FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2004103569> ?p ?o ?g. }

• W2004103569 endingPage "15932" @default.
• W2004103569 startingPage "15926" @default.
• W2004103569 abstract "Several signal transduction events induced by angiotensin II (AngII) binding to the angiotensin II type 1 receptor resemble those evoked by platelet-derived growth factor (PDGF) binding to the PDGF-β receptor (PDGFβ-R). We report here, in agreement with previous data, that AngII and PDGF-B-chain homodimer (PDGF-BB) stimulate tyrosine phosphorylation of the PDGFβ-R. Both AngII and PDGF-BB stimulated the phosphorylation of PDGFβ-R via the binding of tyrosine-phosphorylated Shc to PDGFβ-R. Both PDGF-BB- and AngII-induced phosphorylation of the Shc·PDGFβ-R complex was inhibited by antioxidants such as N-acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGFβ-R by AngII (measured by PDGFβ-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGFβ-R phosphorylation includes differences in the time course of PDGFβ-R phosphorylation, differing effects of inhibitors of the endogenous PDGFβ-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGFβ-R was directly immunoprecipitated (PDGFβ-R-antibody) versuscoimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc·PDGFβ-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand. Several signal transduction events induced by angiotensin II (AngII) binding to the angiotensin II type 1 receptor resemble those evoked by platelet-derived growth factor (PDGF) binding to the PDGF-β receptor (PDGFβ-R). We report here, in agreement with previous data, that AngII and PDGF-B-chain homodimer (PDGF-BB) stimulate tyrosine phosphorylation of the PDGFβ-R. Both AngII and PDGF-BB stimulated the phosphorylation of PDGFβ-R via the binding of tyrosine-phosphorylated Shc to PDGFβ-R. Both PDGF-BB- and AngII-induced phosphorylation of the Shc·PDGFβ-R complex was inhibited by antioxidants such as N-acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGFβ-R by AngII (measured by PDGFβ-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGFβ-R phosphorylation includes differences in the time course of PDGFβ-R phosphorylation, differing effects of inhibitors of the endogenous PDGFβ-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGFβ-R was directly immunoprecipitated (PDGFβ-R-antibody) versuscoimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc·PDGFβ-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand. angiotensin II angiotensin type 1 receptor G-protein coupled receptor vascular smooth muscle cells extracellular signal-regulated kinases janus kinase epidermal growth factor epidermal growth factor receptor platelet-derived growth factor B-chain homodimer platelet-derived growth factor β receptor tumor necrosis factor 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl reactive oxygen species Angiotensin II (AngII),1an octapeptide pressor hormone, activates cellular events that may contribute to the pathogenesis of cardiovascular disease (1.Griendling K.K. Ushio F.-M. Lassegue B. Alexander R.W. Hypertension. 1997; 29: 366-373Crossref PubMed Google Scholar, 2.Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (88) Google Scholar). The physiological actions of AngII are mediated largely via the AngII type 1 receptor (AT1R) (3.Oliverio M.I. Best C.F. Kim H.S. Arendshorst W.J. Smithies O. Coffman T.M. Am. J. Physiol. 1997; 272: F515-F520PubMed Google Scholar), which is a G protein-coupled receptor (GPCR). GPCRs share a common basic structure of seven transmembrane helices connected by alternating cytoplasmic and extracellular loops (4.Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar). AngII-mediated growth effects in target cells such as vascular smooth muscle cells (VSMC) and cardiac myocytes require the rapid activation of several mitogen-activated protein kinases including the extracellular signal-regulated kinases (ERK1/2) (5.Weber H. Taylor D.S. Molloy C.J. J. Clin. Invest. 1994; 93: 788-798Crossref PubMed Scopus (145) Google Scholar). The signal transduction pathway that leads to ERK1/2 activation upon AngII binding to the AT1R is still incompletely characterized. There is evidence that binding of AngII to the AT1R stimulates tyrosine phosphorylation of several proteins including Shc and GRB-2 prior to activation of members of the Ras family (6.Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Crossref PubMed Scopus (232) Google Scholar). Rapid tyrosine phosphorylation is likely mediated by several tyrosine kinases including Src family tyrosine kinases, JAK2 and PYK2 (7.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar, 8.Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Interestingly, certain aspects of the signal transduction pathways induced by AngII resemble those evoked by classic mitogenic growth factors (9.Berk B.C. Alexander R.W. Brock T.A. Gimbrone M.A. Webb Jr., R.C. Science. 1986; 232: 87-90Crossref PubMed Scopus (340) Google Scholar). In fact, recent studies show that GPCRs, including the AT1R, transactivate growth factor receptors, which possess intrinsic tyrosine kinase activity, including the EGF receptor (EGF-R) and PDGF receptor (PDGF-R) (10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12.Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar). It has been suggested that the EGF-R and the PDGF-R mediate several of the cellular effects of AngII (8.Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 11.van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar). In cardiac fibroblasts, Moriguchi et al. (13.Moriguchi Y. Matsubara H. Mori Y. Murasawa S. Masaki H. Maruyama K. Tsutsumi Y. Shibasaki Y. Tanaka Y. Circ. Res. 1999; 84: 1073-1084Crossref PubMed Scopus (109) Google Scholar) showed recently that ERK1/2 activation by AngII was mediated via EGF-R. Further elucidation of the mechanisms of transactivation of the EGF-R by AngII has revealed a Ca2+/calmodulin-dependent process that involves the endogenous EGF-R tyrosine kinase (12.Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar). Phosphorylation of the PDGFβ-R by AngII in VSMC has been described previously by Linseman (10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), but the mechanisms for PDGFβ-R phosphorylation remain poorly defined. In the present study, we compared the signaling events involved in AngII-induced PDGFβ-R transactivation with events involved in PDGF-B-chain homodimer (PDGF-BB)-mediated tyrosine phosphorylation of the PDGFβ-R. We show that AngII stimulates phosphorylation of the PDGFβ-R via the adaptor protein Shc more rapidly than PDGF itself and that PDGFβ-R phosphorylation by AngII is not dependent on calcium. Both Shc and PDGFβ-R phosphorylation induced by PDGF-BB and AngII were completely abolished by the antioxidants Tiron and N-acetylcysteine. However, phosphorylation of the PDGFβ-R by AngII and PDGF-BB occurred via different pathways as shown by different subcellular location and sensitivity to kinase inhibitors. Cell culture media and protein G-agarose were from Life Technologies, Inc. Polyclonal antibody against Shc, monoclonal anti-phosphotyrosine antibody (4G10), and polyclonal antibody against PDGFβ-R were from Upstate Biotechnology (Lake Placid, NY). Polyclonal anti-phospho-specific ERK1/2 antibody was from New England Biolabs. Recombinant (human) TNF and recombinant (human) PDGF-BB were from Sigma. BAPTA-AM, AG1296 (PDGF-R tyrosine kinase inhibitor), and PP-1 were from Calbiochem. Rat aortic VSMC were isolated from the thoracic aorta of 200–250-g male Harlan Sprague-Dawley rats and maintained in Dulbecco's modified Eagle's medium supplemented with 10% serum as described (14.Ishida M. Ishida T. Thomas S. Berk B.C. Circ. Res. 1998; 82: 7-12Crossref PubMed Scopus (150) Google Scholar). Passages 8–14 at 60–70% confluence were growth arrested by incubation in Dulbecco's modified Eagle's medium with 0.1% serum for 48 h before use. The methods for immunoprecipitation and immunoblot analysis were described previously (15.Schmitz U. Ishida T. Ishida M. Surapisitchat J. Hasham M.I. Pelech S. Berk B.C. Circ. Res. 1998; 82: 1272-1278Crossref PubMed Scopus (84) Google Scholar). In brief, growth-arrested VSMC were stimulated with AngII or PDGF-BB as indicated in each experiment. Cells were lysed in Triton/Nonidet P-40 lysis buffer (0.5% Triton, 0.5% Nonidet P-40, 10 mm Tris (pH 7.5), 2.5 mm KCl, 150 mm NaCl, 20 mm β-glycerol phosphate, 50 mm NaF, 1 mm orthovanadate, 10 μg/ml leupeptin, 1 mm dithiothreitol, 10 μg/ml soybean trypsin inhibitor, and 200 mm benzamidine), scraped off the dish, and centrifuged. Lysates containing equal amounts of protein were precleared and incubated with antibodies overnight at 4 °C. Antibody complexes were collected by incubation with protein G-agarose for 3 h at 4 °C. Precipitates were washed four times with the Triton/Nonidet P-40 lysis buffer and then resuspended in SDS sample buffer. Samples were separated by SDS-polyacrylamide gel electrophoresis (8–12%) and transferred to nitrocellulose membranes. After incubation in blocking solution (1% bovine serum albumin, 10 mm Tris (pH 7.5), 100 mm NaCl, 0.1% Tween 20), membranes were incubated with primary antibodies. After washing (Tris-buffered saline, 0.03% Tween 20), the blots were incubated with the appropriate secondary antibodies. The membranes were washed and proteins were