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. 1995 Mar;15(3):1162–1168. doi: 10.1128/mcb.15.3.1162

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

N al-Alawi 1, D W Rose 1, C Buckmaster 1, N Ahn 1, U Rapp 1, J Meinkoth 1, J R Feramisco 1
PMCID: PMC230338  PMID: 7862110

Abstract

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.

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Selected References

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  1. Ahn N. G., Seger R., Krebs E. G. The mitogen-activated protein kinase activator. Curr Opin Cell Biol. 1992 Dec;4(6):992–999. doi: 10.1016/0955-0674(92)90131-u. [DOI] [PubMed] [Google Scholar]
  2. Ambesi-Impiombato F. S., Parks L. A., Coon H. G. Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3455–3459. doi: 10.1073/pnas.77.6.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ball R. L., Tanner K. D., Carpenter G. Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells. J Biol Chem. 1990 Aug 5;265(22):12836–12845. [PubMed] [Google Scholar]
  4. Berra E., Diaz-Meco M. T., Dominguez I., Municio M. M., Sanz L., Lozano J., Chapkin R. S., Moscat J. Protein kinase C zeta isoform is critical for mitogenic signal transduction. Cell. 1993 Aug 13;74(3):555–563. doi: 10.1016/0092-8674(93)80056-k. [DOI] [PubMed] [Google Scholar]
  5. Bruder J. T., Heidecker G., Rapp U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 1992 Apr;6(4):545–556. doi: 10.1101/gad.6.4.545. [DOI] [PubMed] [Google Scholar]
  6. Burgering B. M., Pronk G. J., van Weeren P. C., Chardin P., Bos J. L. cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J. 1993 Nov;12(11):4211–4220. doi: 10.1002/j.1460-2075.1993.tb06105.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bushman W. A., Wilson L. K., Luttrell D. K., Moyers J. S., Parsons S. J. Overexpression of c-src enhances beta-adrenergic-induced cAMP accumulation. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7462–7466. doi: 10.1073/pnas.87.19.7462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cai H., Erhardt P., Troppmair J., Diaz-Meco M. T., Sithanandam G., Rapp U. R., Moscat J., Cooper G. M. Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction. Mol Cell Biol. 1993 Dec;13(12):7645–7651. doi: 10.1128/mcb.13.12.7645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cook S. J., McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science. 1993 Nov 12;262(5136):1069–1072. doi: 10.1126/science.7694367. [DOI] [PubMed] [Google Scholar]
  10. Dent P., Haser W., Haystead T. A., Vincent L. A., Roberts T. M., Sturgill T. W. 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. doi: 10.1126/science.1326789. [DOI] [PubMed] [Google Scholar]
  11. Dominguez I., Diaz-Meco M. T., Municio M. M., Berra E., García de Herreros A., Cornet M. E., Sanz L., Moscat J. Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes. Mol Cell Biol. 1992 Sep;12(9):3776–3783. doi: 10.1128/mcb.12.9.3776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Downes C. P., Carter A. N. Phosphoinositide 3-kinase: a new effector in signal transduction? Cell Signal. 1991;3(6):501–513. doi: 10.1016/0898-6568(91)90027-r. [DOI] [PubMed] [Google Scholar]
  13. Dumont J. E., Lamy F., Roger P., Maenhaut C. Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors. Physiol Rev. 1992 Jul;72(3):667–697. doi: 10.1152/physrev.1992.72.3.667. [DOI] [PubMed] [Google Scholar]
  14. Dérijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994 Mar 25;76(6):1025–1037. doi: 10.1016/0092-8674(94)90380-8. [DOI] [PubMed] [Google Scholar]
  15. Frost J. A., Geppert T. D., Cobb M. H., Feramisco J. R. A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3844–3848. doi: 10.1073/pnas.91.9.3844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Frödin M., Peraldi P., Van Obberghen E. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J Biol Chem. 1994 Feb 25;269(8):6207–6214. [PubMed] [Google Scholar]
  17. Gille H., Sharrocks A. D., Shaw P. E. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature. 1992 Jul 30;358(6385):414–417. doi: 10.1038/358414a0. [DOI] [PubMed] [Google Scholar]
