Signal Transduction Pathways Activated by RET Oncoproteins in PC12 Pheochromocytoma Cells
1998; Elsevier BV; Volume: 273; Issue: 9 Linguagem: Inglês
10.1074/jbc.273.9.4909
ISSN1083-351X
AutoresShunhua Xing, Tara L. Furminger, Qiang Tong, Sissy Jhiang,
Tópico(s)Cancer-related molecular mechanisms research
ResumoGene alterations in the retproto-oncogene, which encodes a receptor tyrosine kinase, have been found to associate with several human diseases. In this study, we showed that induction of the vgf promoter activity is a good molecular indicator for RET activation in PC12 cells, a rat pheochromocytoma cell line. We demonstrated that all forms of RET oncoprotein, including RET chimeric oncoproteins found in human papillary thyroid carcinomas (RET/PTC) as well as RET oncoproteins found in patients with multiple endocrine neoplasia type 2A and 2B (2A/RET and 2B/RET) can induce vgf promoter activity in PC12 cells. In contrast, a RET mutant found in a patient with Hirschsprung's disease, as well as a RET/PTC1 mutant with deletion of the dimerization domain, failed to induce vgf promoter activity in PC12 cells. We further determined that the signaling events mediated by phosphorylated Tyr294 and phosphorylated Tyr451 binding sites are essential for RET/PTC1 to inducevgf promoter activity in PC12 cells. We also showed that RET/PTC1, 2A/RET, and 2B/RET induce ELK-, cAMP-responsive element binding protein (CREB), or JUN-mediated gene expression in PC12 cells, and these three signaling events are mediated by phosphorylated Tyr294 and phosphorylated Tyr451 binding sites in RET/PTC1. Gene alterations in the retproto-oncogene, which encodes a receptor tyrosine kinase, have been found to associate with several human diseases. In this study, we showed that induction of the vgf promoter activity is a good molecular indicator for RET activation in PC12 cells, a rat pheochromocytoma cell line. We demonstrated that all forms of RET oncoprotein, including RET chimeric oncoproteins found in human papillary thyroid carcinomas (RET/PTC) as well as RET oncoproteins found in patients with multiple endocrine neoplasia type 2A and 2B (2A/RET and 2B/RET) can induce vgf promoter activity in PC12 cells. In contrast, a RET mutant found in a patient with Hirschsprung's disease, as well as a RET/PTC1 mutant with deletion of the dimerization domain, failed to induce vgf promoter activity in PC12 cells. We further determined that the signaling events mediated by phosphorylated Tyr294 and phosphorylated Tyr451 binding sites are essential for RET/PTC1 to inducevgf promoter activity in PC12 cells. We also showed that RET/PTC1, 2A/RET, and 2B/RET induce ELK-, cAMP-responsive element binding protein (CREB), or JUN-mediated gene expression in PC12 cells, and these three signaling events are mediated by phosphorylated Tyr294 and phosphorylated Tyr451 binding sites in RET/PTC1. The ret proto-oncogene (c-ret) encodes a receptor tyrosine kinase with an extracellular domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The ligands for c-RET have been recently identified as glial cell line-derived neurotrophic factor (1Trupp M. Arenas E. Fainzilber M. Nilsson A. Sieber B. Grigoriou M. Kilkenny C. Salazar-Grueso E. Pachnis V. Arumae U. Sariola H. Saarma M. Ibanez C.F. Nature. 1996; 381: 785-789Google Scholar, 2Durbec P. Marcos-Gutierrez C.V. Kilkenny C. Grigoriou M. Wartiowaara K. Suvanto P. Smith D. Ponder B. Costantini F. Saarma M. Sariola H. Pachnis V. Nature. 1996; 381: 789-793Google Scholar, 3Jing S. Wen D. Yu Y. Holst P.L. Luo Y. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Google Scholar) and neurturin (4Klein R.D. Sherman D. Ho W. Stone D. Bennett G.L. Moffat B. Vandlen R. Simmons L. Gu Q. Hongo J. Devaux B. Poulsen K. Armanini M. Nozaki C. Asai N. Goddard A. Phillips H. Henderson C.E. Takahashi M. Rosenthal A. Nature. 