Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Sep;15(9):4930–4938. doi: 10.1128/mcb.15.9.4930

Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

R Zinck 1, M A Cahill 1, M Kracht 1, C Sachsenmaier 1, R A Hipskind 1, A Nordheim 1
PMCID: PMC230739  PMID: 7651411

Abstract

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction.

Full Text

The Full Text of this article is available as a PDF (772.8 KB).

Selected References

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

  1. Alessi D. R., Gomez N., Moorhead G., Lewis T., Keyse S. M., Cohen P. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr Biol. 1995 Mar 1;5(3):283–295. doi: 10.1016/s0960-9822(95)00059-5. [DOI] [PubMed] [Google Scholar]
  2. Almendral J. M., Sommer D., Macdonald-Bravo H., Burckhardt J., Perera J., Bravo R. Complexity of the early genetic response to growth factors in mouse fibroblasts. Mol Cell Biol. 1988 May;8(5):2140–2148. doi: 10.1128/mcb.8.5.2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  4. Baccarini M., Sabatini D. M., App H., Rapp U. R., Stanley E. R. Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the RAF-1 proto-oncogene product. EMBO J. 1990 Nov;9(11):3649–3657. doi: 10.1002/j.1460-2075.1990.tb07576.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cano E., Hazzalin C. A., Mahadevan L. C. Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol Cell Biol. 1994 Nov;14(11):7352–7362. doi: 10.1128/mcb.14.11.7352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Düring K. Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology. Anal Biochem. 1991 Aug 1;196(2):433–438. doi: 10.1016/0003-2697(91)90490-k. [DOI] [PubMed] [Google Scholar]
  8. Edwards D. R., Mahadevan L. C. Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors. EMBO J. 1992 Jul;11(7):2415–2424. doi: 10.1002/j.1460-2075.1992.tb05306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fort P., Rech J., Vie A., Piechaczyk M., Bonnieu A., Jeanteur P., Blanchard J. M. Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation. Nucleic Acids Res. 1987 Jul 24;15(14):5657–5667. doi: 10.1093/nar/15.14.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Greenberg M. E., Hermanowski A. L., Ziff E. B. Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription. Mol Cell Biol. 1986 Apr;6(4):1050–1057. doi: 10.1128/mcb.6.4.1050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  13. Herrera R. E., Shaw P. E., Nordheim A. Occupation of the c-fos serum response element in vivo by a multi-protein complex is unaltered by growth factor induction. Nature. 1989 Jul 6;340(6228):68–70. doi: 10.1038/340068a0. [DOI] [PubMed] [Google Scholar]
  14. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993 Nov;7(11):2135–2148. doi: 10.1101/gad.7.11.2135. [DOI] [PubMed] [Google Scholar]
  15. Hipskind R. A., Baccarini M., Nordheim A. Transient activation of RAF-1, MEK, and ERK2 coincides kinetically with ternary complex factor phosphorylation and immediate-early gene promoter activity in vivo. Mol Cell Biol. 1994 Sep;14(9):6219–6231. doi: 10.1128/mcb.14.9.6219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hipskind R. A., Büscher D., Nordheim A., Baccarini M. Ras/MAP kinase-dependent and -independent signaling pathways target distinct ternary complex factors. Genes Dev. 1994 Aug 1;8(15):1803–1816. doi: 10.1101/gad.8.15.1803. [DOI] [PubMed] [Google Scholar]
  17. Hipskind R. A., Rao V. N., Mueller C. G., Reddy E. S., Nordheim A. Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF. Nature. 1991 Dec 19;354(6354):531–534. doi: 10.1038/354531a0. [DOI] [PubMed] [Google Scholar]
  18. Janknecht R., Ernst W. H., Pingoud V., Nordheim A. Activation of ternary complex factor Elk-1 by MAP kinases. EMBO J. 1993 Dec 15;12(13):5097–5104. doi: 10.1002/j.1460-2075.1993.tb06204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Janknecht R., Nordheim A. Affinity purification of histidine-tagged proteins transiently produced in HeLa cells. Gene. 1992 Nov 16;121(2):321–324. doi: 10.1016/0378-1119(92)90137-e. [DOI] [PubMed] [Google Scholar]
  20. Karin M. Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors. Curr Opin Cell Biol. 1994 Jun;6(3):415–424. doi: 10.1016/0955-0674(94)90035-3. [DOI] [PubMed] [Google Scholar]
  21. Keyse S. M., Emslie E. A. Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase. Nature. 1992 Oct 15;359(6396):644–647. doi: 10.1038/359644a0. [DOI] [PubMed] [Google Scholar]
