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Growth Factors, Cytokines and Signal Transduction

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism

Leukemia volume 14, pages 642–656 (2000)Cite this article

Abstract

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (Δ) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous β-estradiol. The vast majority of ΔRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of ΔRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from ΔA-Raf:ER than either ΔRaf-1:ER or ΔB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the ΔB-Raf:ER and ΔRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in ΔRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the ΔRaf:ER proteins as removal of β-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.

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References

  1. Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, Wang XY, Algate PA, Franklin RA, Oberhaus SM, Steelman LS, McCubrey JA . Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs Leukemia 1999 13: 1109–1166

    Article  CAS  Google Scholar 

  2. Wang XY, Steelman LS, McCubrey JA . Abnormal activation of cytokine gene expression by intracisternal type A particle transposition: effects of mutations which result in autocrine growth factor stimulation and malignant transformation Cytok Cell Mol Ther 1997 3: 3–19

    CAS  Google Scholar 

  3. Lotem J, Sachs L . Control of apoptosis in hematopoiesis and leukemia by cytokines, tumor suppressor and oncogenes Leukemia 1996 10: 925–931

    CAS  Google Scholar 

  4. Wang XY, McCubrey J . Regulation of interleukin 3 expression in normal and autocrine transformed hematopoietic cells Int J Oncol 1997 10: 989–1001

    CAS  Google Scholar 

  5. Algate PA, Steelman LS, Mayo MW, Miyajima A, McCubrey JA . Regulation of the interleukin-3 (IL-3) receptor by IL-3 in the fetal liver-derived FL5.12 cell line Blood 1994 83: 2450–2468

    Google Scholar 

  6. Steelman LS, Algate PA, Blalock WL, Wang XY, Prevost KD, Hoyle PE, McCubrey JA . Oncogenic effects of overexpression of the interleukin-3 receptor on hematopoietic cells Leukemia 1996 10: 528–542

    CAS  Google Scholar 

  7. Kinoshita T, Yokata T, Arai K, Miyajima A . Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors EMBO J 1995 14: 265–275

    Article  Google Scholar 

  8. Dexter TM, Garland J, Scott D, Scolnick E, Metcalf, D . Growth factor-dependent hemopoietic precursor cell lines J Exp Med 1980 152: 1036–1047

    Article  CAS  Google Scholar 

  9. McKearn JP, McCubrey JA, Fagg B . Enrichment of hematopoietic precursor cells and cloning of multipotential B lymphocyte precursors Proc Natl Acad Sci USA 1985 82: 7414–7418

    Article  CAS  Google Scholar 

  10. McCubrey JA, Holland G, McKearn J, Risser R . Abrogation of factor-dependence in 2 IL-3-dependent cell lines can occur by two distinct mechanisms Oncogene Res 1989 4: 97–109

    CAS  Google Scholar 

  11. McCubrey JA, Smith SR, Algate PA, deVente JE, White MK, Steelman LS . Retroviral infection can abrogate the factor-dependency of hematopoietic cells by autocrine and non-autocrine mechanisms depending on the presence of a functional viral oncogene Oncogene 1993 8: 2905–2915

    CAS  Google Scholar 

  12. McCubrey JA, Steelman LS, Wang XY, Algate PA, Hoyle PE, White C, Davidian EW, Prevost KD, Robbins P, Mylott D, White MK . Differential effects of viral and cellular oncogenes on the growth factor-dependency of hematopoietic cells Int J Oncol 1995 7: 285–310

    Google Scholar 

  13. Kitamura T, Sato M, Arai K-I, Miyajima A . Expression cloning of the human IL-3 receptor cDNA reveals a shared β subunit of the human IL-3 and GM-CSF receptors Cell 1991 66: 1165–1174

    Article  CAS  Google Scholar 

  14. McCubrey JA, May WS, Duronio V, Mufson A . Serine/threonine phosphorylation in cytokine signal transduction Leukemia 2000 (in press)

  15. McCubrey JA, Steelman LS, Hoyle PA, Blalock WL, Weinstein-Oppenheimer CR, Franklin RA, Cherwinski H, Bosch E, McMahon M . Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells Leukemia 1998 12: 1903–1929