1997; 387: 717-721Google Scholar, 5Buj-Bello A. Adu J. Pinon L.G.P. Horton A. Thompson J. Rosenthal A. Chinchetru M. Buchman V.L. Davies A.M. Nature. 1997; 387: 721-724Google Scholar). Gene alterations in c-ret have been found to associate with five different diseases, including papillary thyroid carcinoma (PC), 1The abbreviations used are: PC, papillary thyroid carcinoma; MEN, multiple endocrine neoplasia; HSCR, Hirschsprung's disease; PTC, papillary thyroid carcinomas; PLC, phospholipase C; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; CREB, cAMP-responsive element binding protein. multiple endocrine neoplasia type 2A (MEN2A), MEN2B, familial medullary thyroid carcinoma, and a congenital developmental defect, Hirschsprung's disease (HSCR). The ret/PTC oncogenes encoding chimeric oncoproteins with the RET tyrosine kinase domain fused to the N terminus of other genes are frequently detected in human papillary thyroid carcinomas (see review, see Ref. 6Jhiang S.M. Mazzaferri E.L. J. Lab. Clin. Med. 1994; 123: 331-337Google Scholar). Missense ret mutations found in patients with MEN2 syndromes are clustered in specific codons, whereasret mutations found in patients with HSCR are scattered along the gene without any hot spots (for review, see Ref. 7Eng C. Mulligan L.M. Hum. Mutat. 1997; 9: 97-109Google Scholar). It was reported that both ret/PTC and ret with MEN2A or MEN2B mutations (2A/ret or 2B/ret) function as dominant oncogenes with transforming activity in NIH/3T3 cells (8Ishizaka Y. Shima H. Sugimura T. Nagao M. Oncogene. 1992; 7: 1441-1444Google Scholar, 9Bongarzone I. Monzini N. Borrello M.G. Carcano C. Ferraresi G. Arighi E. Mondellini P. Della Porta G. Pierotti M.A. Mol. Cell. Biol. 1993; 13: 358-366Google Scholar, 10Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Google Scholar, 11Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Google Scholar, 12Xing S. Smanik S.A. Oglesbee M.J. Trosko J.E. Mazzaferri E.L. Jhiang S.M. Endocrinology. 1996; 137: 1512-1519Google Scholar), whereas ret with HSCR mutations (HSCR/ret) encodes nonfunctional proteins (13Carlomagno F. Vita G.D. Berlingieri M.T. de Franciscis V. Melillo R.M. Colantuoni V. Kraus M.H. Di Fiore P.P. Fusco A. Santoro M. EMBO J. 1996; 15: 2717-2725Google Scholar). It has been shown that mutations found in MEN2A lead to formation of disulfide-linked 2A/RET homodimers (10Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Google Scholar, 11Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Google Scholar, 12Xing S. Smanik S.A. Oglesbee M.J. Trosko J.E. Mazzaferri E.L. Jhiang S.M. Endocrinology. 1996; 137: 1512-1519Google Scholar), whereas the mutation found in MEN2B changes the substrate specificity of 2B/RET from a receptor tyrosine kinase to a cytoplasmic tyrosine kinase (14Songyang Z. Carraway III, K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Google Scholar). We recently demonstrated that RET/PTC1 oncoprotein, the major form of RET/PTC found in human papillary thyroid carcinoma, forms constitutive dimers through the leucine zipper motif in the N terminus of H4 (15Tong Q. Li Y. Smanik P.A. Fithian L.J. Xing S. Mazzaferri E.L. Jhiang S.M. Oncogene. 1995; 10: 1781-1787Google Scholar, 16Tong Q. Xing S. Jhiang S.M. J. Biol. Chem. 1997; 272: 9043-9047Google Scholar). The intracellular domain of c-RET consists of 14 tyrosine residues, and the long isoform of c-RET has two extra tyrosine residues at the C terminus (see review, see Ref. 17Itoh F. Ishizaka Y. Tahira T. Ikeda I. Imai K. Yachi A. Sugimura T. Nagao M. Tumor Res. 1989; 24: 1-13Google Scholar). Among those tyrosine residues, Tyr905(P), Tyr1015(P), and Tyr1062(P) of c-RET (corresponding to Tyr294(P), Tyr404(P), and Tyr451(P) of RET/PTC1 or Tyr429(P), Tyr539(P), and Tyr586(P) of RET/PTC2) were identified as the docking sites for Grb7/Grb10, PLCγ, and Shc/Enigma, respectively (18Pandey A. Liu X. Dixon J.E. Di Fiore P.P. Dixit V.M. J. Biol. Chem. 1996; 271: 10607-10610Google Scholar, 19Borrello M.G. Alberti L. Arighi E. Bongarzone I. Battistini C. Bardelli A. Pasini B. Piutti C. Rizzetti M.G. Mondellini P. Radice M.T. Pierotti M.A. Mol. Cell. Biol. 1996; 16: 2151-2163Google Scholar, 20Borrello M.G. Pelicci G. Arighi E. De Filippis L. Greco A. Bongarzone I. Rizzetti M. Pelicci P.G. Pierotti M.A. Oncogene. 