  22. Kortenjann M., Thomae O., Shaw P. E. Inhibition of v-raf-dependent c-fos expression and transformation by a kinase-defective mutant of the mitogen-activated protein kinase Erk2. Mol Cell Biol. 1994 Jul;14(7):4815–4824. doi: 10.1128/mcb.14.7.4815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kracht M., Shiroo M., Marshall C. J., Hsuan J. J., Saklatvala J. Interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide T669. Biochem J. 1994 Sep 15;302(Pt 3):897–905. doi: 10.1042/bj3020897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kracht M., Truong O., Totty N. F., Shiroo M., Saklatvala J. Interleukin 1 alpha activates two forms of p54 alpha mitogen-activated protein kinase in rabbit liver. J Exp Med. 1994 Dec 1;180(6):2017–2025. doi: 10.1084/jem.180.6.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  26. Lau L. F., Nathans D. Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1182–1186. doi: 10.1073/pnas.84.5.1182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mahadevan L. C., Edwards D. R. Signalling and superinduction. Nature. 1991 Feb 28;349(6312):747–748. doi: 10.1038/349747c0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Minden A., Lin A., McMahon M., Lange-Carter C., Dérijard B., Davis R. J., Johnson G. L., Karin M. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science. 1994 Dec 9;266(5191):1719–1723. doi: 10.1126/science.7992057. [DOI] [PubMed] [Google Scholar]
  30. Morello D., Lavenu A., Babinet C. Differential regulation and expression of jun, c-fos and c-myc proto-oncogenes during mouse liver regeneration and after inhibition of protein synthesis. Oncogene. 1990 Oct;5(10):1511–1519. [PubMed] [Google Scholar]
  31. Pingoud V., Zinck R., Hipskind R. A., Janknecht R., Nordheim A. Heterogeneity of ternary complex factors in HeLa cell nuclear extracts. J Biol Chem. 1994 Sep 16;269(37):23310–23317. [PubMed] [Google Scholar]
  32. Sachsenmaier C., Radler-Pohl A., Zinck R., Nordheim A., Herrlich P., Rahmsdorf H. J. Involvement of growth factor receptors in the mammalian UVC response. Cell. 1994 Sep 23;78(6):963–972. doi: 10.1016/0092-8674(94)90272-0. [DOI] [PubMed] [Google Scholar]
  33. Sassone-Corsi P., Sisson J. C., Verma I. M. Transcriptional autoregulation of the proto-oncogene fos. Nature. 1988 Jul 28;334(6180):314–319. doi: 10.1038/334314a0. [DOI] [PubMed] [Google Scholar]
  34. Schönthal A., Büscher M., Angel P., Rahmsdorf H. J., Ponta H., Hattori K., Chiu R., Karin M., Herrlich P. The Fos and Jun/AP-1 proteins are involved in the downregulation of Fos transcription. Oncogene. 1989 May;4(5):629–636. [PubMed] [Google Scholar]
  35. Schönthal A., Tsukitani Y., Feramisco J. R. Transcriptional and post-transcriptional regulation of c-fos expression by the tumor promoter okadaic acid. Oncogene. 1991 Mar;6(3):423–430. [PubMed] [Google Scholar]
  36. Stewart A. F., Herrera R. E., Nordheim A. Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities. Cell. 1990 Jan 12;60(1):141–149. doi: 10.1016/0092-8674(90)90724-s. [DOI] [PubMed] [Google Scholar]
  37. Subramaniam M., Schmidt L. J., Crutchfield C. E., 3rd, Getz M. J. Negative regulation of serum-responsive enhancer elements. Nature. 1989 Jul 6;340(6228):64–66. doi: 10.1038/340064a0. [DOI] [PubMed] [Google Scholar]
  38. Sun H., Charles C. H., Lau L. F., Tonks N. K. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell. 1993 Nov 5;75(3):487–493. doi: 10.1016/0092-8674(93)90383-2. [DOI] [PubMed] [Google Scholar]
  39. Sánchez I., Hughes R. T., Mayer B. J., Yee K., Woodgett J. R., Avruch J., Kyriakis J. M., Zon L. I. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature. 1994 Dec 22;372(6508):794–798. doi: 10.1038/372794a0. [DOI] [PubMed] [Google Scholar]
  40. Treier M., Staszewski L. M., Bohmann D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell. 1994 Sep 9;78(5):787–798. doi: 10.1016/s0092-8674(94)90502-9. [DOI] [PubMed] [Google Scholar]
  41. Treisman R. Ternary complex factors: growth factor regulated transcriptional activators. Curr Opin Genet Dev. 1994 Feb;4(1):96–101. doi: 10.1016/0959-437x(94)90097-3. [DOI] [PubMed] [Google Scholar]
  42. Treisman R. The serum response element. Trends Biochem Sci. 1992 Oct;17(10):423–426. doi: 10.1016/0968-0004(92)90013-y. [DOI] [PubMed] [Google Scholar]
  43. Wilson T., Treisman R. Fos C-terminal mutations block down-regulation of c-fos transcription following serum stimulation. EMBO J. 1988 Dec 20;7(13):4193–4202. doi: 10.1002/j.1460-2075.1988.tb03316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yan M., Dai T., Deak J. C., Kyriakis J. M., Zon L. I., Woodgett J. R., Templeton D. J. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature. 1994 Dec 22;372(6508):798–800. doi: 10.1038/372798a0. [DOI] [PubMed] [Google Scholar]
  45. Zinck R., Hipskind R. A., Pingoud V., Nordheim A. c-fos transcriptional activation and repression correlate temporally with the phosphorylation status of TCF. EMBO J. 1993 Jun;12(6):2377–2387. doi: 10.1002/j.1460-2075.1993.tb05892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES