goat anti-mouse secondary antibodies. The following antibodies and kits were obtained from Upstate Biotechnology (Lake Placid, NY): monoclonal anti-Src (clone GD11), anti-Ras (clone RAS10), anti-Grb2, and anti-phospho-EGFR (Tyr1173) (clone 9H2) antibodies, polyclonal anti-Shc and anti-EGFR antibodies, Src kinase activity and Ras activation kits, and protein G-agarose. The polyclonal anti-Src (pY418) and anti-Src (pY529) antibodies were from BIOSOURCE. The monoclonal anti-α1 antibody (α6F) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA). The Optitran nitrocellulose membranes used for Western blotting were obtained from Schleicher and Schuell. Rat A7r5, pig LLC-PK1, and mouse SYF and SYF + c-Src cells were obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). When cell cultures reached about 80% confluence, A7r5 and LLC-PK1 cells were serum-starved for 24 h and used for the experiments, whereas SYF and SYF + c-Src cells were cultured in medium containing 0.5% fetal bovine serum for 24 h before being used for the experiments. Immunoblotting was performed to identify increases in tyrosine phosphorylation and activation of p42/44 MAPK using the antibodies described under "Materials" (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Dilutions of these antibodies were done as recommended by the manufacturer. After the indicated treatment, the incubation medium was rapidly replaced with 5 ml of ice-cold phosphate-buffered saline. The washed cells were then lysed in 200 μl of ice-cold RIPA containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 1 mm NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 50 mm Tris-HCl (pH 7.4). Cell lysates were centrifuged at 16,000 × g for 10 min, and the supernatants were used for Western blot analysis. Samples were separated by SDS-PAGE (60 μg/lane) and transferred to an Optitran membrane as described previously (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). The membranes were then probed with an anti-phosphotyrosine monoclonal antibody or an anti-phospho-MAPK monoclonal antibody. The anti-phospho-MAPK monoclonal antibody was then stripped, and the membrane was reprobed with a polyclonal antibody that recognizes the total amount of MAPK to account for equal loading as reported previously (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Autoradiograms were scanned with a Bio-Rad densitometer to quantitate MAPK signals as described previously (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). To assay Src kinase activity, cells were lysed in RIPA at 4 °C. The insoluble material was removed by centrifugation at 16,000 × g for 10 min, and the cell lysates were immunoprecipitated using the Src monoclonal antibody and protein G-agarose. The immunoprecipitate was washed twice with RIPA and twice with ice-cold phosphate-buffered saline. Src kinase activity was assayed as described previously (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Cell lysates made in RIPA were immunoprecipitated using a polyclonal anti-EGFR antibody that has established reactivity with the rat receptor. The immunoprecipitate was dissolved in sample buffer, separated on 10% SDS-PAGE, and blotted with the monoclonal anti-phosphotyrosine antibody. To determine whether EGFR binds to Src, Shc, and Grb2, the same blots were stripped and reprobed with the monoclonal anti-Src antibody, the polyclonal anti-Shc antibody, and the monoclonal anti-Grb2 antibody. To determine whether the EGFR is autophosphorylated at its major phosphorylation site (Tyr1173), the lysates made in RIPA were immunoprecipitated using a polyclonal anti-EGFR antibody. The immunoprecipitate was dissolved in SDS sample buffer, separated by 10% SDS-PAGE, and blotted with a monoclonal anti-phospho-EGFR antibody raised against peptide NAE[pY]LRV that corresponds to the protein sequence around Tyr1173 of the EGFR. RIPA cell lysates were immunoprecipitated with either a polyclonal anti-α1antibody or a monoclonal anti-Src antibody as described in the preceding paragraph. Immunoprecipitates were then separated by SDS-PAGE and immunoblotted for α1 and Src. To assay for Ras activation, cells were collected in a Mg2+-containing lysis buffer (MLB) containing 150 mm NaCl, 1% Igepal CA-630, 0.25% sodium deoxycholate, 