1994; 9: 1661-1668Google Scholar, 21Arighi E. Alberti L. Torriti F. Ghizzoni S. Rizzetti M.G. Pelicci G. Pasini B. Bongarzone I. Piutti C. Pierotti M.A. Borrello M.G. Oncogene. 1997; 14: 773-782Google Scholar, 22Durick K. Wu R. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Google Scholar). The roles of these phosphotyrosine residues in RET-induced mitogenic activity and transforming activity have been studied vigorously in mouse fibroblasts. The docking site for PLCγ was reported to be essential for full oncogenic activation of RET/PTC2 in NIH/3T3 cells (19Borrello M.G. Alberti L. Arighi E. Bongarzone I. Battistini C. Bardelli A. Pasini B. Piutti C. Rizzetti M.G. Mondellini P. Radice M.T. Pierotti M.A. Mol. Cell. Biol. 1996; 16: 2151-2163Google Scholar). The docking site for Grb7/Grb10 was reported to be essential for the transforming activity of 2A/RET in NIH/3T3 cells (23Iwashita T. Asai N. Murakami H. Matsuyama M. Takahashi M. Oncogene. 1996; 12: 481-487Google Scholar). However, both phosphotyrosine residues are not essential for mitogenic signaling of RET/PTC2 in mouse fibroblasts (22Durick K. Wu R. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Google Scholar). The docking site for Shc/Enigma was reported to be essential not only for the mitogenic activity of RET/PTC2 in mouse fibroblast cells (22Durick K. Wu R. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Google Scholar) but also for the transforming activity of 2A/RET and 2B/RET in NIH/3T3 cells (24Asai N. Murakami H. Iwashita T. Takahashi M. J. Biol. Chem. 1996; 271: 17644-17649Google Scholar). RET-induced signaling pathways appear to be cell type-specific. Santoroet al. (25Santoro M. Wong W.T. Aroca P. Santos E. Matoskova B. Grieco M. Fusco A. Di Fiore P.P. Mol. Cell. Biol. 1994; 14: 663-675Google Scholar) reported that RET activation induces Ras activation but is not able to activate the MAPK signaling pathway or to induce phosphatidylinositol 3-kinase activity in NIH/3T3 transfectants. However, other investigators have reported that RET activation not only induces the activation of the Ras-MAPK pathway but also induces phosphatidylinositol 3-kinase activity in human neuroblastoma cells that express either exogenous or endogenous c-RET (26van Weering D.H.J. Medema J.P. Puijenbroek A.V. Burgering B.M.T. Baas P.D. Bos J.L. Oncogene. 1995; 11: 2207-2214Google Scholar, 27van Weering D.H.J. Bos J.L. J. Biol. Chem. 1997; 272: 249-254Google Scholar, 28Worby C.A. Vega Q. Zhao Y. Chao H.H.-J. Seasholtz A.F. Dixon J.E. J. Biol. Chem. 1996; 271: 23619-23622Google Scholar). In this paper, we report our studies of RET-induced signaling pathways in a physiologically relevant cell line, PC12 cells. For patients with MEN2A or MEN2B, 10–50% develop pheochromocytomas. PC12 cells derived from a rat pheochromocytoma are able to differentiate into sympathetic neuron-like cells with neurite outgrowth by stimulation with NGF (29Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Google Scholar). It has been shown that expression of RET/PTC1, 2A/RET, or 2B/RET in PC12 cells can also induce the expression of various neuronal markers and lead to morphological changes with neurite outgrowth (30Califano D. Monaco C. Vita G.D. D'Alessio A. Dathan N.A. Possenti R. Vecchio G. Fusco A. Santoro M. de Franciscis V. Oncogene. 