  18. Gonzalez F. A., Seth A., Raden D. L., Bowman D. S., Fay F. S., Davis R. J. Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus. J Cell Biol. 1993 Sep;122(5):1089–1101. doi: 10.1083/jcb.122.5.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Graves L. M., Bornfeldt K. E., Raines E. W., Potts B. C., Macdonald S. G., Ross R., Krebs E. G. Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10300–10304. doi: 10.1073/pnas.90.21.10300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gross M., Sweet R. W., Sathe G., Yokoyama S., Fasano O., Goldfarb M., Wigler M., Rosenberg M. Purification and characterization of human H-ras proteins expressed in Escherichia coli. Mol Cell Biol. 1985 May;5(5):1015–1024. doi: 10.1128/mcb.5.5.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heidecker G., Kölch W., Morrison D. K., Rapp U. R. The role of Raf-1 phosphorylation in signal transduction. Adv Cancer Res. 1992;58:53–73. doi: 10.1016/s0065-230x(08)60290-0. [DOI] [PubMed] [Google Scholar]
  22. Howe L. R., Leevers S. J., Gómez N., Nakielny S., Cohen P., Marshall C. J. Activation of the MAP kinase pathway by the protein kinase raf. Cell. 1992 Oct 16;71(2):335–342. doi: 10.1016/0092-8674(92)90361-f. [DOI] [PubMed] [Google Scholar]
  23. Hunter T., Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. doi: 10.1016/0092-8674(92)90162-6. [DOI] [PubMed] [Google Scholar]
  24. Häfner S., Adler H. S., Mischak H., Janosch P., Heidecker G., Wolfman A., Pippig S., Lohse M., Ueffing M., Kolch W. Mechanism of inhibition of Raf-1 by protein kinase A. Mol Cell Biol. 1994 Oct;14(10):6696–6703. doi: 10.1128/mcb.14.10.6696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kikuchi A., Demo S. D., Ye Z. H., Chen Y. W., Williams L. T. ralGDS family members interact with the effector loop of ras p21. Mol Cell Biol. 1994 Nov;14(11):7483–7491. doi: 10.1128/mcb.14.11.7483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kodaki T., Woscholski R., Hallberg B., Rodriguez-Viciana P., Downward J., Parker P. J. The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol. 1994 Sep 1;4(9):798–806. doi: 10.1016/s0960-9822(00)00177-9. [DOI] [PubMed] [Google Scholar]
  27. Kolch W., Heidecker G., Lloyd P., Rapp U. R. Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature. 1991 Jan 31;349(6308):426–428. doi: 10.1038/349426a0. [DOI] [PubMed] [Google Scholar]
  28. Kolch W., Weissinger E., Mischak H., Troppmair J., Showalter S. D., Lloyd P., Heidecker G., Rapp U. R. Probing structure and function of the raf protein kinase domain with monoclonal antibodies. Oncogene. 1990 May;5(5):713–720. [PubMed] [Google Scholar]
  29. Kupperman E., Wen W., Meinkoth J. L. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase. Mol Cell Biol. 1993 Aug;13(8):4477–4484. doi: 10.1128/mcb.13.8.4477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kyriakis J. M., App H., Zhang X. F., Banerjee P., Brautigan D. L., Rapp U. R., Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. doi: 10.1038/358417a0. [DOI] [PubMed] [Google Scholar]
  31. Lamy F., Wilkin F., Baptist M., Posada J., Roger P. P., Dumont J. E. Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway. J Biol Chem. 1993 Apr 25;268(12):8398–8401. [PubMed] [Google Scholar]
  32. Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
  33. Lenormand P., Sardet C., Pagès G., L'Allemain G., Brunet A., Pouysségur J. Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts. J Cell Biol. 1993 Sep;122(5):1079–1088. doi: 10.1083/jcb.122.5.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. MacNicol A. M., Muslin A. J., Williams L. T. 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. doi: 10.1016/0092-8674(93)90143-e. [DOI] [PubMed] [Google Scholar]
  35. Mansour S. J., Resing K. A., Candi J. M., Hermann A. S., Gloor J. W., Herskind K. R., Wartmann M., Davis R. J., Ahn N. G. Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis. J Biochem. 1994 Aug;116(2):304–314. doi: 10.1093/oxfordjournals.jbchem.a124524. [DOI] [PubMed] [Google Scholar]
  36. 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. doi: 10.1016/0092-8674(93)90237-k. [DOI] [PubMed] [Google Scholar]
  37. McClain D. A., Maegawa H., Lee J., Dull T. J., Ulrich A., Olefsky J. M. A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem. 1987 Oct 25;262(30):14663–14671. [PubMed] [Google Scholar]