detected by the ECL system (Amersham Pharmacia Biotech). To compare phosphorylation of the PDGβ-R by AngII and PDGF-BB, the PDGFβ-R was immunoprecipitated from cell lysates treated with AngII (100 nm) or PDGF-BB (30 ng/ml) for 2 min. Immunoblotting with an antibody against tyrosine-phosphorylated proteins showed phosphorylation of a ∼180-kDa band (Fig.1 A, upper panel) which was identified as the PDGFβ-R by stripping and reprobing with PDGFβ-R antibody (Fig. 1 A, lower panel). Phosphorylation of the PDGFβ-R by AngII at 2 min was ∼50% of phosphorylation by PDGF-BB itself. The adaptor protein Shc exists as three isoforms, 66 kDa, 52 kDa, and 46 kDa, and is a likely candidate to mediate cross-talk between the PDGFβ-R and the AT1R by assembling a signal transduction complex at the PDGFβ-R (7.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar, 10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Therefore, AngII-induced PDGFβ-R phosphorylation may be studied by coimmunoprecipitation of Shc and the PDGFβ-R. AngII and PDGF-BB both stimulated tyrosine phosphorylation of only the 66-kDa isoform of Shc (Fig. 1 B, upper panel), whereas the two smaller Shc isoforms were not involved in PDGFβ-R transactivation in our cell system. Furthermore, tyrosine-phosphorylated Shc was part of a signal transduction complex that included the PDGFβ-R as shown by coimmunoprecipitation of the PDGFβ-R with Shc (Fig. 1 B, lower panel). To determine whether the signaling events induced by AngII and PDGF-BB which cause PDGFβ-R phosphorylation were similar, the time course for tyrosine phosphorylation of the PDGFβ-R was studied. AngII caused a more rapid and transient phosphorylation of the PDGFβ-R than did PDGF-BB (Fig. 2). AngII-induced phosphorylation of the PDGFβ-R peaked at 1 min (23.4 ± 3.8-fold) and returned to base line after 10 min, when immunoprecipitated with antibody against the PDGFβ-R. A similar increase (24.6 ± 3.4-fold) in PDGFβ-R phosphorylation was observed when coimmunoprecipitated with antibody against Shc (Fig. 2). Thus, it appeared that 50% of the PDGFβ-R phosphorylated in response to AngII was bound to Shc, and 50% was not bound. In contrast, PDGF-BB maximally phosphorylated the PDGFβ-R at 2 min, and phosphorylation was sustained up to 30 min (Fig. 2, and data not shown). Phosphorylation of the 66-kDa isoform of Shc by AngII was also more rapid than phosphorylation of the PDGFβ-R (data not shown). Because AngII increases intracellular calcium (16.Balla T. Varnai P. Tian Y. Smith R.D. Endocr. Res. 1998; 24: 335-344Crossref PubMed Scopus (25) Google Scholar, 17.Dixon B.S. Sharma R.V. Dickerson T. Fortune J. Am. J. Physiol. 1994; 266: C1406-C1420Crossref PubMed Google Scholar) and the transactivation of the EGF-R by AngII was reported to be calcium-dependent (12.Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar), we determined the role of calcium in PDGFβ-R phosphorylation. Treatment with BAPTA-AM, a chelator of intracellular calcium, did not alter phosphorylation of the PDGFβ-R by either AngII or PDGF-BB when coimmunoprecipitated with an antibody against Shc (Fig. 3,B and C) or when immunoprecipitated with an antibody against PDGFβ-R (Fig. 3 A, upper panel, and 3 B). Furthermore, tyrosine phosphorylation of the 66-kDa Shc isoform was also not significantly inhibited (Fig. 3 C). Activation of ERK1/2 by both AngII and PDGF-BB was also not calcium-dependent in these VSMC (Fig. 3 A,lower panel). BAPTA-AM treatment stimulated ERK1/2 activation to a small extent in control cells. Because chelation of intracellular calcium blocks activation of the MAP kinase phosphatase-1 (18.Cook S.J. Beltman J. Cadwallader K.A. McMahon M. McCormick F. J. Biol. Chem. 1997; 272: 13309-13319Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), we presume that activation of ERK1/2 in unstimulated control cells is a consequence of changes in MAP kinase phosphatase-1 activity. As a positive control for calcium chelation, VSMC were also treated with TNF. BAPTA-AM completely inhibited ERK1/2 activation by TNF, confirming that pretreatment with BAPTA-AM inhibits calcium-regulated signaling events (Fig. 3 A, lower panel). It is well known that AngII stimulates reactive oxygen species (ROS) production in vitro and in vivo (19.Griendling K.K. Harrison D.G. Circ. Res. 1999; 85: 562-563Crossref PubMed Scopus (125) Google Scholar). Furthermore, previous studies have shown that ROS are required for PDGF-BB-mediated signal transduction (20.Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar). To determine the role of ROS in AngII-induced Shc·PDGFβ-R phosphorylation, VSMC were preincubated with the antioxidants, N-acetylcysteine and Tiron. Both antioxidants completely abolished AngII-stimulated tyrosine phosphorylation of the Shc·PDGFβ-R complex (Fig. 4). To evaluate the role of the endogenous PDGFβ-R tyrosine kinase in phosphorylation of the Shc·PDGFβ-R complex by AngII, we used tyrphostin AG1296, a potent and specific inhibitor of this kinase (21.Kovlenko M. Ronnstrand L. Heldin C.H. Loubtchenkov M. Gazit A. Levitzki A. Bohmer F.D. Biochemistry. 1997; 36: 6260-6269Crossref PubMed Scopus (121) Google Scholar). First, we studied the effect of AG1296 on phosphorylation of PDGFβ-R that was associated with Shc as measured by immunoprecipitation with anti-Shc antibody. At concentrations reported to inhibit PDGF-mediated events (21.Kovlenko M. Ronnstrand L. Heldin C.H. Loubtchenkov M. Gazit A. Levitzki A. Bohmer F.D. Biochemistry. 1997; 36: 6260-6269Crossref PubMed Scopus (121) Google Scholar), AG1296 (10 μm) completely inhibited tyrosine phosphorylation of PDGFβ-R and Shc induced by PDGF-BB (Fig.5 A). In contrast, AG1296 did not affect AngII-induced phosphorylation of the Shc·PDGFβ-R complex (Fig. 5 A). Second, we studied the effect of AG1296 on total PDGFβ-R phosphorylation by immunoprecipitation with anti-PDGFβ-R antibody (Fig. 5 B). Under these experimental conditions, AG1296 caused complete inhibition of the AngII-induced tyrosine phosphorylation of the PDGFβ-R. These results suggest that two different populations of the PDGFβ-R exist, differentiated by whether the PDGFβ-R is bound to the 66-kDa Shc isoform or is not bound. The endogenous PDGFβ-R tyrosine kinase is therefore responsible for phosphorylation of the uncomplexed PDGFβ-R but not for phosphorylation of the Shc·PDGFβ-R complex (Fig.5 B). Previous studies from our laboratory and others showed that the Src tyrosine kinase family is required for AngII- and PDGF-BB-induced signaling events in VSMC, such as cytoskeletal reorganization at focal adhesions (22.Ishida T. Ishida M. Suero J. Takahashi M. Berk B.C. J. Clin. Invest. 1999; 103: 789-797Crossref PubMed Scopus (144) Google Scholar), migration (23.Waltenberger J. Uecker A. Kroll J. Frank H. Mayr U. Bjorge J.D. Fujita D. Gazit A. Hombach V. Levitzki A. Bohmer F.D. Circ. Res. 1999; 85: 12-22Crossref PubMed Scopus (107) Google Scholar), and proliferation (23.Waltenberger J. Uecker A. Kroll J. Frank H. Mayr U. Bjorge J.D. Fujita D. Gazit A. Hombach V. Levitzki A. Bohmer F.D. Circ. Res. 1999; 85: 12-22Crossref PubMed Scopus (107) Google Scholar). To determine the role of Src in AngII- and PDGF-BB-mediated phosphorylation of the Shc·PDGFβ-R complex, we used PP-1, a pyrazolopyrimidine that interacts specifically with Src family kinases and is a competitive inhibitor of ATP (24.Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brisette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar). PP-1 did not inhibit AngII-induced phosphorylation of the PDGFβ-R (Fig.6, upper panel), nor AngII phosphorylation of Shc (Fig. 6 A, bottom panel). In contrast, PP-1 completely inhibited PDGF-BB-mediated PDGFβ-R phosphorylation and Shc phosphorylation (Fig. 6 A), in agreement with findings of Waltenberger et al. (23.Waltenberger J. Uecker A. Kroll J. Frank H. Mayr U. Bjorge J.D. Fujita D. Gazit A. Hombach V. Levitzki A. Bohmer F.D. Circ. Res. 1999; 85: 12-22Crossref PubMed Scopus (107) Google Scholar). PP-1 also completely inhibited the tyrosine phosphorylation of PDGFβ-R not bound to Shc that was stimulated by AngII (Fig. 6 B). These results further support the concept that PDGF-BB and AngII activate the PDGFβ-R via two different pathways in VSMC, differentiated by their interaction with Shc. To define further the kinases involved in AngII-mediated Shc·PDGFβ-R complex phosphorylation, we investigated JAK2, which is rapidly activated by AngII (26.Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-648Crossref PubMed Scopus (848) Google Scholar). AG490 (60 μm), a specific JAK2 inhibitor (25.Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26.Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-648Crossref PubMed Scopus (848) Google Scholar), did not inhibit phosphorylation of the PDGFβ-R induced by PDGF-BB (Fig.7). AG490 also had no effect on AngII-mediated PDGFβ-R phosphorylation measured by Shc coimmunoprecipitation (Fig. 7). These data demonstrate that JAK2 activity is not responsible for PDGFβ-R phosphorylation by AngII. A possible explanation for different pathways of Shc·PDGFβ-R complex phosphorylation by AngII and PDGF-BB could be that the Shc·PDGFβ-R complex is localized to a different subcellular compartment when stimulated with AngII. This hypothesis is supported by the findings of Lotti et al. (27.Lotti L.V. Lanfrancone