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. 1994 Oct;14(10):6522–6530. doi: 10.1128/mcb.14.10.6522

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

R R Vaillancourt 1, A M Gardner 1, G L Johnson 1
PMCID: PMC359182  PMID: 7935374

Abstract

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boulton T. G., Nye S. H., Robbins D. J., Ip N. Y., Radziejewska E., Morgenbesser S. D., DePinho R. A., Panayotatos N., Cobb M. H., Yancopoulos G. D. ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 1991 May 17;65(4):663–675. doi: 10.1016/0092-8674(91)90098-j. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Carter A. N., Downes C. P. Phosphatidylinositol 3-kinase is activated by nerve growth factor and epidermal growth factor in PC12 cells. J Biol Chem. 1992 Jul 25;267(21):14563–14567. [PubMed] [Google Scholar]
  5. 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]
  6. Crews C. M., Alessandrini A., Erikson R. L. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. doi: 10.1126/science.1411546. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fling S. P., Gregerson D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. doi: 10.1016/0003-2697(86)90228-9. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Gardner A. M., Vaillancourt R. R., Johnson G. L. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase by G protein and tyrosine kinase oncoproteins. J Biol Chem. 1993 Aug 25;268(24):17896–17901. [PubMed] [Google Scholar]
  11. Ginty D. D., Glowacka D., DeFranco C., Wagner J. A. Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type II cAMP-dependent protein kinase activities. J Biol Chem. 1991 Aug 15;266(23):15325–15333. [PubMed] [Google Scholar]
  12. 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]
  13. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heasley L. E., Johnson G. L. Detection of nerve growth factor and epidermal growth factor-regulated protein kinases in PC12 cells with synthetic peptide substrates. Mol Pharmacol. 1989 Mar;35(3):331–338. [PubMed] [Google Scholar]
  15. Heasley L. E., Johnson G. L. The beta-PDGF receptor induces neuronal differentiation of PC12 cells. Mol Biol Cell. 1992 May;3(5):545–553. doi: 10.1091/mbc.3.5.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Hempstead B. L., Rabin S. J., Kaplan L., Reid S., Parada L. F., Kaplan D. R. Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation. Neuron. 1992 Nov;9(5):883–896. doi: 10.1016/0896-6273(92)90241-5. [DOI] [PubMed] [Google Scholar]
  18. Huff K., End D., Guroff G. Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor. J Cell Biol. 1981 Jan;88(1):189–198. doi: 10.1083/jcb.88.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jaiswal R. K., Murphy M. B., Landreth G. E. Identification and characterization of a nerve growth factor-stimulated mitogen-activated protein kinase activator in PC12 cells. J Biol Chem. 1993 Apr 5;268(10):7055–7063. [PubMed] [Google Scholar]
  20. Kovacina K. S., Yonezawa K., Brautigan D. L., Tonks N. K., Rapp U. R., Roth R. A. Insulin activates the kinase activity of the Raf-1 proto-oncogene by increasing its serine phosphorylation. J Biol Chem. 1990 Jul 25;265(21):12115–12118. [PubMed] [Google Scholar]
  21. 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]
  22. Lange-Carter C. A., Pleiman C. M., Gardner A. M., Blumer K. J., Johnson G. L. A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science. 1993 Apr 16;260(5106):315–319. doi: 10.1126/science.8385802. [DOI] [PubMed] [Google Scholar]
  23. Li B. Q., Kaplan D., Kung H. F., Kamata T. Nerve growth factor stimulation of the Ras-guanine nucleotide exchange factor and GAP activities. Science. 1992 Jun 5;256(5062):1456–1459. doi: 10.1126/science.1604323. [DOI] [PubMed] [Google Scholar]
  24. Macdonald S. G., Crews C. M., Wu L., Driller J., Clark R., Erikson R. L., McCormick F. Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. Mol Cell Biol. 1993 Nov;13(11):6615–6620. doi: 10.1128/mcb.13.11.6615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miyasaka T., Chao M. V., Sherline P., Saltiel A. R. Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2. J Biol Chem. 1990 Mar 15;265(8):4730–4735. [PubMed] [Google Scholar]
  26. Morrison D. K., Kaplan D. R., Rapp U., Roberts T. M. Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8855–8859. doi: 10.1073/pnas.85.23.8855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Muroya K., Hattori S., Nakamura S. Nerve growth factor induces rapid accumulation of the GTP-bound form of p21ras in rat pheochromocytoma PC12 cells. Oncogene. 1992 Feb;7(2):277–281. [PubMed] [Google Scholar]