1995; 11: 107-112Google Scholar, 31Califano D. D'Alessio A. Colucci-D'Amato G.L. De Vita G. Monacom C. Santelli G. Di Fiore P.P. Vecchio G. Fusco A. Santoro M. de Franciscis V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7933-7937Google Scholar, 32Rossel M. Pasini A. Chappuis S. Geneste O. Fournier L. Schuffenecker I. Takahashi M. van Grunsven L.A. Urdiales J.L. Rudkin B.B. Lenoir G.M. Billaud M. Oncogene. 1997; 14: 265-275Google Scholar). Among these changes, the induction of vgf promoter activity in PC12 cells appears to be specific to NGF stimulation and RET activation, as both epidermal growth factor and bovine fibroblast growth factor are unable to stimulate the vgf promoter activity. PC12-N21 cell line (33Burry R.W. Perrone-Bizzozero N.I. J. Neurosci. Res. 1993; 36: 241-251Google Scholar), kindly provided by Dr. Richard Burry at the Ohio State University, is a subclone of PC12 cells. The cells were grown in DMEM (Life Technologies, Inc.) supplemented with 5% horse serum and 5% fetal bovine serum. COS-7 cells (ATCC 1651) were grown in DMEM supplemented with 10% fetal bovine serum. LA-N-5 cells, a neuroblastoma cell line that was kindly provided by Dr. Jack Dixon at University of Michigan, were cultured in DMEM medium supplemented with 15% fetal bovine serum. The DNA fragment containing the vgf promoter and the 5′ noncoding region upstream of the first methionine (from −178 to +710) (34Possenti R. Di Rocco G. Nasi S. Levi A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3815-3819Google Scholar) was PCR-amplified from the genomic DNA isolated from PC12 cells. The DNA fragment was then inserted into pGL3Basic vector (Promega) to generate the pvgf-luc plasmid. Mutations changing Tyr294, Tyr404, or Tyr451 to Phe were introduced into PTC1 cDNA by site-directed mutagenesis (Muta-gene M13 in vitro mutagenesis kit, Version 2, Bio-Rad). These mutated DNA fragments were then subcloned into the pRcCMV expression vector. 2A/RET, 2B/RET, and HSCR/RET carry mutations Cys-634 → Arg, Met-918 → Thr, and Arg-897 → Glu, respectively (12Xing S. Smanik S.A. Oglesbee M.J. Trosko J.E. Mazzaferri E.L. Jhiang S.M. Endocrinology. 1996; 137: 1512-1519Google Scholar). RET/PTC1ΔN (49 kDa) had the N terminus of H4 (amino acid residues 3–52) deleted but retained the leucine zipper motif. RET/PTC1ΔZip (46 kDa) had the leucine zipper motif of H4 (amino acid residues 56–102) deleted (16Tong Q. Xing S. Jhiang S.M. J. Biol. Chem. 1997; 272: 9043-9047Google Scholar). The PathDetect in vivo signal transduction pathway reporting systems containing fusion activator plasmids and a reporter plasmid were purchased from Stratagene. pCH110 (Pharmacia Biotech Inc.) is a plasmid encoding β-galactosidase. For the COS-7 transient transfection assay, cells (1 × 106) were plated in 100-mm Petri dishes 24 h before transfection. Each plasmid (10 μg) was transfected into COS-7 cells using the calcium phosphate transfection method (Life Technologies, Inc.). Twenty-four hours after transfection, fresh growth medium was added, and the cells were grown for an additional 48 h before harvest. For the vgf promoter assay, PC12 cells (3 × 105 cells) were plated in 35-mm Petri dishes 24 h before transfection. Various RET plasmids (500 ng), 1 μg of pvgf-luc, and 0.8 μg of pCH110 (Pharmacia) were incubated with 40 μl of Superfect transfection reagent (Qiagen) in 400 μl of DMEM. The total DNA amount used for transfection was 8 μg, which was obtained by adding various amounts of salmon sperm DNA (Life Technologies, Inc.). After incubation for 10 min at room temperature, the transfection mixture was then divided into four aliquots and added onto four Petri dishes with plated cells. Three h after transfection, the medium was aspirated, and cells