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• W2148997157 abstract "The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1β acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1β acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2β acting through the same heterodimeric receptor species. Surprisingly, NRG1β and NRG2β also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes. The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1β acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1β acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2β acting through the same heterodimeric receptor species. Surprisingly, NRG1β and NRG2β also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes. receptor tyrosine kinase immediate early gene average difference neuregulin cAMP-responsive element-binding protein protein kinase C epidermal growth factor epidermal growth factor receptor signal transducers and activators of transcription phosphoinositide 3-kinase mitogen-activated protein kinase c-Jun N-terminal kinase tumor necrosis factor platelet-activating factor breast cancer Polypeptide growth factor hormones act on individual cells within tissues or on pluripotent stem cells to induce responses that contribute to development, tissue maintenance and repair, or disease state. Depending on cell type and the identity of the growth factor presented to the cell, a variety of responses are possible, including proliferation, differentiation, apoptosis, survival, migration, and fate specification. A fundamental question in growth factor signaling concerns the mechanisms by which specificity is generated: how do different growth factors elicit different responses within a single cell type, and how do different cell types respond differently to a single growth factor? Cellular responses to growth factors are mediated by cell surface receptor tyrosine kinases (RTKs)1 that possess an intrinsic protein tyrosine kinase activity. Growth factor binding stimulates receptor dimerization and autophosphorylation on several tyrosine residues, and phosphorylated tyrosines provide docking sites for intracellular signaling proteins that contain Src homology 2 or protein tyrosine binding domains (1Margolis B. Cell Growth Differ. 1992; 3: 73-80PubMed Google Scholar, 2van der Geer P. Pawson T. Trends Biochem. Sci. 1995; 20: 277-280Abstract Full Text PDF PubMed Scopus (234) Google Scholar). The sequence-specific recruitment of signaling proteins couples activated RTKs to intracellular signaling cascades that propagate signals to the nucleus to elicit initial changes in gene expression. Growth factor-stimulated expression of immediate early genes (IEGs), or genes whose induction does not require protein synthesis, then lays the foundation for the ultimate cellular response (3Woodgett J.R. Br. Med. Bull. 1989; 45: 529-540Crossref PubMed Scopus (20) Google Scholar, 4Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar, 5Winkles J.A. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 41-78Crossref PubMed Scopus (80) Google Scholar). Over the past decade, several signaling cascades connecting activated RTKs to the nucleus have been characterized (6Pawson T. Saxton T.M. Cell. 1999; 97: 675-678Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). For example, activation of the Erk serine/threonine kinases follows the recruitment of the Grb2/SOS complex to receptors and the stimulation of the Ras GTPase. Erk phosphorylation of ternary complex factors leads to the stimulation of genes containing serum response elements in their promoters. Phosphoinositide 3′-kinase (PI3K) activation by growth factors leads to the phosphorylation of the forkhead and CREB transcription factors through stimulation of the serine/threonine kinase Akt, as well as the activation of nuclear factor κB through regulation of IκB kinase. These factors are thought to play a prominent role in the regulation of genes involved in cellular survival and apoptosis. Members of the STAT family of transcription factors may be phosphorylated directly by growth factor receptors to regulate cytokine-inducible genes, and phospholipase C-γ1 can mediate the response of calcium-sensitive genes and protein kinase C (PKC) targets to growth factor stimulation. Through the phosphorylation of multiple tyrosine residues, each RTK has the capacity to stimulate several different signaling cascades. By independently targeting different subsets of genes or by acting in a