Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Oct 17;13(20):4787–4797. doi: 10.1002/j.1460-2075.1994.tb06804.x

Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases.

M F Dubois 1, V T Nguyen 1, M E Dahmus 1, G Pagès 1, J Pouysségur 1, O Bensaude 1
PMCID: PMC395417  PMID: 7957047

Abstract

The largest subunit of RNA polymerase (RNAP) II contains at it C-terminus an unusual domain comprising tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain (CTD) can undergo phosphorylation at multiple sites giving rise to a form of the enzyme designated RNAP IIO. The unphosphorylated form is designated RNAP IIA. The largest subunits of RNAPs IIO and IIA are designated IIo and IIa, respectively. In quiescent NIH 3T3 fibroblasts, subunits IIo and IIa are present in comparable amounts. Upon serum stimulation, the amount of subunit IIo increases markedly and remains elevated for several hours. The increase of subunit IIo also occurs in transcription-inhibited cells and, therefore, is not a consequence of serum-activated transcription. This observation suggests that serum stimulation activates a CTD kinase and/or inhibits a CTD phosphatase. This hypothesis is supported by the finding that serum stimulates phosphorylation of a beta-galactosidase-CTD fusion protein expressed in these cells. Furthermore, an enhanced CTD kinase activity was discovered in lysates from serum-stimulated fibroblasts and was found to copurify with MAP kinases on a Mono Q column and to bind to anti-MAP kinase antibodies. The idea that MAP kinases phosphorylate the CTD in vivo is supported by the observation that subunit IIa, but not subunit IIb which lacks the CTD, is phosphorylated at multiple sites by purified MAP kinase. Consequently, the MAP kinases are a new class of CTD kinases which appear to be involved in the phosphorylation of RNAP II following serum stimulation. This phosphorylation may contribute to the transcriptional activation of serum-stimulated genes.

Full text

PDF
4787

Images in this article

4788Image
on p.4788
4789Image
on p.4789
4789Image
on p.4789
4790Image
on p.4790
4791Image
on p.4791
4792Image
on p.4792
4793Image
on p.4793
4793Image
on p.4793