were washed with phosphate-buffered saline once before regular PC12 growth medium was added. Twenty-four hours after transfection, NGF (100 ng/ml) (Boehringer Mannheim) was added to two of the four parallel cultures for 48 h, and the total proteins were then isolated for luciferase assay. All experiments were repeated three times. To investigate the induction of ELK-, CREB-, or JUN-mediated gene expression by RET in PC12 cells and LA-N-5 cells, PC12 cells or LA-N-5 cells (5 × 105) were plated in 35-mm Petri dishes 24 h before transfection. Various RET plasmids or pcDNA3 vector (150 ng), 3 μg of pFR-luc, 150 ng of chimeric transactivator plasmid, and 360 ng of pCH110 were mixed in 120 μl DMEM and incubated with 20 μl of Superfect transfection reagent for 10 min at room temperature. The transfection mixture was then divided into three aliquots and added onto three Petri dishes with plated cells. Three h after transfection, the medium was aspirated, and cells were washed with phosphate-buffered saline once before regular growth medium was added. Twenty-four h after transfection, the medium was replaced with DMEM medium containing 0.5% fetal bovine serum. Cells were grown for an additional 24 h before the total proteins were isolated for luciferase assay. The experiment was repeated three times. To ensure the proper expression of these RET proteins from the corresponding DNA constructs, various DNA constructs were transfected into COS-7 cells, and total proteins were extracted for Western blot analysis using antibody against the cytoplasmic domain of c-RET. COS-7 cells were lysed in TN1 buffer (150 mm NaCl, 25 mmTris-HCl, pH 8.0, 1% Triton X-100, 20 mm EDTA, 20 mm sodium pyrophospate, 20 mm NaF, 3 mm Na3VO4, 2 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Immunoprecipitation and Western blot analysis were carried out as described previously (12Xing S. Smanik S.A. Oglesbee M.J. Trosko J.E. Mazzaferri E.L. Jhiang S.M. Endocrinology. 1996; 137: 1512-1519Google Scholar). The antibody against RET used in immunoprecipitation was first conjugated to protein-A beads before use. For Western blot analysis, a monoclonal antibody against c-RET was used. Anti-phosphotyrosine antibody 4G10 is a commercially available monoclonal antibody (Upstate Biotechnology Inc.). Various forms of RET used in this study are schematically shown in Fig. 1 a. All of these RET proteins can be expressed at comparable levels in COS-7 cells (Fig. 1,b and c). RET/PTC1, RET/PTC2, and RET/PTC3 represent the three forms of RET/PTC oncoproteins found in papillary thyroid carcinoma. We demonstrated that mutations changing Tyr294, Tyr404, or Tyr451 to Phe in RET/PTC1 did not affect the expression levels of these three mutated proteins in COS-7 cells. We also showed that the tyrosine phosphorylation levels of RET/PTC1-Y294F, RET/PTC1-Y404F, and RET/PTC1-Y451F were not affected by the corresponding mutations (Fig. 1 c). It has been shown previously that 2A/RET, 2B/RET, RET/PTC1, RET/PTC2, and RET/PTC3 oncoproteins are constitutively phosphorylated on tyrosine residues (8Ishizaka Y. Shima H. Sugimura T. Nagao M. Oncogene. 1992; 7: 1441-1444Google Scholar, 9Bongarzone I. Monzini N. Borrello M.G. Carcano C. Ferraresi G. Arighi E. Mondellini P. Della Porta G. Pierotti M.A. Mol. Cell. Biol. 1993; 13: 358-366Google Scholar, 10Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Google Scholar, 11Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Google Scholar, 12Xing S. Smanik S.A. Oglesbee M.J. Trosko J.E. Mazzaferri E.L. Jhiang S.M. Endocrinology. 1996; 137: 1512-1519Google Scholar), whereas HSCR/RET is a kinase-defective mutant (13Carlomagno F. Vita G.D. Berlingieri M.T. de Franciscis V. Melillo R.M. Colantuoni V. Kraus M.H. Di Fiore P.P. Fusco A. Santoro M. EMBO J. 1996; 15: 2717-2725Google Scholar). 