    Article  CAS  Google Scholar 

  16. Morrison D . Mechanisms regulating Raf-1 activity in signal transduction pathways Mol Repro Dev 1995 42: 507–514

    Article  CAS  Google Scholar 

  17. Dent P, Reardon DB, Morrison DK, Sturgill TW . Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro Mol Cell Biol 1995 15: 4125–4135

    Article  CAS  Google Scholar 

  18. Dent P, Jarvis WD, Birrer MJ, Fisher PB, Schmidt-Ullrich RK, Grant S . The roles of signaling by the p42/p44 mitogen-activated protein (MAP) kinase pathway; a potential route to radio- and chemo-sensitization of tumor cells resulting in the induction of apoptosis and loss of clonogenicity Leukemia 1998 12: 1843–1850

    Article  CAS  Google Scholar 

  19. Stancato LF, Sakatsume M, David M, Dent P, Dong F, Petricoin EF, Krolewski JJ, Silvennoinen O, Saharinen P, Pierce J, Marshall CJ, Sturgill T, Finbloom DS, Larner AC . Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway Mol Cell Biol 1997 17: 3833–3840

    Article  CAS  Google Scholar 

  20. Ruvolo PP, Deng X, Ito T, Carr BK, May WS . Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A J Biol Chem 1999 274: 20296–20300

    Article  CAS  Google Scholar 

  21. Sakamoto KM, Mignacca RC, Gasson JC . Signal transduction by granulocyte–macrophage colony-stimulating factor and interleukin-3 receptors Receptors Channels 1994 2: 175–181

    CAS  PubMed  Google Scholar 

  22. Kwon EM, Sakamoto KM . The molecular mechanism of action of granulocyte–macrophage/colony-stimulating factor J Invest Med 1996 44: 442–446

    CAS  Google Scholar 

  23. Lee HJ, Mignacca RC, Sakamoto KM . Transcriptional activation of egr-1 by granulocyte–macrophage colony-stimulating factor but not interleukin 3 requires phosphorylation of cAMP response element-binding protein (CREB) on serine 133 J Biol Chem 1995 270: 15979–15983

    Article  CAS  Google Scholar 

  24. Wong A, Sakamoto KM . Granulocyte–macrophage colony-stimulating factor induces the transcriptional activation of egr-1 through a protein kinase A-independent signaling pathway J Biol Chem 1995 270: 30271–30273

    Article  CAS  Google Scholar 

  25. Watanabe S, Kubota H, Sakamoto KM, Arai K . Characterization of cis-acting sequences and trans-acting signals regulating early growth response 1 and c-fos promoters through the granulocyte–macrophage colony-stimulating factor receptor in BA/F3 cells Blood 1997 89: 1197–1206

    CAS  PubMed  Google Scholar 

  26. Mignacca RC, Lee HJ, Kwon EM, Sakamoto KM . Mechanism of transcriptional activation of the immediate early gene Egr-1 in response to PIXY321 Blood 1996 88: 848–854

    CAS  PubMed  Google Scholar 

  27. Sakamoto KM, Fraser JK, Lee HJ, Lehman E, Gasson JC . Granulocyte–macrophage colony-stimultating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter Mol Cell Biol 1994 14: 5975–5985

    Article  CAS  Google Scholar 

  28. Mark GE, Mac IR, Digan ME, Ambrosio L, Perrimon N . Drosophila melanogaster homologs of the raf oncogene Mol Cell Biol 1987 7: 2134–2140

    Article  CAS  Google Scholar 

  29. Pritchard CA, Bolin L, Slattery R, Murray RL, McMahon M . Post-natal lethality and neurological and gastrointestinal defects in mice with targeted disruption of the A-Raf protein kinase Curr Biol 1996 6: 614–617

    Article  CAS  Google Scholar 

  30. Pritchard C, McMahon M . Raf revealed in life-or-death decisions Nat Genet 1997 16: 214–215

    Article  CAS  Google Scholar 

  31. Wojnowski L, Zimmer AM, Beck TW, Hahn H, Bernal R, Rapp UR, Zimmer A . Endothelial apoptosis in Braf-deficient mice Nat Genet 1997 16: 293–297