combinatorial manner to regulate gene expression, the different signaling pathways have the potential to mediate a variety of cellular responses. However, many different RTKs utilize identical signaling pathways in mediating diverse responses to growth factors. Hence, a major question has become how signaling cascades couple growth factor receptors with specific gene expression patterns to mediate a diverse array of cellular responses. One model suggests that RTKs send general signals through a limited number of pathways, and that these signals are interpreted in the target cell by context-specific transcription factors. A series of recent studies with invertebrate model systems support this view. Analysis of the regulatory regions of marker genes for specific late stage developmental events in Drosophila melanogastersuggests that gene expression is markedly dependent on the immediate environment and developmental history of the target cell (7Halfon M.S. Carmena A. Gisselbrecht S. Sackerson C.M. Jimenez F. Baylies M.K. Michelson A.M. Cell. 2000; 103: 63-74Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 8Flores G.V. Duan H. Yan H. Nagaraj R. Fu W. Zou Y. Noll M. Banerjee U. Cell. 2000; 103: 75-85Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 9Xu C. Kauffmann R.C. Zhang J. Kladny S. Carthew R.W. Cell. 2000; 103: 87-97Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In addition, a single RTK in Caenorhabditis elegans uses a different signaling pathway to mediate the development of two different tissues (10Clandinin T.R. DeModena J.A. Sternberg P.W. Cell. 1998; 92: 523-533Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), suggesting that some pathways may be dispensable depending on cellular context. In a mammalian system utilizing immortalized mouse fibroblasts, activation of fibroblast growth factor receptor-1 and the PDGFβ receptor led to identical profiles of gene expression (11Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). These observations point to a model whereby pathways emanating from growth factor-activated RTKs are generic and simply provide a “go” signal to pre-primed tissue precursor cells to alter gene expression and initiate tissue development (12Simon M.A. Cell. 2000; 103: 13-15Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, this model does not easily explain how multipotent stem cells differentially respond to different RTK-binding growth factors, a common theme in mammalian systems. For example, epidermal growth factor (EGF) receptor ligands act as survival/proliferation factors for many neural precursor cells whereas other growth factors such as nerve growth factor, brain-derived neurotrophic factor, and the neuregulins stimulate their differentiation into glial cells or neurons (13Cameron H.A. Hazel T.G. McKay R.D. J. Neurobiol. 1998; 36: 287-306Crossref PubMed Scopus (462) Google Scholar). Cultured rat PC12 pheochromacytoma cells are a biochemical model for neural differential RTK signaling. It has been suggested that a quantitative difference in signaling pathway usage, the prolonged activation of the Erk1/2 pathway by nerve growth factor relative to EGF, accounts for the differentiation activity of this factor toward these cells (14Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar). To begin to explore the link between receptor tyrosine kinase activation and gene expression in mammalian cells, we have focused on the ErbB RTK signaling network, a model for the generation of specificity and diversity in growth factor signaling. Signaling through ErbB receptor family members has been observed to play roles in a variety of developmental processes (15Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar), including cardiac and neural development (16Gassmann M. Casagranda F. Orioli D. Simon H. Lai C. Klein R. Lemke G. Nature. 1995; 378: 390-394Crossref PubMed Scopus (951) Google Scholar, 17Lee K.F. Simon H. Chen H. Bates B. Hung M.C. Hauser C. Nature. 1995; 378: 394-398Crossref PubMed Scopus (1100) Google Scholar, 18Meyer D. Birchmeier C. Nature. 1995; 378: 386-390Crossref PubMed Scopus (1058) Google Scholar, 19Gassmann M. Lemke G. Curr. Opin. Neurobiol. 1997; 7: 87-92Crossref PubMed Scopus (108) Google Scholar), glial cell development (20Marchionni M.A. Goodearl A.D. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. et al.Nature. 1993; 362: 312-318Crossref PubMed Scopus (681) Google Scholar, 21Canoll P.D. Musacchio J.M. Hardy R. Reynolds R. Marchionni M.A. Salzer J.L. Neuron. 