L. Migliaccio E. Zompetta C. Pelicci G. Salcini A.E. Falini B. Pelicci P.G. Torrisi M.R. Mol. Cell. Biol. 1996; 16: 1946-1954Crossref PubMed Scopus (68) Google Scholar), who showed that Shc proteins were localized to endoplasmic reticulum membranes and redistributed after activation of tyrosine kinase receptors. To determine whether separate populations of Shc·PDGFβ-R complexes could be purified, we performed cell fractionation studies. Tyrosine-phosphorylated PDGFβ-R was present in the supernatant after centrifugation of lysates at 3,500 ×g for 15 min from cells stimulated with both AngII and PDGF-BB (Fig. 8 A, left panel). In contrast, tyrosine-phosphorylated PDGFβ-R was no longer present in the supernatant of lysates prepared from AngII-stimulated cells after centrifugation at 22,500 ×g for 10 min (Fig. 8 A, right panel). However, when cells were stimulated with PDGF-BB, tyrosine-phosphorylated PDGFβ-R was still present in the 22,500 × g supernatant (Fig. 8 A, right panel). To determine the location of Shc after AngII and PDGF-BB stimulation, we dissolved the 22,500 × g pellet in 2% SDS-Triton/Nonidet P-40 lysis buffer and immunoprecipitated Shc with an anti-Shc antibody. As shown in Fig. 8 B, the Shc·PDGFβ-R complex was only present in the pellet of cells stimulated with AngII. In PDGF-BB-stimulated lysates only a small amount of Shc was present in the 22,500 × g pellet. These results suggest that the PDGFβ-R phosphorylated in response to AngII (present as a Shc·PDGFβ-R complex) was located in a compartment physically different from the PDGFβ-R phosphorylated in response to PDGF-BB. We measured the relative amounts of caveolin-1 present in the Shc·PDGFβ-R complexes purified from AngII- and PDGF-BB-stimulated cells. Because there was no difference in caveolin-1 coprecipitated (data not shown) we do not believe that translocation to caveolae explains the difference between AngII and PDGF-BB. The major findings of the present study are that AngII and PDGF-BB stimulate tyrosine phosphorylation of the PDGFβ-R via different pathways (Fig. 9). Although transactivation of the PDGFβ-R and EGF-R by AngII has been reported previously (10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12.Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar), the novel aspect of the present study is the demonstration of two different pathways that are mechanistically separate as shown by the presence of two populations of the PDGFβ-R. One population of PDGFβ-R exists in which Shc is bound to the receptor in the basal state, whereas in the other population the PDGFβ-R is not complexed to Shc. Evidence to support different mechanisms for PDGFβ-R phosphorylation related to these separate populations includes differences in the time course of PDGFβ-R phosphorylation (Fig. 2), differing effects of inhibitors of the endogenous PDGFβ-R tyrosine kinase and Src family tyrosine kinases (Figs. 4 and 5), differing results when the PDGFβ-R was directly immunoprecipitated (PDGFβ-R-antibody) versuscoimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the PDGFβ-R phosphorylated by AngII and PDGF-BB is located in separate subcellular compartments (Fig. 8). These studies are the first to suggest that transactivation of tyrosine kinase receptors by GPCRs involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand (Fig. 9). In the discussion below, the different mechanisms for AngII- and PDGF-mediated PDGFβ-R tyrosine phosphorylation are examined based on the model shown in Fig. 9. It has become clear that ROS mediate many of the rapid responses of VSMC to growth-promoting agents (19.Griendling K.K. Harrison D.G. Circ. Res. 1999; 85: 562-563Crossref PubMed Scopus (125) Google Scholar, 28.Baas A.S. Berk B.C. Circ. Res. 1995; 77: 29-36Crossref PubMed Scopus (370) Google Scholar, 29.Rao G.N. Berk B.C. Circ. Res. 1992; 70: 593-599Crossref PubMed Google Scholar, 30.Griendling K.K. Ushio-Fukai M. J. Lab. Clin. Med. 1998; 132: 9-15Abstract Full Text PDF PubMed Scopus (185) Google Scholar). ROS are essential for both PDGF-mediated stimulation of VSMC DNA synthesis (20.Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar) and for AngII-mediated increases in VSMC hypertrophy (32.Ushio-Fukai M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). In addition, ROS are necessary for increases in signal events related to cell survival and protein synthesis such as activation of the Akt/protein kinase B pathway (33.Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Finally, inhibiting ROS-dependent signal transduction events by overexpression of catalase (34.Brown M.R. Miller Jr., F.J. Li W.G. Ellingson A.N. Mozena J.D. Chatterjee P. Engelhardt J.F. Zwacka R.M. Oberley L.W. Fang X. Spector A.A. Weintraub N.L. Circ. Res. 1999; 85: 524-533Crossref PubMed Scopus (188) Google Scholar) or by treatment with antioxidants such asN-acetylcysteine (35.Tsai J.C. Jain M. Hsieh C.M. Lee W.S. Yoshizumi M. Patterson C. Perrella M.A. Cooke C. Wang H. Haber E. Schlegel R. Lee M.E. J. Biol. Chem. 1996; 271: 3667-3670Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) inhibits VSMC DNA synthesis and cell survival. In the present study we observed that AngII-dependent phosphorylation of both the PDGFβ-R and Shc required ROS generation (Fig. 4). Similar results have been shown by others for PDGF and its receptor (20.Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2313) Google Scholar). What is the nature of the ROS-stimulated tyrosine kinase that phosphorylates the PDGFβ-R and Shc in response to AngII? In the present study we show that the kinase is not PYK2 (no calcium requirement, Fig. 3), not Src (no effect of the Src kinase inhibitor PP1, Fig. 6), and not JAK2 (no effect of the JAK2 inhibitor AG490, Fig.7). The PDGFβ-R tyrosine kinase plays a role in AngII-mediated tyrosine phosphorylation of the PDGFβ-R but not of Shc (Fig. 5). Specifically, we found that the population of PDGFβ-R which is not bound to Shc is completely dependent on the endogenous PDGFβ-R tyrosine kinase as shown by inhibition with AG1296 (Fig.5 B). In contrast, tyrosine phosphorylation of the PDGFβ-R that is complexed to Shc was not altered by AG1296 (Fig.5 B). As expected, PDGF-BB-mediated tyrosine phosphorylation of both populations of the PDGFβ-R was inhibited by AG1296 (Fig. 5). A candidate tyrosine kinase that could transactivate the PDGFβ-R is Syk. Syk is a nonreceptor 72-kDa tyrosine kinase, first isolated from porcine spleen and capable of phosphorylating Shc (36.Qin S. Inazu T. Yamamura H. Biochem. J. 1995; 308: 347-352Crossref PubMed Scopus (51) Google Scholar). There are reports showing that Syk is involved in ERK1/2 activation following activation of GPCRs (37.Wan Y. Bence K. Hata A. Kurosaki T. Veillette A. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (258) Google Scholar). Syk is expressed mainly in cells of the lymphoid system, although high level expression has been observed in mouse heart (38.Fluck M. Zurcher G. Andres A.C. Ziemiecki A. Biochem. Biophys. Res. Commun. 1995; 213: 273-281Crossref PubMed Scopus (38) Google Scholar). Future studies will be required to determine whether Syk is expressed in VSMC and is activated by AngII. The adaptor protein Shc appears to be central to the differences in AngII- and PDGF-mediated phosphorylation of the PDGFβ-R. Shc proteins are adaptors, involved in the signaling events of GPCRs, as well as receptor tyrosine kinases (39.Sudgen P.H. Clerk A. Cell. Signal. 1997; 9: 337-351Crossref PubMed Scopus (282) Google Scholar, 40.van der Geer P. Pawson T. Trends Biochem. Sci. 1995; 20: 277-280Abstract Full Text PDF PubMed Scopus (233) Google Scholar). In the present study, we found that association of Shc with the PDGFβ-R in VSMC determines the nature of PDGFβ-R phosphorylation and subcellular location. Stimulation of PDGFβ-R that is not complexed to Shc by either AngII or PDGF-BB causes tyrosine phosphorylation of the PDGFβ-R, and the receptor remains in a low molecular weight complex. We characterize this population of PDGFβ-R as residing in compartment I, possibly cytosol (Fig. 9). PDGF-BB binding to the PDGFβ-R causes Shc tyrosine phosphorylation, and the Shc-phosphorylated PDGFβ-R complex (when stimulated by PDGF-BB) also resides in compartment I as shown by our inability to sediment the Shc·PDGFβ-R complex (Fig. 8, Aand B). In contrast, AngII has a very different effect as shown by the finding that high speed centrifugation after AngII stimulation sedimented the Shc-phosphorylated PDGFβ-R complex (Fig.8 B, compartment II). After solubilizing the pellet resulting from the high speed centrifugation at 22,500 × g we could recover all of the Shc-phosphorylated PDGFβ-R in that pellet, which leads to two possible conclusions. One is that the yet unidentified “Shc-kinase” phosphorylates the PDGFβ-R bound to Shc on different tyrosine residues than the intrinsic PDGFβ-R kinase, and this results in the translocation to a different compartment. A second explanation is that the Shc-kinase itself forms a multiprotein complex with the Shc-phosphorylated PDGFβ-R complex, which then leads to translocation to a different compartment. Further studies in our laboratory will be done to address these possibilities. Candidate locations for compartments I and II are the cytosol and endoplasmic reticulum membranes, respectively, based on work of