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Mol Cell Biol. 1993 October; 13(10): 6241–6252.
PMCID: PMC364683

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.


We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

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  • Ahn NG, Robbins DJ, Haycock JW, Seger R, Cobb MH, Krebs EG. Identification of an activator of the microtubule-associated protein 2 kinases ERK1 and ERK2 in PC12 cells stimulated with nerve growth factor or bradykinin. J Neurochem. 1992 Jul;59(1):147–156. [PubMed]
  • Ambrosio L, Mahowald AP, Perrimon N. Requirement of the Drosophila raf homologue for torso function. Nature. 1989 Nov 16;342(6247):288–291. [PubMed]
  • Anderson NG, Li P, Marsden LA, Williams N, Roberts TM, Sturgill TW. Raf-1 is a potential substrate for mitogen-activated protein kinase in vivo. Biochem J. 1991 Jul 15;277(Pt 2):573–576. [PubMed]
  • Ashworth A, Nakielny S, Cohen P, Marshall C. The amino acid sequence of a mammalian MAP kinase kinase. Oncogene. 1992 Dec;7(12):2555–2556. [PubMed]
  • Beck TW, Huleihel M, Gunnell M, Bonner TI, Rapp UR. The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus. Nucleic Acids Res. 1987 Jan 26;15(2):595–609. [PMC free article] [PubMed]
  • Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5889–5892. [PubMed]
  • Boehmelt G, Walker A, Kabrun N, Mellitzer G, Beug H, Zenke M, Enrietto PJ. Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression. EMBO J. 1992 Dec;11(12):4641–4652. [PubMed]
  • Brown DJ, Gordon JA. The stimulation of pp60v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme. J Biol Chem. 1984 Aug 10;259(15):9580–9586. [PubMed]
  • Burk O, Klempnauer KH. Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein. EMBO J. 1991 Dec;10(12):3713–3719. [PubMed]
  • Charles CH, Sun H, Lau LF, Tonks NK. The growth factor-inducible immediate-early gene 3CH134 encodes a protein-tyrosine-phosphatase. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5292–5296. [PubMed]
  • Chen RH, Sarnecki C, Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol. 1992 Mar;12(3):915–927. [PMC free article] [PubMed]
  • Christopherson KS, Mark MR, Bajaj V, Godowski PJ. Ecdysteroid-dependent regulation of genes in mammalian cells by a Drosophila ecdysone receptor and chimeric transactivators. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6314–6318. [PubMed]
  • Cobb MH, Robbins DJ, Boulton TG. ERKs, extracellular signal-regulated MAP-2 kinases. Curr Opin Cell Biol. 1991 Dec;3(6):1025–1032. [PubMed]
  • Crews CM, Alessandrini A, Erikson RL. Erks: their fifteen minutes has arrived. Cell Growth Differ. 1992 Feb;3(2):135–142. [PubMed]
  • Crews CM, Alessandrini A, Erikson RL. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. [PubMed]
  • Dent P, Haser W, Haystead TA, Vincent LA, Roberts TM, Sturgill TW. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science. 1992 Sep 4;257(5075):1404–1407. [PubMed]
  • Dickson B, Sprenger F, Morrison D, Hafen E. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature. 1992 Dec 10;360(6404):600–603. [PubMed]
  • Eilers M, Picard D, Yamamoto KR, Bishop JM. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature. 1989 Jul 6;340(6228):66–68. [PubMed]
  • Gallego C, Gupta SK, Heasley LE, Qian NX, Johnson GL. Mitogen-activated protein kinase activation resulting from selective oncogene expression in NIH 3T3 and rat 1a cells. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7355–7359. [PubMed]
  • Godowski PJ, Picard D, Yamamoto KR. Signal transduction and transcriptional regulation by glucocorticoid receptor-LexA fusion proteins. Science. 1988 Aug 12;241(4867):812–816. [PubMed]
  • Gupta SK, Gallego C, Johnson GL, Heasley LE. MAP kinase is constitutively activated in gip2 and src transformed rat 1a fibroblasts. J Biol Chem. 1992 Apr 25;267(12):7987–7990. [PubMed]
  • Han M, Golden A, Han Y, Sternberg PW. C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation. Nature. 1993 May 13;363(6425):133–140. [PubMed]
  • Haystead TA, Sim AT, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. [PubMed]
  • Hill CS, Marais R, John S, Wynne J, Dalton S, Treisman R. Functional analysis of a growth factor-responsive transcription factor complex. Cell. 1993 Apr 23;73(2):395–406. [PubMed]
  • Howe LR, Leevers SJ, Gómez N, Nakielny S, Cohen P, Marshall CJ. Activation of the MAP kinase pathway by the protein kinase raf. Cell. 1992 Oct 16;71(2):335–342. [PubMed]