1996; 17: 229-243Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 22Adlkofer K. Lai C. Glia. 2000; 29: 104-111Crossref PubMed Scopus (158) Google Scholar), the remodeling of mammary tissue during pregnancy (23Yang Y. Spitzer E. Meyer D. Sachs M. Niemann C. Hartmann G. Weidner K.M. Birchmeier C. Birchmeier W. J. Cell Biol. 1995; 131: 215-226Crossref PubMed Scopus (281) Google Scholar, 24Jones F.E. Jerry D.J. Guarino B.C. Andrews G.C. Stern D.F. Cell Growth Differ. 1996; 7: 1031-1038PubMed Google Scholar), and the development of the neuromuscular junction (25Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (244) Google Scholar, 26Fischbach G.D. Rosen K.M. Annu. Rev. Neurosci. 1997; 20: 429-458Crossref PubMed Scopus (254) Google Scholar, 27Lin W. Sanchez H.B. Deerinck T. Morris J.K. Ellisman M. Lee K.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1299-1304Crossref PubMed Scopus (156) Google Scholar). The EGF-like ligand NRG has been demonstrated to promote cellular proliferation (28Carraway III, K.L. Soltoff S.P. Diamonti A.J. Cantley L.C. J. Biol. Chem. 1995; 270: 7111-7116Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), differentiation (29Bacus S.S. Huberman E. Chin D. Kiguchi K. Simpson S. Lippman M. Lupu R. Cell Growth Differ. 1992; 3: 401-411PubMed Google Scholar), migration (30Rio C. Rieff H.I. Qi P. Khurana T.S. Corfas G. Neuron. 1997; 19: 39-50Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar), apoptosis (31Daly J.M. Jannot C.B. Beerli R.R. Graus-Porta D. Maurer F.G. Hynes N.E. Cancer Res. 1997; 57: 3804-3811PubMed Google Scholar), survival (32Syroid D.E. Maycox P.R. Burrola P.G. Liu N. Wen D. Lee K.F. Lemke G. Kilpatrick T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9229-9234Crossref PubMed Scopus (217) Google Scholar), and fate (33Shah N.M. Marchionni M.A. Isaacs I. Stroobant P. Anderson D.J. Cell. 1994; 77: 349-360Abstract Full Text PDF PubMed Scopus (460) Google Scholar), depending on cell type and the NRG isoform used in stimulation. The broad range of cellular responses to EGF-like growth factors makes the ErbB network uniquely suited for addressing fundamental questions pertaining to RTK signaling specificity and cellular response. The network consists of four known RTKs (EGF receptor, ErbB2, ErbB3, and ErbB4) and more than a dozen EGF-like ligands. Each of the receptors is predicted to couple to unique complements of signaling cascades (34Carraway III, K.L. Cantley L.C. Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 35Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar, 36Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (657) Google Scholar). This, together with their ability to undergo an extensive array of ligand-induced receptor homo- and heterodimerization events, has been proposed to underlie the diverse array of cellular and developmental responses attributed to the EGF-like growth factors in mammals (15Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 36Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (657) Google Scholar, 37Riese D.J., II Stern D.F. Bioessays. 1998; 20: 41-48Crossref PubMed Scopus (698) Google Scholar). In this study we use different EGF-like ligands to differentially stimulate ErbB receptors. We observe that that different ErbB dimeric pairs differentially stimulate intracellular signaling pathways and gene expression. Interestingly, the same dimeric receptor pair, when activated by two different growth factors, also leads to differences in gene expression. MDA-MB-361 and MDA-MB-453 cells were from ATCC and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Immunoprecipitation experiments were carried out as described previously (38Crovello C.S. Lai C. Cantley L.C. Carraway III, K.L. J. Biol. Chem. 1998; 273: 26954-26961Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 39Sweeney C. Lai C. Riese D.J., II Diamonti A.J. Cantley L.C. Carraway III, K.L. J. Biol. Chem. 2000; 275: 19803-19807Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly, cells at 50–60% confluence were serum-starved overnight in Dulbecco's modified Eagle's medium, 0.1% fetal bovine serum, and then treated with 30 nm growth factor for 2 min at 37 °C. After rinsing twice with ice-cold phosphate-buffered saline, cells were lysed in co-immunoprecipitation buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mm Na3VO4, 1 mm NaF, 1 mm ZnCl2, 10 mmβ-glycerophosphate, 5 mm tetrasodium pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, and 4 μg/ml each aprotinin, leupeptin, and pepstatin), and cleared lysates were immunoprecipitated with 1.5 μg of antibodies. Precipitating receptor monoclonal antibodies used were: anti-epidermal growth factor receptor Ab-1 (clone 528), anti-ErbB2 Ab-4, anti-ErbB3 Ab-4, and anti-ErbB4 Ab-1, all from NeoMarkers. Precipitating signaling protein antibodies used were: rabbit anti-Grb2 and rabbit anti-Cbl from Santa Cruz, rabbit anti-Shc and mouse anti-SHP2 from Transduction Labs, and rabbit anti-p85 described previously (40Soltoff S.P. Carraway III, K.L. Prigent S.A. Gullick W.G. Cantley L.C. Mol. Cell. Biol. 1994; 14: 3550-3558Crossref PubMed Scopus (461) Google Scholar). Precipitates were washed three times in wash buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 1 mm Na3VO4, 1 mm NaF, 10 mm β-glycerophosphate, 5 mm tetrasodium pyrophosphate, 0.2 mm phenylmethylsulfonyl fluoride, and 4 μg/ml each aprotinin, leupeptin, and pepstatin). Precipitated proteins were resolved by 6–10% gradient SDS-polyacrylamide gel electrophoresis and immunoblotted using enhanced chemiluminescence for detection. Blotting antibodies used were: anti-phosphotyrosine RC20, mouse anti-SHP2, and mouse anti-Shc from Transduction Laboratories, rabbit anti-Grb2 and rabbit anti-Cbl from Santa Cruz, and rabbit anti-p85 described previously (40Soltoff S.P. Carraway III, K.L. Prigent S.A. Gullick W.G. Cantley L.C. Mol. Cell. Biol. 1994; 14: 3550-3558Crossref PubMed Scopus (461) Google Scholar). Cells in 60-mm dishes were grown to 50–60% confluence, serum-starved overnight, and then treated with 30 nm growth factor for various times at 37 °C. Cells were rinsed twice in ice-cold phosphate-buffered saline and lysed in 1 ml of 1× sample buffer. 100-μl lysates were resolved by 6–10% gradient SDS-polyacrylamide gel electrophoresis and blotted with antibodies to phosphorylated kinases, kinase targets, and transcription factors. All antibodies to phosphoproteins were from New England Biolabs and Cell Signaling Technology, and those used were: rabbit anti-pErk1/2(Thr-202,Tyr-204), rabbit anti-pAkt(Ser-473), rabbit anti-pp70(Thr-389,Thr-421,Ser-424), rabbit anti-pp90(Ser-381), rabbit anti-pPKC(pan), rabbit anti-phospho-c-Myc(Thr-58, Ser-62), rabbit anti-phospho-c-Jun(Ser-63,Ser-73), and rabbit anti-pCREB(Ser-133). Filters were stripped and re-probed with antibodies to actin (Sigma). Cells in 100-mm dishes were grown to 50–60% confluence, serum-starved overnight, and treated for 1 h with 30 nm growth factor. RNA was harvested from treated cells using Tri Reagent (Molecular Research Center) according to the directions of the manufacturer. cRNA target was prepared from 20 μg of total RNA. Preparation of cRNA target and hybridization to HuGeneFL chips (Affymetrix) has been described previously (11Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 41Tamayo P. Slonim D. Mesirov J. Zhu Q. Kitareewan S. Dmitrovsky E. Lander E.S. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2907-2912Crossref PubMed Scopus (2414) Google Scholar), and is detailed at the Whitehead/MIT Genome Center's Molecular Pattern Recognition web site. Fluorescence intensities were obtained with a laser confocal scanner (Hewlett Packard), and analyzed using GeneChip software (Affymetrix). Each gene sequence is represented on the chips by 20 25-mer oligonucleotides identical to the cDNA of interest and a corresponding control 20 25-mer oligonucleotides containing a mismatch at the thirteenth residue. Expression of each gene sequence is reflected in the fluorescence of the identical series relative to the mismatch series, and is reported by GeneChip software as an “average difference” (AD) value (for a further discussion, see Ref. 11Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). AD values reported for genes on chips corresponding to growth factor-treated samples were normalized to control sample chips by scaling to equivalent total average differences as described previously (41Tamayo P. Slonim D. Mesirov J. Zhu Q. Kitareewan S. Dmitrovsky E. Lander E.S. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2907-2912Crossref PubMed Scopus (2414) Google Scholar). A threshold value of 30 was assigned to genes on all chips with a reported AD value of less than 30. Genes were then sorted for growth factor response according to these normalized, thresholded AD values. Genes that exhibited a difference in AD values between growth factor treatment and control of less than 100 in at least one of duplicate experiments were discarded as either not expressed or not reproducibly regulated by growth factor. To be considered regulated by growth factor, the AD value for a gene must have been 2.5-fold higher or lower with growth factor treatment relative to control in both of duplicate experiments. To be considered preferentially regulated by a given growth factor, the -fold stimulation or suppression by one growth factor must have been 1.5-fold greater or lesser than by the other in both of duplicate experiments. To begin to explore the relationship between activation of RTKs and gene expression, we assessed growth factor-stimulated gene expression in two different cultured human mammary tumor cell lines. MDA-MB-361 cells express modest levels of three of the four ErbB receptor family members. In these cells the activation of different receptor dimeric pairs may be achieved by treating cells with different EGF-like growth factors. MDA-MB-453 cells express abundant ErbB2 and ErbB3, but very little EGF receptor or ErbB4. The stimulation of these cells with the ligands NRG1 and NRG2 leads to the activation of the same receptor dimeric pair, the ErbB2/ErbB3 heterodimer. Numerous studies using model transfected cell systems suggest that different EGF-like growth factors preferentially induce the formation and activation of different ErbB receptor dimeric pairs (42Riese D.J., II van Raaij T.M. Plowman G.D. Andrews G.C. Stern D.F. Mol. Cell. Biol. 1995; 15: 5770-5776Crossref PubMed Scopus (347) Google Scholar, 43Pinkas-Kramarski R. Shelly M. Glathe S. Ratzkin B.J. Yarden Y. J. Biol. Chem. 1996; 271: 19029-19032Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 44Pinkas-Kramarski R. Shelly M. Guarino B.C. Wang L.M. Lyass L. Alroy I. Alimandi M. Kuo A. Moyer J.D. Lavi S. Eisenstein M. Ratzkin B.J. Seger R. Bacus S.S. Pierce J.H. Andrews G.C. Yarden Y. Alamandi M. Mol. Cell. Biol. 1998; 18: 6090-6101Crossref PubMed Scopus (127) Google Scholar). Two ligands that exhibit markedly different receptor-activating properties are EGF and NRG1β. While EGF binds directly to EGF receptor to stimulate EGF receptor homodimers and EGF receptor/ErbB2 heterodimers, NRG1β binds to either ErbB3 or ErbB4 to induce ErbB2/ErbB3 and ErbB2/ErbB4 heterodimers. In MDA-MB-361 cells EGF strongly stimulated the tyrosine phosphorylation of the 170-kDa EGF receptor and the 185-kDa ErbB2 protein, and had little effect on ErbB3. NRG1β strongly stimulated the tyrosine phosphorylation of ErbB2 and ErbB3 (185 kDa) and had little effect on EGF receptor (Fig.1 A, upper panel). These cells express very low levels of ErbB4, which with prolonged exposure was observed to be stimulated exclusively by NRG1β (data not shown). The initial impact of differential receptor activation is on the first step in signal transduction, the recruitment of Src homology 2 and protein tyrosine binding domain-containing proteins to activated receptors. We examined the association of signaling proteins with activated receptors first by blotting receptor immunoprecipitates with antibodies to signaling proteins (Fig. 1 A, lower two panels). As expected, EGF preferentially stimulated the recruitment of the adaptor protein Grb2 to EGF receptor whereas NRG1β preferentially stimulated the recruitment of this protein to ErbB3. NRG1β also preferentially stimulated the association of p85, the 85-kDa subunit of PI3K, with ErbB3, as demonstrated previously (40Soltoff S.P. Carraway III, K.L. Prigent S.A. Gullick W.G. Cantley L.C. Mol. Cell. Biol. 1994; 14: 3550-3558Crossref PubMed Scopus (461) Google Scholar). We also examined the growth factor-stimulated recruitment of tyrosine-phosphorylated proteins to complexes with the adaptor proteins Grb2 and Shc, the protein-tyrosine phosphatase SHP2, p85, and the negative regulatory protein Cbl (Fig. 1 B). In general each of the signaling proteins associated with the EGF receptor upon EGF treatment, and with 185-kDa ErbB receptors upon NRG1β treatment. The exception was Cbl, which, as suggested previously (45Levkowitz G. Klapper L.N. Tzahar E. Freywald A. Sela M. Yarden Y. Oncogene. 