10% glycerol, 25 mm NaF, 10 mmMgCl2, 1 mm EDTA, 1 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 25 mm HEPES (pH 7.5). Active Ras was then affinity-precipitated by adding 20 μl of the Raf-1 Ras binding domain-agarose conjugate to the cell lysates and gently rocking them for 30 min at 4 °C. The agarose beads were washed three times with MLB, dissolved in 50 μl of 2× SDS sample buffer, and boiled for 5 min. The samples were separated by 10% SDS-PAGE, transferred to a supported nitrocellulose membrane, and blotted with a monoclonal anti-Ras antibody. Whole cell total Na+/K+-ATPase activity was assayed in the presence of alamethicin as described previously (20Xie Z. Wang Y. Ganjeizadeh M. McGee R. Askari A Anal. Biochem. 1989; 183: 215-219Crossref PubMed Scopus (62) Google Scholar). Briefly, cells were collected from the cultures and treated with alamethicin at a concentration of 0.1 mg/mg protein. Na+/K+-ATPase activity was measured by the determination of the initial rate of release of Pi from ATP. The reaction was carried out in a final volume of 1 ml of reaction mixture containing 100 mm NaCl, 20 mm KCl, 3 mm MgCl2, 1 mm EGTA, 5 mm NaN3, and 2 mm[γ-32P]ATP. To determine the ouabain concentration curves, whole cell lysates were preincubated with different concentrations of ouabain for 15 min. The reaction was started by the addition of an aliquot of cell lysate (50 μg). Intracellular Na+ concentration was measured using sodium binding benzofuran isophthalate as a probe as described previously (10Liu J. Tian J. Haas M. Shapiro J.I. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27838-27844Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Data are given as the mean ± SE. Statistical analysis was performed using Student's t test, and significance was accepted at p < 0.05. Each presented immunoblot is representative of the similar results of at least three separate experiments. We have shown previously that ouabain activates Src and increases tyrosine phosphorylation of the EGFR in several cell types (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Although it is well established that Na+/K+-ATPase is the specific receptor for cardiotonic steroids such as ouabain, it is always possible that some of the novel effects of ouabain may be due to its binding to other cellular proteins, especially at higher ouabain concentrations. To address this issue, we compared the dose-dependent effects of ouabain on Na+/K+-ATPase activity and MAPK activation. When Na+/K+-ATPase activity was measured in A7r5 and LLC-PK1 cells as a function of ouabain concentration, it is clear that LLC-PK1 cells express a highly ouabain-sensitive isoform of Na+/K+-ATPase (Fig. 1). The enzyme was inhibited 50% in LLC-PK1 cells when the cells were exposed to about 1 μmouabain. In contrast, 100 μm ouabain was required to cause the same degree of inhibition of Na+/K+-ATPase activity in A7r5 cells. Interestingly, when Src and MAPKs were measured in these cells as a function of ouabain concentration, we found that the effects of ouabain on both MAPKs and Src correlated well with the inhibition curves of ouabain on Na+/K+-ATPase in these cells (Figs. 2 and3). To ensure that ouabain-induced activation of MAPKs and Src is not a result of significant changes in intracellular Na+ or depolarization of the membrane potential due to inhibition of the ion pumping function of Na+/K+-ATPase, we measured the effects of ouabain on intracellular Na+ as a function of time in A7r5 cells loaded with sodium binding benzofuran isophthalate. Consistent with our prior studies (10Liu J. Tian J. Haas M. Shapiro J.I. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27838-27844Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), exposure of A7r5 cells to 100 μm ouabain for 20 min did not change the intracellular Na+ concentration (17.1 ± 1.2 versus17.2 ± 1.3 mm). Therefore, it is unlikely that the effects of ouabain on MAPKs are due to changes in either intracellular Na+ or membrane potential. This notion is further supported by the following observations. First, although the addition of monensin caused a large increase in intracellular Na+, it failed to activate MAPKs (99.7 ± 9.6% of control; n = 3). Second, increasing extracellular K+ from 5 to 20 mm, which should cause partial depolarization of the plasma membrane, also showed no significant effect on MAPKs (114.8 ± 5.4% of control; n = 3).Figure 2Dose-dependent effects of ouabain on MAPKs in both A7r5 and LLC-PK1 cells. Cells were treated with different concentrations of ouabain for 5 min and assayed for MAPKs as described under "Experimental Procedures." The values are the mean ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of ouabain on Src kinase activity in both A7r5 and LLC-PK1 cells. A7r5 and LLC-PK1 cells were exposed to 100 and 1 μm ouabain, respectively, for the indicated times. The cells were lysed, immunoprecipitated with a monoclonal anti-Src antibody, and assayed for Src kinase activity as described under "Experimental Procedures." The values are the mean ± S.E. of at least three independent experiments. Data are expressed relative to a control value of 1. ∗, p < 0.05; ∗∗, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We showed that inhibition of protein tyrosine kinases by a nonspecific tyrosine kinase inhibitor (genistein) or a relatively selective Src inhibitor (herbimycin A) abolished ouabain-induced stimulation of p42/44 MAPKs (9Haas M. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27832-27837Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). To obtain additional evidence that Src is involved in ouabain-induced activation of MAPKs, A7r5 cells were pretreated with another Src-specific inhibitor, PP2, and then exposed to 100 μm ouabain and assayed for MAPK activation. As depicted in Fig. 4, inhibition of Src completely abrogated ouabain-induced but not EGF-induced activation of MAPKs. The data supported our previous proposition that Src plays a key role in ouabain-activated signaling pathways. They also indicate that ouabain and EGF signal differently in A7r5 cells. Because both PP2 and herbimycin A might have nonspecific effects on other kinases, we employed both SYF and SYF + c-Src cells in the experiments of Fig. 5 to further test the hypothesis that Src relays the signal from ouabain to MAPKs. The SYF cells are derived from mouse embryos harboring functional null mutations in both alleles of the Src family kinases Src, Yes, and Fyn. The SYF + c-Src cells are the stable transfectants of SYF cells that express c-Src. As shown in Fig. 5, ouabain activated p42/44 MAPKs in a dose-dependent manner in the SYF + c-Src cells. However, under the same experimental conditions, ouabain failed to stimulate MAPKs in the SYF cells. Clearly, activation of Src is critical for ouabain to stimulate MAPKs.Figure 5Dose-dependent effects of ouabain on MAPKs in SYF and SYF + c-Src cells. Cells were treated with different concentrations of ouabain for 5 min and assayed for MAPKs as described in the Fig. 2 legend. Control experiments showed that ouabain-induced activation of MAPKs reached maximal levels after the cells were exposed to ouabain for 5 min. A, a representative Western blot is shown. B, quantitation of the effects of ouabain on p44 MAPK measured in three independent experiments. Data are expressed relative to a control value of 1. ∗,p < 0.05; ∗∗, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because receptor tyrosine kinases are involved in transmitting signals from H2O2 and angiotensin II to MAPKs, the effects of either AG1478 or AG1295 on ouabain-induced activation of MAPKs were studied in the experiments of Fig. 6. Both AG1478 and AG1295 belong to the tyrphostin family of receptor tyrosine kinase inhibitors. They are ATP analogues and specific toward the ATP binding sites of receptor tyrosine kinases. The selectivity and dose-dependent effects of these inhibitors on MAPK activation induced by either EGF or PDGF had been assessed previously in rat vascular smooth muscle cells (25Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). These studies showed that AG1478 completely blocked the effects of EGF (50 ng/ml) on MAPKs at 250 nm, whereas AG1295 abolished the effects of PDGF (30 ng/ml) at 25 μm. Control experiments showed that both AG1478 (250 nm) and AG1295 (25 μm) had no effect on cell morphology and viability (assessed by trypan blue exclusion) when A7r5 cells were exposed to them for 1 h. Therefore, A7r5 cells were exposed to either 250 nm