  38. Meinkoth J. L., Goldsmith P. K., Spiegel A. M., Feramisco J. R., Burrow G. N. Inhibition of thyrotropin-induced DNA synthesis in thyroid follicular cells by microinjection of an antibody to the stimulatory G protein of adenylate cyclase, Gs. J Biol Chem. 1992 Jul 5;267(19):13239–13245. [PubMed] [Google Scholar]
  39. Minden A., Lin A., Smeal T., Dérijard B., Cobb M., Davis R., Karin M. c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases. Mol Cell Biol. 1994 Oct;14(10):6683–6688. doi: 10.1128/mcb.14.10.6683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nakanishi H., Exton J. H. Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. J Biol Chem. 1992 Aug 15;267(23):16347–16354. [PubMed] [Google Scholar]
  41. Pagès G., Lenormand P., L'Allemain G., Chambard J. C., Meloche S., Pouysségur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8319–8323. doi: 10.1073/pnas.90.18.8319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pelech S. L. Networking with protein kinases. Curr Biol. 1993 Aug 1;3(8):513–515. doi: 10.1016/0960-9822(93)90043-n. [DOI] [PubMed] [Google Scholar]
  43. Polakis P., McCormick F. Interactions between p21ras proteins and their GTPase activating proteins. Cancer Surv. 1992;12:25–42. [PubMed] [Google Scholar]
  44. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994 Aug 18;370(6490):527–532. doi: 10.1038/370527a0. [DOI] [PubMed] [Google Scholar]
  45. Santoro M., Wong W. T., Aroca P., Santos E., Matoskova B., Grieco M., Fusco A., di Fiore P. P. An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway. Mol Cell Biol. 1994 Jan;14(1):663–675. doi: 10.1128/mcb.14.1.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Seth A., Gonzalez F. A., Gupta S., Raden D. L., Davis R. J. Signal transduction within the nucleus by mitogen-activated protein kinase. J Biol Chem. 1992 Dec 5;267(34):24796–24804. [PubMed] [Google Scholar]
  47. Sevetson B. R., Kong X., Lawrence J. C., Jr Increasing cAMP attenuates activation of mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10305–10309. doi: 10.1073/pnas.90.21.10305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sjölander A., Yamamoto K., Huber B. E., Lapetina E. G. Association of p21ras with phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7908–7912. doi: 10.1073/pnas.88.18.7908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
  50. Suarez H. G., du Villard J. A., Caillou B., Schlumberger M., Parmentier C., Monier R. gsp mutations in human thyroid tumours. Oncogene. 1991 Apr;6(4):677–679. [PubMed] [Google Scholar]
  51. Suarez H. G., du Villard J. A., Severino M., Caillou B., Schlumberger M., Tubiana M., Parmentier C., Monier R. Presence of mutations in all three ras genes in human thyroid tumors. Oncogene. 1990 Apr;5(4):565–570. [PubMed] [Google Scholar]
  52. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  53. Tominaga T., Dela Cruz J., Burrow G. N., Meinkoth J. L. Divergent patterns of immediate early gene expression in response to thyroid-stimulating hormone and insulin-like growth factor I in Wistar rat thyrocytes. Endocrinology. 1994 Sep;135(3):1212–1219. doi: 10.1210/endo.135.3.8070365. [DOI] [PubMed] [Google Scholar]
  54. Vaillancourt R. R., Gardner A. M., Johnson G. L. B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP. Mol Cell Biol. 1994 Oct;14(10):6522–6530. doi: 10.1128/mcb.14.10.6522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vassart G., Dumont J. E. The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev. 1992 Aug;13(3):596–611. doi: 10.1210/edrv-13-3-596. [DOI] [PubMed] [Google Scholar]
  56. Williams N. G., Paradis H., Agarwal S., Charest D. L., Pelech S. L., Roberts T. M. Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5772–5776. doi: 10.1073/pnas.90.12.5772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wu J., Dent P., Jelinek T., Wolfman A., Weber M. J., Sturgill T. W. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science. 1993 Nov 12;262(5136):1065–1069. doi: 10.1126/science.7694366. [DOI] [PubMed] [Google Scholar]
  58. Young S. W., Dickens M., Tavaré J. M. Differentiation of PC12 cells in response to a cAMP analogue is accompanied by sustained activation of mitogen-activated protein kinase. Comparison with the effects of insulin, growth factors and phorbol esters. FEBS Lett. 1994 Jan 31;338(2):212–216. doi: 10.1016/0014-5793(94)80367-6. [DOI] [PubMed] [Google Scholar]

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