    Article  CAS  Google Scholar 

  32. Wojnowski L, Stancato LF, Zimmer AM, Hahn H, Beck TW, Larner AC, Rapp UR, Zimmer A . c-raf-1 protein kinase is essential for mouse development Mech Dev 1998 76: 141–149

    Article  CAS  Google Scholar 

  33. Samuels ML, Weber MJ, Bishop JM, McMahon M . Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase Mol Cell Biol 1993 13: 6241–6252

    Article  CAS  Google Scholar 

  34. Pritchard CA, Samuels ML, Bosch E, McMahon M . Conditionally oncogenic forms of the A-raf and B-raf protein kinases display different biological and biochemical properties in NIH 3T3 cells Mol Cell Biol 1995 15: 9430–9442

    Article  Google Scholar 

  35. Bosch E, Cherwinski H, Peterson D, McMahon M . Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1 Oncogene 1997 11: 1021–1034

    Article  Google Scholar 

  36. Blalock WL, Steelman LS, Pearce M, Franklin RA, McCarthy SA, Cherwinski H, McMahon M, McCubrey JA . A conditionally active form of MEK1 results in autocrine transformation of human and mouse hematopoietic cells Oncogene (in press)

  37. McCubrey JA, Steelman LS, Moye PW, Hoyle PE, Weinstein-Oppenheimer C, Chang F, Pearce M, White MK, Franklin R, Blalock WL . Effects of deregulated Raf and MEK1 expression on the cytokine dependency of hematopoietic cells Adv Enzyme Regl 2000 40: (in press)

    Article  CAS  Google Scholar 

  38. Fabian JR, Daar IO, Morrison DK . Critical tyrosine residues regulate the enzymatic and biological activity or raf-1 kinase Mol Cell Biol 1993 13: 7170–7179

    Article  CAS  Google Scholar 

  39. Marais R, Light Y, Paterson H, Marshall CJ . Ras recruits raf-1 to the plasma membrane for activation by tyrosine phosphorylation EMBO J 1995 14: 101–110

    Article  Google Scholar 

  40. Jaiswal RK, Moodie SA, Wolfman A, Landreth GE . The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras Mol Cell Biol 1994 14: 6944–6953

    Article  CAS  Google Scholar 

  41. Nair APK, Diamantis ID, Conscience JF, Kindler V, Hofer P, Moroni C . A v-Ha-ras dependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production Mol Cell Biol 1993 9: 1183–1190

    Article  Google Scholar 

  42. Rapp UR, Troppmair J, Carroll M, May WS . Role of raf-1 protein kinase in IL-3 and GM-CSF mediated signal transduction Curr Top Microbiol Immunol 1990 166: 129–139

    CAS  PubMed  Google Scholar 

  43. Cleveland JL, Troppmair J, Packham G, Askew DS, Lloyd P, Gonzalez-Garcia M, Nuñez G, Ihle JN, Rapp UR . v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells Oncogene 1994 9: 2227–2233

    Google Scholar 

  44. Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ . Bad, a heterodimeric partner for Bcl-xL and Bcl-2 displaces Bax and promotes cell death Cell 1995 80: 285–291

    Article  CAS  Google Scholar 

  45. Wang HG, Miyashita T, Takayama S, Sato T, Torigoe T, Krajewski S, Tanaka S, Hovey L III, Troppmair J, Rapp UR, Reed JC . Apoptosis regulation by interaction of Bcl-2 and Raf-1 kinase Oncogene 1994 9: 2751–2756

    CAS  PubMed  Google Scholar 

  46. Wang HG, Takayama S, Rapp UR, Reed JC . Bcl-2 interacting protein, Bag-1 binds to and activates the kinase Raf-1 Proc Natl Acad Sci USA 1996 93: 7063–7068

    Article  CAS  Google Scholar 

  47. Wang HG, Rapp UR, Reed JC . Bcl-2 targets the protein kinase Raf-1 to mitochondria Cell 1996 87: 629–638

    Article  CAS  Google Scholar 

  48. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ . Serine phosphorylation of death agonist Bad in response to survival factor results in binding to 14–3-3 not Bcl-xL Cell 1996 87: 619–628

    Article  CAS  Google Scholar 

  49. Morgenstern JP, Land H . Advanced mammalian gene transfer: high titer retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line Nucleic Acid Res 1990 18: 3587–3596

    Article  CAS  Google Scholar 

  50. Woods D, Parry D, Chewinski H, Bosch E, Lees E, McMahon M . Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1 Mol Cell Biol 1997 17: 5598–5611

    Article  CAS  Google Scholar 

  51. Nori M, Shawver LK, Weber MJ . A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src Mol Cell Biol 1990 10: 4155–4162

    Article  CAS  Google Scholar 

  52. Schwartz RC, Stanton LW, Riley SC, Marcu KB, Witte ON . Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells Mol Cell Biol 1986 6: 3321–3331

    Article  Google Scholar 

  53. Miller AD, Rosman CJ . Improved retroviral vectors for gene transfer and expression Biotechniques 1989 7: 980–990

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Dudley DT, Pung L, Decker SJ, Bridges AJ, Saltiel AR . A synthetic inhibitor of the mitogen-activated protein kinases cascade Proc Natl Acad Sci USA 1995 92: 7686–7689

    Article  CAS  Google Scholar 

  55. Pumiglia KM, Decker SJ . Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway Proc Natl Acad Sci USA 1997 94: 448–452

    Article  CAS  Google Scholar 

  56. Alessandrini A, Greulich H, Huang W, Erikson RL . MEK1 phosphorylation site mutants activate Raf-1 in NIH 3T3 cells J Biol Chem 1996 271: 31612–31618

    Article  CAS  Google Scholar 

  57. Wang XY, Hoyle PE, McCubrey JA . Characterization of proteins binding specifically to the IL-3 mRNA 3′untranslated region in IL-3-dependent and autocrine transformed cells Leukemia 1998 12: 520–531

    Article  CAS  Google Scholar 

  58. Wang XY, McCubrey JA . Differential effects of retroviral long terminal repeats on interleukin-3 gene expression and autocrine transformation Leukemia 1997 11: 1711–1725

    Article  CAS  Google Scholar 

  59. Kabarowski JH, Allen PB, Wiedemann LM . A temperature sensitive p210 BCR-ABL mutant defines the primary consequences of BCR-ABL tyrosine kinase expression in growth factor dependent cells EMBO J 1995 13: 5887–5895

    Article  Google Scholar 

  60. McCarthy SA, Samuels ML, Pritchard CA, Abraham JA, McMahon M . Rapid induction of heparin binding epidermal growth factor/diphteria toxic receptor by Raf and Ras oncogenes Genes Dev 1995 9: 1953–1964

    Article  CAS  Google Scholar 

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Acknowledgements

We appreciate the artwork done by Ms Catherine Spruill and the staff of the ECU Center for Medical Communications. JAM was supported in part by a grant (R01CA51025) from the National Cancer Institute and the North Carolina Biotechnology Center (9805-ARG-0006). RAF was supported in part by grants from the American Cancer Society (IRG-97–149), American Heart Association (9930099N) and the North Carolina Biotechnology Center (9705-ARG-0009). DNAX Research Institute is supported by the Schering Plough Corporation.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC, USA

    PE Hoyle, PW Moye, LS Steelman, WL Blalock, RA Franklin, M Pearce & JA McCubrey

  2. Leo Jenkins Cancer Center, East Carolina University School of Medicine, Greenville, NC, USA

    RA Franklin & JA McCubrey

  3. Department of Cell Signaling, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA, USA

    H Cherwinski, E Bosch & M McMahon

  4. Sentinel Bioscience, California Avenue, Palo Alto, CA 94304, USA

    E Bosch

  5. 2340 Sutter Street, S-329, UCSF/Mt Zion Cancer Center, Box 0128, San Francisco, CA 94115, USA

    M McMahon

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Hoyle, P., Moye, P., Steelman, L. et al. Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism. Leukemia 14, 642–656 (2000). https://doi.org/10.1038/sj.leu.2401720

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