2S. Xing and S. M. Jhiang, unpublished results. We have also previously demonstrated that RET/PTC1ΔN is constitutively phosphorylated on tyrosine residues, whereas RET/PTC1ΔZip is not (17Itoh F. Ishizaka Y. Tahira T. Ikeda I. Imai K. Yachi A. Sugimura T. Nagao M. Tumor Res. 1989; 24: 1-13Google Scholar). In this study, as expected, tyrosine phosphorylation of RET/PTC1-HSCR was undetectable (Fig. 1 c). The vgf gene is specifically expressed in neuroendocrine cells upon NGF stimulation (34Possenti R. Di Rocco G. Nasi S. Levi A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3815-3819Google Scholar). It is known that c-RET is also predominantly expressed in cells or tissues of neuroendocrine origin (35Santoro M. Rosati R. Grieco M. Berlingieri M.T. D'Amato G.L.-C. de Franciscis V. Fusco A. Oncogene. 1990; 5: 1595-1598Google Scholar, 36Tsuzuki T. Takahashi M. Asai N. Iwashita T. Matsuyama M. Asai J. Oncogene. 1995; 10: 191-198Google Scholar, 37Pachnis V. Monkoo B. Costantini F. Development. 1993; 119: 1005-1017Google Scholar). In this study, we showed that ret/PTC1, ret/PTC2, ret/PTC3, 2A/ret, and 2B/ret oncogenes all can inducevgf promoter activity at a rate greater than six times that of the basal activity of pvgf-luc in PC12 cells (Fig. 2 a). As expected, pcDNA3 vector and HSCR/ret cannot induce vgf promoter activity. This result suggests that 2A/RET, 2B/RET, and RET/PTC activate common signaling pathways in PC12 cells, leading to the induction of vgf expression regardless of the nature of mutations as well as the cellular localization of these oncoproteins. Therefore, the vgf promoter activity is a good molecular marker to monitor RET activation in PC12 cells. Consistent with a previous report (30Califano D. Monaco C. Vita G.D. D'Alessio A. Dathan N.A. Possenti R. Vecchio G. Fusco A. Santoro M. de Franciscis V. Oncogene. 1995; 11: 107-112Google Scholar), NGF treatment can only inducevgf promoter activity about three times higher than the basal activity of pvgf-luc in PC12 cells. However, in PC12 cells that express RET oncoproteins, NGF treatment did not significantly further induce vgf promoter activity. This result is consistent with the previous finding that vgfpromoter activity induced by RET/PTC1 can not be further induced by NGF treatment (30Califano D. Monaco C. Vita G.D. D'Alessio A. Dathan N.A. Possenti R. Vecchio G. Fusco A. Santoro M. de Franciscis V. Oncogene. 1995; 11: 107-112Google Scholar). This finding suggests that RET activation and NGF stimulation utilize overlapping signaling pathways in PC12 cells. A small increase in the vgf promoter activity was observed in the NGF-treated PC12 cells expressing RET oncoproteins compared with those without NGF stimulation. A possible explanation could be that a subpopulation of cells, which were not transfected by RET, remained fully responsive to NGF treatment. Taken together, these results indicated that vgf promoter activity induced by NGF or RET was not additive nor synergistic in PC12 cells. To test whether the leucine zipper motif is important for the induction of vgf promoter activity by RET/PTC1, we co-transfected ret/PTC1ΔZip with pvgf-luc into PC12 cells. Unlike RET/PTC1, RET/PTC1ΔZip can not induce vgf promoter activity (Fig. 2 b). As a control, RET/PTC1ΔN, with deletion of the extreme N-terminal amino acid residues but retention of the leucine zipper motif, was able to induce the vgf promoter activity at a similar rate to RET/PTC1. This result indicates that leucine zipper-mediated dimerization is essential for RET/PTC1 to induce vgfpromoter activity in PC12 cells. We introduced mutations changing Tyr294, Tyr404, or Tyr451 to Phe into RET/PTC1 and investigated the effect of these mutations on RET/PTC1-induced vgf promoter activity (Fig. 2 c). RET/PTC1 with the mutation