Lottiet al. (27.Lotti L.V. Lanfrancone L. Migliaccio E. Zompetta C. Pelicci G. Salcini A.E. Falini B. Pelicci P.G. Torrisi M.R. Mol. Cell. Biol. 1996; 16: 1946-1954Crossref PubMed Scopus (68) Google Scholar). These investigators showed that in quiescent NIH/3T3 cells, Shc is localized to the rough endoplasmic reticulum (27.Lotti L.V. Lanfrancone L. Migliaccio E. Zompetta C. Pelicci G. Salcini A.E. Falini B. Pelicci P.G. Torrisi M.R. Mol. Cell. Biol. 1996; 16: 1946-1954Crossref PubMed Scopus (68) Google Scholar). Upon receptor tyrosine kinase stimulation, Shc redistributed toward the cell periphery (plasma membrane and endocytic structures) (27.Lotti L.V. Lanfrancone L. Migliaccio E. Zompetta C. Pelicci G. Salcini A.E. Falini B. Pelicci P.G. Torrisi M.R. Mol. Cell. Biol. 1996; 16: 1946-1954Crossref PubMed Scopus (68) Google Scholar). Shc redistribution was lost in cells transfected with a phosphorylation-defective mutant of the EGF-R, suggesting that the interaction of Shc with specific phosphotyrosine residues in the cytoplasmic tail of the activated receptor is required to redistribute Shc to the cell periphery. The mechanism for Shc redistribution by GPCRs, however, remains to be elucidated. There is evidence that the different Shc proteins (66-, 52-, and 46-kDa isoforms) are localized in different compartments of the cell (41.Migliaccio E. Mele S. Salcini A.E. Pelicci G. Lai K.M.V. Superti-Furga G. Pawson T. Di Fiore P.P. Lanfrancone L. Pelicci P.G. EMBO J. 1997; 16: 706-716Crossref PubMed Scopus (362) Google Scholar). Clark et al. (42.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Biol. Cell. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar) showed that in 3T3-L1 adipocytes, the 66-kDa isoform was localized mainly to the endoplasmic reticulum, the 46-kDa isoform to the plasma membrane, and the 52-kDa isoform was distributed evenly throughout the cytosol (42.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Biol. Cell. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar). It is possible that GPCRs and tyrosine kinase receptors couple preferentially to a specific Shc isoform. However, in the present study, both AngII and PDGF-BB stimulated phosphorylation predominantly of 66-kDa Shc with minimal phosphorylation of the 52- and 46-kDa isoforms. In addition, the different nature of receptor phosphorylation by either a cytosolic tyrosine kinase (in the case of AngII) or an intrinsic receptor tyrosine kinase (PDGF-BB) may influence the subcellular location of Shc. Transactivation of the EGF-R by AngII in cardiac fibroblasts has been shown to be important in the regulation of fibronectin and transforming growth factor-β synthesis (13.Moriguchi Y. Matsubara H. Mori Y. Murasawa S. Masaki H. Maruyama K. Tsutsumi Y. Shibasaki Y. Tanaka Y. Circ. Res. 1999; 84: 1073-1084Crossref PubMed Scopus (109) Google Scholar). The importance of PDGFβ-R phosphorylation by AngII remains unclear, but the mechanism differs clearly from EGF-R phosphorylation. One model for EGF-R-mediated transactivation by AngII involves an increase in intracellular calcium that stimulates tyrosine phosphorylation of the tyrosine kinase PYK2. PYK2 then forms a complex with c-Src, and phosphorylation of EGF-R occurs via interactions with c-Src and/or PYK2 (12.Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar, 43.Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar). EGF-R transactivation by AngII as reported by Eguchi et al. (31.Eguchi 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) involves an increase in intracellular calcium and leads to the EGFR-dependent Ras activation and subsequent p70s6K kinase activation via two parallel pathways. However, the activation of p70s6K kinase via these two pathways was similar whether AngII or EGF was the agonist. In contrast, in the present study, we show that PDGFβ-R phosphorylation by AngII did not require calcium or c-Src. Furthermore, we show that PDGFβ-R phosphorylation by AngII involves a mechanism distinct from that activated by PDGF-BB. The phosphorylation, localization, and binding of Shc to the PDGFβ-R seem to be important for the signal transduction pathway identified here. Thus, Shc appears to be an important mediator to study the functional significance of receptor tyrosine kinase transactivation by AngII." @default.
• W2004103569 created "2016-06-24" @default.
• W2004103569 creator A5001520142 @default.
• W2004103569 creator A5017817427 @default.
• W2004103569 creator A5038661951 @default.
• W2004103569 creator A5072801244 @default.
• W2004103569 creator A5079583545 @default.
• W2004103569 date "2000-05-01" @default.
• W2004103569 modified "2023-09-30" @default.