1996; 12: 1117-1125PubMed Google Scholar), responded preferentially to EGF stimulation. However, other ligand-dependent differences in recruitment were also observed. A notable example was p85, which associated similarly with tyrosine-phosphorylated proteins at 47, 55, and 185 kDa after EGF and NRG1β treatment, but preferentially associated with other tyrosine-phosphorylated proteins in the 90–130-kDa range in response to EGF. These observations confirm that a major outcome of differential ErbB receptor activation by different EGF-like growth factors is the differential recruitment of intracellular signaling proteins into complexes with activated receptors and other tyrosine-phosphorylated proteins. As expected, differential ErbB receptor stimulation by EGF and NRG1β in MDA-MB-361 cells resulted in the differential stimulation of intracellular serine/threonine kinase cascades, as determined by the phosphorylation of these enzymes in response to growth factor treatment (Fig. 2). The extent and kinetics of activation of Erk1 and Erk2 were similar for the two growth factors, perhaps reflecting the similar levels of Grb2 recruitment to activated receptors. EGF more potently stimulated the phosphorylation of two PKC isoforms, consistent with its preferential recruitment of phospholipase Cγ (46Fedi P. Pierce J.H. di Fiore P.P. Kraus M.H. Mol. Cell. Biol. 1994; 14: 492-500Crossref PubMed Google Scholar). In contrast, NRG1β was reproducibly stronger in stimulating the phosphorylation of Akt at serine 473, consistent with the stronger recruitment of p85 to ErbB3 in response to this factor. Interestingly, EGF preferentially stimulated the phosphorylation of p70S6 kinase. Although Akt and p70S6 kinase phosphorylation have both been demonstrated to depend on PI3K, the regulation of p70S6K is complex and not yet fully understood (47Toker A. Mol. Pharmacol. 2000; 57: 652-658Crossref PubMed Scopus (284) Google Scholar). The ultimate outcome of RTK stimulation by growth factors is the phosphorylation of nuclear factors to elicit changes in transcriptional regulation. To determine whether differential signaling through ErbB receptors might influence transcription factor regulation, we examined the phosphorylation state of three transcription factors, Myc, Jun, and CREB, in response to EGF and NRG1β in MDA-MB-361 cells. Although the phosphorylation of these factors was stimulated by both growth factors, EGF reproducibly elicited a stronger response than did NRG1β (Fig.3 A). Since EGF and NRG1β stimulated overlapping yet distinct patterns of intracellular signaling events in MDA-MB-361 cells, we examined whether the two growth factors also stimulated differences in gene expression. To test this we used Affymetrix HuGeneFL Array chips to simultaneously assess changes in transcript levels of ∼5600 genes after 1 h of growth factor treatment. We observed that, although the levels of a host of mRNAs were similarly elevated by both EGF and NRG1β, many were preferentially elevated by one or the other growth factor. The -fold stimulations observed for a subset of genes is plotted in Fig.3 B. Our analysis revealed 133 genes whose transcript abundance reproducibly changed in response to one or both growth factors. 92 mRNAs were enhanced at least 2.5-fold with growth factor treatment, whereas in two separate experiments 41 mRNAs were suppressed by at least 2.5-fold. Interestingly, despite its weaker potency in stimulating several signaling cascades, NRG1β was the stronger factor in regulating mRNA levels. 44 mRNAs were preferentially elevated by NRG1β, where the -fold induction by this growth factor was at least 1.5-fold greater than that of EGF, compared with 18 mRNAs that were preferentially elevated by EGF. Likewise, 20 mRNAs were preferentially suppressed by NRG1β by at least a 1.5-fold margin compared with 10 mRNAs that were preferentially suppressed by EGF. Table I shows an abbreviated list of genes regulated by growth factors in MDA-MB-361 cells. (A full list of growth factor-responsive genes is provided as supplementary material in the on-line edition of this article.) Particularly noteworthy is the distribution of known IEGs, indicated in Table I and in Fig.3 B by an asterisk. mRNA levels of most IEGs were similarly stimulated by both EGF and NRG1β, suggesting that overlapping pathways elicited by the two growth factors may be responsible for the r" @default.
• W2148997157 created "2016-06-24" @default.
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• W2148997157 date "2001-06-01" @default.
• W2148997157 modified "2023-10-14" @default.
• W2148997157 title "Growth Factor-specific Signaling Pathway Stimulation and Gene Expression Mediated by ErbB Receptors" @default.
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