changing Tyr294 or Tyr451 to Phe was no longer able to induce vgf promoter activity. These results indicate that signaling events mediated by the phosphotyrosine binding sites Tyr294(P) and Tyr451(P) are essential for RET/PTC1-induced vgf promoter activity in PC12 cells. The mutation changing Tyr404 to Phe had little effect on the ability of RET/PTC1 to induce vgf promoter activity. This finding is consistent with previous studies showing that PLCγ is not involved in the neuronal differentiation process in PC12 cells (38Spivak-Kroizman T. Mohammadi M. Hu P. Jaye M. Schlessinger J. Lax I. J. Biol. Chem. 1994; 269: 14419-14423Google Scholar). The importance of these three phosphotyrosine binding sites on the biological effects of RET activation has been studied in other cellular systems. The docking site for Shc or Enigma Tyr451(P) appears not only essential for the transforming activity of 2A/RET and 2B/RET in NIH/3T3 cells and the mitogenic activity of RET/PTC2 in mouse fibroblasts but is also essential for RET/PTC1-inducedvgf promoter activity in PC12 cells. However, the roles of the docking site for Grb10 or Grb7 Tyr294(P) and the docking site for PLCγ Tyr404(P) on the various biological effects induced by RET activation appear to be inconsistent among different assay systems in vitro. To investigate the importance of each phosphotyrosine binding sites in RET/PTC1 oncogenicity in vivo, we are currently establishing transgenic mice lines with thyroid-targeted expression of the RET/PTC1 carrying mutations changing Tyr404, Tyr294, or Tyr451 to Phe. A CREB binding site, a JUN binding site, and an SRE element were identified within the vgf promoter region by GenBankTM sequence analysis. It has been shown that CREB and JUN are phosphorylated and activated by protein kinase A and c-Jun NH2-terminal kinase, respectively, and the SRE element is the binding site for ELK, which is phosphorylated and activated by MAPK kinase 1 (39Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Google Scholar, 40Borrelli E. Montmayeur J.P. Foulkes N.S. Sassone-Corsi P. Crit. Rev. Oncog. 1992; 3: 321-338Google Scholar). This leads us to hypothesize that RET may activate ELK-, CREB-, or JUN-mediated gene expression in PC12 cells. The PathDetect signal transduction reporting system was used to test this hypothesis. In this assay system, the gene to be characterized is co-transfected with a reporter plasmid and a plasmid encoding a pathway-specific transactivator (ELK for MAPK pathway, JUN for c-Jun NH2-terminal kinase pathway, and CREB for protein kinase A pathway) fused to GAL4 binding domain. If expression of the gene to be characterized results in direct or indirect phosphorylation of the ELK, JUN, or CREB fusion protein, luciferase expression will be activated above the background level. As shown in Fig. 3, ELK-mediated gene expression was activated by RET/PTC1, 2A/RET, and 2B/RET more than 50 times higher than that activated by pcDNA3 vector. As expected, HSCR/RET could not activate ELK-mediated gene expression. This result is consistent with a previous report showing that c-RET activation upon glial cell line-derived neurotrophic factor stimulation activates the MAPK pathway in a neuroblastoma cell line (28Worby C.A. Vega Q. Zhao Y. Chao H.H.