  • Ikawa S, Fukui M, Ueyama Y, Tamaoki N, Yamamoto T, Toyoshima K. B-raf, a new member of the raf family, is activated by DNA rearrangement. Mol Cell Biol. 1988 Jun;8(6):2651–2654. [PMC free article] [PubMed]
  • Kosako H, Gotoh Y, Matsuda S, Ishikawa M, Nishida E. Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. EMBO J. 1992 Aug;11(8):2903–2908. [PubMed]
  • Kumar V, Green S, Staub A, Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J. 1986 Sep;5(9):2231–2236. [PubMed]
  • Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. [PubMed]
  • Lee RM, Cobb MH, Blackshear PJ. Evidence that extracellular signal-regulated kinases are the insulin-activated Raf-1 kinase kinases. J Biol Chem. 1992 Jan 15;267(2):1088–1092. [PubMed]
  • Li P, Wood K, Mamon H, Haser W, Roberts T. Raf-1: a kinase currently without a cause but not lacking in effects. Cell. 1991 Feb 8;64(3):479–482. [PubMed]
  • Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. [PubMed]
  • MacNicol AM, Muslin AJ, Williams LT. Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF. Cell. 1993 May 7;73(3):571–583. [PubMed]
  • Marais R, Wynne J, Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 1993 Apr 23;73(2):381–393. [PubMed]
  • Mark GE, MacIntyre RJ, Digan ME, Ambrosio L, Perrimon N. Drosophila melanogaster homologs of the raf oncogene. Mol Cell Biol. 1987 Jun;7(6):2134–2140. [PMC free article] [PubMed]
  • Matsuda S, Kosako H, Takenaka K, Moriyama K, Sakai H, Akiyama T, Gotoh Y, Nishida E. Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade. EMBO J. 1992 Mar;11(3):973–982. [PubMed]
  • McMahon M, Schatzman RC, Bishop JM. The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB. Mol Cell Biol. 1991 Sep;11(9):4760–4770. [PMC free article] [PubMed]
  • Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980–990. [PMC free article] [PubMed]
  • Morrison DK. The Raf-1 kinase as a transducer of mitogenic signals. Cancer Cells. 1990 Dec;2(12):377–382. [PubMed]
  • Nishida Y, Hata M, Ayaki T, Ryo H, Yamagata M, Shimizu K, Nishizuka Y. Proliferation of both somatic and germ cells is affected in the Drosophila mutants of raf proto-oncogene. EMBO J. 1988 Mar;7(3):775–781. [PubMed]
  • Payne DM, Rossomando AJ, Martino P, Erickson AK, Her JH, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 1991 Apr;10(4):885–892. [PubMed]
  • Picard D, Salser SJ, Yamamoto KR. A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor. Cell. 1988 Sep 23;54(7):1073–1080. [PubMed]
  • Rapp UR. Role of Raf-1 serine/threonine protein kinase in growth factor signal transduction. Oncogene. 1991 Apr;6(4):495–500. [PubMed]
  • Stanton VP, Jr, Nichols DW, Laudano AP, Cooper GM. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol Cell Biol. 1989 Feb;9(2):639–647. [PMC free article] [PubMed]
  • Storm SM, Cleveland JL, Rapp UR. Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene. 1990 Mar;5(3):345–351. [PubMed]
  • Sturgill TW, Ray LB, Erikson E, Maller JL. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature. 1988 Aug 25;334(6184):715–718. [PubMed]
  • Superti-Furga G, Bergers G, Picard D, Busslinger M. Hormone-dependent transcriptional regulation and cellular transformation by Fos-steroid receptor fusion proteins. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5114–5118. [PubMed]
  • Thomas G. MAP kinase by any other name smells just as sweet. Cell. 1992 Jan 10;68(1):3–6. [PubMed]
  • Thompson EW, Katz D, Shima TB, Wakeling AE, Lippman ME, Dickson RB. ICI 164,384, a pure antagonist of estrogen-stimulated MCF-7 cell proliferation and invasiveness. Cancer Res. 1989 Dec 15;49(24 Pt 1):6929–6934. [PubMed]
  • Tsuda L, Inoue YH, Yoo MA, Mizuno M, Hata M, Lim YM, Adachi-Yamada T, Ryo H, Masamune Y, Nishida Y. A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila. Cell. 1993 Feb 12;72(3):407–414. [PubMed]
  • Umek RM, Friedman AD, McKnight SL. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991 Jan 18;251(4991):288–292. [PubMed]
  • Webster NJ, Green S, Jin JR, Chambon P. The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell. 1988 Jul 15;54(2):199–207. [PubMed]
  • Wiseman LR, Wakeling AE, May FE, Westley BR. Effects of the antioestrogen, ICI 164,384, on oestrogen induced RNAs in MCF-7 cells. J Steroid Biochem. 1989 Jul;33(1):1–6. [PubMed]
  • Wood KW, Sarnecki C, Roberts TM, Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. [PubMed]
  • Wu J, Harrison JK, Vincent LA, Haystead C, Haystead TA, Michel H, Hunt DF, Lynch KR, Sturgill TW. Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):173–177. [PubMed]

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