  28. Ohmichi M., Pang L., Decker S. J., Saltiel A. R. Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene. J Biol Chem. 1992 Jul 25;267(21):14604–14610. [PubMed] [Google Scholar]
  29. Ohmichi M., Pang L., Ribon V., Saltiel A. R. Divergence of signaling pathways for insulin in PC-12 pheochromocytoma cells. Endocrinology. 1993 Jul;133(1):46–56. doi: 10.1210/endo.133.1.7686484. [DOI] [PubMed] [Google Scholar]
  30. Oshima M., Sithanandam G., Rapp U. R., Guroff G. The phosphorylation and activation of B-raf in PC12 cells stimulated by nerve growth factor. J Biol Chem. 1991 Dec 15;266(35):23753–23760. [PubMed] [Google Scholar]
  31. Qiu M. S., Green S. H. NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation. Neuron. 1991 Dec;7(6):937–946. doi: 10.1016/0896-6273(91)90339-2. [DOI] [PubMed] [Google Scholar]
  32. Qui M. S., Green S. H. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron. 1992 Oct;9(4):705–717. doi: 10.1016/0896-6273(92)90033-a. [DOI] [PubMed] [Google Scholar]
  33. Rabin S. J., Cleghon V., Kaplan D. R. SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells. Mol Cell Biol. 1993 Apr;13(4):2203–2213. doi: 10.1128/mcb.13.4.2203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rudkin B. B., Lazarovici P., Levi B. Z., Abe Y., Fujita K., Guroff G. Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation. EMBO J. 1989 Nov;8(11):3319–3325. doi: 10.1002/j.1460-2075.1989.tb08493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Russell M., Winitz S., Johnson G. L. Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity. Mol Cell Biol. 1994 Apr;14(4):2343–2351. doi: 10.1128/mcb.14.4.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rydel R. E., Greene L. A. Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci. 1987 Nov;7(11):3639–3653. doi: 10.1523/JNEUROSCI.07-11-03639.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Satoh T., Endo M., Nakafuku M., Akiyama T., Yamamoto T., Kaziro Y. Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7926–7929. doi: 10.1073/pnas.87.20.7926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schanen-King C., Nel A., Williams L. K., Landreth G. Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells. Neuron. 1991 Jun;6(6):915–922. doi: 10.1016/0896-6273(91)90232-o. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Sithanandam G., Kolch W., Duh F. M., Rapp U. R. Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies. Oncogene. 1990 Dec;5(12):1775–1780. [PubMed] [Google Scholar]
  41. Soltoff S. P., Rabin S. L., Cantley L. C., Kaplan D. R. Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase. J Biol Chem. 1992 Aug 25;267(24):17472–17477. [PubMed] [Google Scholar]
  42. Stephens R. M., Sithanandam G., Copeland T. D., Kaplan D. R., Rapp U. R., Morrison D. K. 95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site. Mol Cell Biol. 1992 Sep;12(9):3733–3742. doi: 10.1128/mcb.12.9.3733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Storm S. M., Cleveland J. L., Rapp U. R. Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene. 1990 Mar;5(3):345–351. [PubMed] [Google Scholar]
  44. Thomas S. M., DeMarco M., D'Arcangelo G., Halegoua S., Brugge J. S. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell. 1992 Mar 20;68(6):1031–1040. doi: 10.1016/0092-8674(92)90075-n. [DOI] [PubMed] [Google Scholar]
  45. Vetter M. L., Martin-Zanca D., Parada L. F., Bishop J. M., Kaplan D. R. Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5650–5654. doi: 10.1073/pnas.88.13.5650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  47. Warne P. H., Viciana P. R., Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul 22;364(6435):352–355. doi: 10.1038/364352a0. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Wood K. W., Qi H., D'Arcangelo G., Armstrong R. C., Roberts T. M., Halegoua S. The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5016–5020. doi: 10.1073/pnas.90.11.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wood K. W., Sarnecki C., Roberts T. M., 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. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Zhang X. F., Settleman J., Kyriakis J. M., Takeuchi-Suzuki E., Elledge S. J., Marshall M. S., Bruder J. T., Rapp U. R., Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 1993 Jul 22;364(6435):308–313. doi: 10.1038/364308a0. [DOI] [PubMed] [Google Scholar]

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