-J. Seasholtz A.F. Dixon J.E. J. Biol. Chem. 1996; 271: 23619-23622Google Scholar). MAPK kinase 1, which directly phosphorylates ELK, could induce ELK-mediated gene expression about 1.6 times higher than RET/PTC1 did. RET/PTC1 carrying a mutation abolishing the phosphotyrosine binding site Tyr451(P) or Tyr294(P) did not significantly activate ELK-mediated gene expression. This result indicates that Tyr451(P)- and Tyr294(P)-mediated signaling cascades involve MAPK activation. As shown in Fig. 4, RET/PTC1 as well as 2A/RET and 2B/RET, but not HSCR/RET, could induce gene expression mediated by CREB. This is the first report showing that RET activation can induce CREB-mediated gene expression. It has been reported that treatment of PC12 cells with NGF, epidermal growth factor, or 12-O-tetradecanoylphorbol-13-acetate induces CREB phosphorylation at Ser133 through the activation of a CREB kinase, RSK2, via Ras-MAPK pathway (41Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Google Scholar). Therefore, we speculate that RET may activate CREB-mediated gene expression through a similar mechanism. CREB-mediated gene expression induced by the positive control protein kinase A, which directly phosphorylates CREB, was 1.6 times higher than that of RET/PTC1. Similar to ELK-mediated gene expression, CREB-mediated gene expression could not be activated by RET/PTC1 carrying a mutation abolishing phosphotyrosine binding site Tyr451 or Tyr294. Finally, we also demonstrated that RET/PTC1 as well as 2A/RET and 2B/RET could induce JUN-mediated gene expression in PC12 cells through the signaling events mediated by phosphotyrosine binding sites Tyr451(P) and Tyr294(P) in RET/PTC1 (Fig. 5). This is in contrast to the study in a neuroblastoma cell line demonstrating that c-Jun NH2-terminal kinase pathway was not able to be induced by the activation of c-RET through glial cell line-derived neurotrophic factor stimulation (28Worby C.A. Vega Q. Zhao Y. Chao H.H.-J. Seasholtz A.F. Dixon J.E. J. Biol. Chem. 1996; 271: 23619-23622Google Scholar). It is possible that the promoter analysis assay system we used in this study is more sensitive than the biochemical characterization assay system used in the study of neuroblastoma cells. Indeed, in PC12 cells, JUN-mediated gene expression induced by RET was much lower than that induced by MAPK kinase kinase, which directly phosphorylates and activates c-Jun NH2-terminal kinase. Alternatively, this discrepancy might suggest that the activation of c-Jun NH2-terminal kinase pathway by RET is restricted to PC12 cells. As a comparison to PC12 cells, we also transfected RET/PTC1 cDNA constructs into a neuroblastoma cell line, LA-N-5 cells. Consistent with the published report (28Worby C.A. Vega Q. Zhao Y. Chao H.H.-J. Seasholtz A.F. Dixon J.E. J. Biol. Chem. 1996; 271: 23619-23622Google Scholar), RET/PTC1 could significantly induce ELK-mediated gene expression in LA-N-5 cells (Fig. 6). However, in contrast to the study carried out in PC12 cells, neither CREB- nor JUN-mediated gene expression could be induced by RET/PTC1 in LA-N-5 cells (Fig. 6). Interestingly, our study showed that protein kinase A could not induce CREB-mediated gene expression in LA-N-5 cells. These results indicate that the stimulation effect of RET on ELK-, CREB-, or JUN-mediated gene expression could be different in different cells. Activation of the MAPK pathway by RET in neuronal cells has been demonstrated by both promoter analysis and detailed biochemical characterization. However, our study is the first report showing that RET activation can induce CREB-mediated gene expression by promoter analysis. Furthermore, we showed that RET activation in PC12 cells could activate JUN-mediated gene expression, despite the fact that our results and those of others showed that JUN-mediated gene expression was not induced by RET activation in LA-N-5 cells. To fully understand how RET activates CREB- or JUN-mediated gene expression in PC12 cells, detailed biochemical characterization of the RET signaling pathways that lead to activation of CREB or JUN in PC12 cells is warranted. The information obtained could provide significant insights into the molecular mechanisms underlying the predisposition of RET activation in the development of MEN2 diseases.
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