AG1478 or 25 μm AG1295 and then treated with 100 μm ouabain. Interestingly, whereas AG1478 blocked the effects of both EGF and ouabain on MAPKs (Fig. 6), AG1295 inhibited the effects of PDGF but not ouabain on MAPKs. To ensure that the effects of AG1478 on ouabain-induced activation of MAPKs are specifically mediated through inhibition of the EGFR, the effects of 10 nm AG1478 on MAPK activation were also assessed. Previous studies on rat vascular smooth muscle cells indicated that 10 nm AG1478 caused about 50% inhibition of EGF-induced MAPK activation (25Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). We found that AG1478 at the same concentration inhibited about 37% of both EGF- and ouabain-induced activation of MAPKs. Taken together, the above findings indicate that ouabain signals differently than both EGF and PDGF and demonstrate an involvement of the EGFR, but not the PDGFR, in ouabain-induced activation of MAPKs. The above findings firmly established the role of Src and the EGFR in ouabain-induced activation of MAPKs. To understand how ouabain transduces signals through Src and the EGFR, we first determined the effects of ouabain on Src tyrosine phosphorylation. It is well established that Src kinase activity is regulated by the phosphorylation of Tyr418 and Tyr529. Either decreases in phosphorylation of Tyr529 by activation of a phosphotyrosine phosphatase or increases in phosphorylation of Tyr418 by binding of Src to a regulatory protein stimulates Src kinase activity. As shown in Fig. 7, ouabain caused a significant increase in Tyr418phosphorylation but had no effect on Tyr529phosphorylation. Because there is evidence that binding of a regulatory protein to either the SH2, SH3, or kinase domain of Src can activate kinase activity by stimulation of Tyr418 phosphorylation, we tested whether ouabain regulates Src by increasing its interaction with Na+/K+-ATPase. In the experiments of Fig. 8, we immunoprecipitated either α1 or Src from both control and ouabain-treated LLC-PK1 cells. LLC-PK1 cells were chosen for the immunoprecipitations because these cells express high levels of ouabain-sensitive Na+/K+-ATPase. Initial experiments with A7r5 cells proved to be difficult because each immunoprecipitation needed more than 2 mg of cell lysates to give a detectable signal of α1 by Western blot. As shown in Figs. 8 and 9, ouabain increased Src binding to Na+/K+-ATPase in a dose- and time-dependent manner. The maximal effects of ouabain on Src binding were observed after the cells were incubated with ouabain for 2–5 min and decreased thereafter.Figure 8Time-dependent effects of ouabain on Src binding to Na+/K+-ATPase.A, LLC-PK1 cells were treated with 1 μmouabain for the times indicated. The cells were then lysed in RIPA, immunoprecipitated with an anti-α1 antibody, and assayed for Src binding by Western blot using an anti-Src antibody. A representative immunoblot is shown. B, quantitation data from four independent experiments. Data are expressed relative to a control value of 1. C, LLC-PK1 cells were treated as described in A. The cells were then lysed in RIPA, immunoprecipitated with an anti-Src antibody, and assayed for α1 binding by Western blot using the α6F antibody. A representative immunoblot is shown. D, quantitation data from four independent experiments. Data are expressed relative to a control value of 1. ∗, p < 0.05; ∗∗,p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Dose-dependent effects of ouabain on Src binding to the Na+/K+-ATPase. LLC-PK1 cells were treated with various concentrations of ouabain for 5 min. The cells were then lysed in RIPA, immunoprecipitated with an anti-α1 antibody, and assayed for Src binding by Western blot using an anti-Src antibody. A representative immunoblot from four independent experiments is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because Src can mediate inter-receptor communications, the above findings prompted us to hypothesize that the Src-mediated inter-receptor cross-talk between Na+/K+-ATPase and the EGFR is essential for ouabain-induced activation of MAPKs. To test this hypothesis, we assessed the role of Src in ouabain-induced transactivation of the EGFR. In the experim
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