• W2004103569 title "Angiotensin II Induces Transactivation of Two Different Populations of the Platelet-derived Growth Factor β Receptor" @default.
• W2004103569 cites W103992419 @default.
• W2004103569 cites W1965526093 @default.
• W2004103569 cites W1966439052 @default.
• W2004103569 cites W1968529156 @default.
• W2004103569 cites W1978231615 @default.
• W2004103569 cites W1985873763 @default.
• W2004103569 cites W1992965538 @default.
• W2004103569 cites W2005342628 @default.
• W2004103569 cites W2007349364 @default.
• W2004103569 cites W2020631276 @default.
• W2004103569 cites W2029259343 @default.
• W2004103569 cites W2029652412 @default.
• W2004103569 cites W2031665308 @default.
• W2004103569 cites W2039049403 @default.
• W2004103569 cites W2042154282 @default.
• W2004103569 cites W2048569323 @default.
• W2004103569 cites W2050420714 @default.
• W2004103569 cites W2057181580 @default.
• W2004103569 cites W2059215487 @default.
• W2004103569 cites W2069785923 @default.
• W2004103569 cites W2072881410 @default.
• W2004103569 cites W2074535435 @default.
• W2004103569 cites W2079051863 @default.
• W2004103569 cites W2090895449 @default.
• W2004103569 cites W2097665925 @default.
• W2004103569 cites W2101413460 @default.
• W2004103569 cites W2101622006 @default.
• W2004103569 cites W2102014126 @default.
• W2004103569 cites W2102564220 @default.
• W2004103569 cites W2103301305 @default.
• W2004103569 cites W2114648315 @default.
• W2004103569 cites W2124355115 @default.
• W2004103569 cites W2127048642 @default.
• W2004103569 cites W2127281109 @default.
• W2004103569 cites W2137199906 @default.
• W2004103569 cites W2141388886 @default.
• W2004103569 cites W2145545657 @default.
• W2004103569 cites W2153899018 @default.
• W2004103569 cites W2157760569 @default.
• W2004103569 cites W2166056434 @default.
• W2004103569 cites W2419626516 @default.
• W2004103569 cites W79302672 @default.
• W2004103569 doi "https://doi.org/10.1074/jbc.m909616199" @default.
• W2004103569 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10748142" @default.
• W2004103569 hasPublicationYear "2000" @default.
• W2004103569 type Work @default.
• W2004103569 sameAs 2004103569 @default.
• W2004103569 citedByCount "157" @default.
• W2004103569 countsByYear W20041035692012 @default.
• W2004103569 countsByYear W20041035692013 @default.
• W2004103569 countsByYear W20041035692014 @default.
• W2004103569 countsByYear W20041035692015 @default.
• W2004103569 countsByYear W20041035692016 @default.
• W2004103569 countsByYear W20041035692017 @default.
• W2004103569 countsByYear W20041035692018 @default.
• W2004103569 countsByYear W20041035692019 @default.
• W2004103569 countsByYear W20041035692020 @default.
• W2004103569 countsByYear W20041035692021 @default.
• W2004103569 countsByYear W20041035692022 @default.
• W2004103569 countsByYear W20041035692023 @default.
• W2004103569 crossrefType "journal-article" @default.
• W2004103569 hasAuthorship W2004103569A5001520142 @default.
• W2004103569 hasAuthorship W2004103569A5017817427 @default.
• W2004103569 hasAuthorship W2004103569A5038661951 @default.
• W2004103569 hasAuthorship W2004103569A5072801244 @default.
• W2004103569 hasAuthorship W2004103569A5079583545 @default.
• W2004103569 hasBestOaLocation W20041035691 @default.
• W2004103569 hasConcept C104317684 @default.
• W2004103569 hasConcept C104849204 @default.
• W2004103569 hasConcept C123915805 @default.
• W2004103569 hasConcept C126322002 @default.
• W2004103569 hasConcept C1292079 @default.
• W2004103569 hasConcept C134018914 @default.
• W2004103569 hasConcept C170493617 @default.
• W2004103569 hasConcept C185592680 @default.
• W2004103569 hasConcept C2908929049 @default.
• W2004103569 hasConcept C55493867 @default.
• W2004103569 hasConcept C71924100 @default.
• W2004103569 hasConcept C86339819 @default.
• W2004103569 hasConcept C86803240 @default.
• W2004103569 hasConcept C89560881 @default.
• W2004103569 hasConcept C95444343 @default.
• W2004103569 hasConceptScore W2004103569C104317684 @default.
• W2004103569 hasConceptScore W2004103569C104849204 @default.
• W2004103569 hasConceptScore W2004103569C123915805 @default.
• W2004103569 hasConceptScore W2004103569C126322002 @default.
• W2004103569 hasConceptScore W2004103569C1292079 @default.
• W2004103569 hasConceptScore W2004103569C134018914 @default.

• next