Selected References

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

  1. Allison L. A., Ingles C. J. Mutations in RNA polymerase II enhance or suppress mutations in GAL4. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2794–2798. doi: 10.1073/pnas.86.8.2794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allison L. A., Wong J. K., Fitzpatrick V. D., Moyle M., Ingles C. J. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol Cell Biol. 1988 Jan;8(1):321–329. doi: 10.1128/mcb.8.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baskaran R., Dahmus M. E., Wang J. Y. Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11167–11171. doi: 10.1073/pnas.90.23.11167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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. doi: 10.1073/pnas.90.13.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cadena D. L., Dahmus M. E. Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II. J Biol Chem. 1987 Sep 15;262(26):12468–12474. [PubMed] [Google Scholar]
  6. Chesnut J. D., Stephens J. H., Dahmus M. E. The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa. J Biol Chem. 1992 May 25;267(15):10500–10506. [PubMed] [Google Scholar]
  7. Choder M., Young R. A. A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival. Mol Cell Biol. 1993 Nov;13(11):6984–6991. doi: 10.1128/mcb.13.11.6984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chodosh L. A., Fire A., Samuels M., Sharp P. A. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. J Biol Chem. 1989 Feb 5;264(4):2250–2257. [PubMed] [Google Scholar]
  9. Chou S. Y., Baichwal V., Ferrell J. E., Jr Inhibition of c-Jun DNA binding by mitogen-activated protein kinase. Mol Biol Cell. 1992 Oct;3(10):1117–1130. doi: 10.1091/mbc.3.10.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clark-Lewis I., Sanghera J. S., Pelech S. L. Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. J Biol Chem. 1991 Aug 15;266(23):15180–15184. [PubMed] [Google Scholar]
  11. Corden J. L. RNA polymerase II transcription cycles. Curr Opin Genet Dev. 1993 Apr;3(2):213–218. doi: 10.1016/0959-437x(93)90025-k. [DOI] [PubMed] [Google Scholar]
  12. Corden J. L. Tails of RNA polymerase II. Trends Biochem Sci. 1990 Oct;15(10):383–387. doi: 10.1016/0968-0004(90)90236-5. [DOI] [PubMed] [Google Scholar]
  13. Crews C. M., Erikson R. L. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell. 1993 Jul 30;74(2):215–217. doi: 10.1016/0092-8674(93)90411-i. [DOI] [PubMed] [Google Scholar]
  14. Dahmus M. E. Phosphorylation of eukaryotic DNA-dependent RNA polymerase. Identification of calf thymus RNA polymerase subunits phosphorylated by two purified protein kinases, correlation with in vivo sites of phosphorylation in HeLa cell RNA polymerase II. J Biol Chem. 1981 Apr 10;256(7):3332–3339. [PubMed] [Google Scholar]
  15. Dahmus M. E. The role of multisite phosphorylation in the regulation of RNA polymerase II activity. Prog Nucleic Acid Res Mol Biol. 1994;48:143–179. doi: 10.1016/s0079-6603(08)60855-7. [DOI] [PubMed] [Google Scholar]
  16. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  17. Dubois M. F., Bellier S., Seo S. J., Bensaude O. Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in HeLa cells. J Cell Physiol. 1994 Mar;158(3):417–426. doi: 10.1002/jcp.1041580305. [DOI] [PubMed] [Google Scholar]
  18. Dubois M. F., Bensaude O. MAP kinase activation during heat shock in quiescent and exponentially growing mammalian cells. FEBS Lett. 1993 Jun 14;324(2):191–195. doi: 10.1016/0014-5793(93)81391-c. [DOI] [PubMed] [Google Scholar]
  19. Dubois M. F., Nguyen V. T., Bellier S., Bensaude O. Inhibitors of transcription such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8 and H-7) promote dephosphorylation of the carboxyl-terminal domain of RNA polymerase II largest subunit. J Biol Chem. 1994 May 6;269(18):13331–13336. [PubMed] [Google Scholar]
  20. 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]
  21. Feaver W. J., Gileadi O., Li Y., Kornberg R. D. CTD kinase associated with yeast RNA polymerase II initiation factor b. Cell. 1991 Dec 20;67(6):1223–1230. doi: 10.1016/0092-8674(91)90298-d. [DOI] [PubMed] [Google Scholar]
  22. Fraser N. W., Sehgal P. B., Darnell J. E. DRB-induced premature termination of late adenovirus transcription. Nature. 1978 Apr 13;272(5654):590–593. doi: 10.1038/272590a0. [DOI] [PubMed] [Google Scholar]
  23. Giardina C., Lis J. T. Polymerase processivity and termination on Drosophila heat shock genes. J Biol Chem. 1993 Nov 15;268(32):23806–23811. [PubMed] [Google Scholar]
  24. 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]
  25. Greenleaf A. L. A positive addition to a negative tail's tale. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10896–10897. doi: 10.1073/pnas.90.23.10896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gupta S., Seth A., Davis R. J. Transactivation of gene expression by Myc is inhibited by mutation at the phosphorylation sites Thr-58 and Ser-62. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3216–3220. doi: 10.1073/pnas.90.8.3216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hall C. V., Jacob P. E., Ringold G. M., Lee F. Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet. 1983;2(1):101–109. [PubMed] [Google Scholar]
  28. 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]
  29. Kamada S., Toyoshima K., Akiyama T. Serum-independent phosphorylation of c-Jun and alterations in AP-1 components by transformation with various oncogenes. J Biol Chem. 1994 Feb 11;269(6):4565–4570. [PubMed] [Google Scholar]
  30. Kang M. E., Dahmus M. E. RNA polymerases IIA and IIO have distinct roles during transcription from the TATA-less murine dihydrofolate reductase promoter. J Biol Chem. 1993 Nov 25;268(33):25033–25040. [PubMed] [Google Scholar]
  31. Kelly W. G., Dahmus M. E., Hart G. W. RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc. J Biol Chem. 1993 May 15;268(14):10416–10424. [PubMed] [Google Scholar]
  32. Kim W. Y., Dahmus M. E. Purification of RNA polymerase IIO from calf thymus. J Biol Chem. 1988 Dec 15;263(35):18880–18885. [PubMed] [Google Scholar]
  33. Krumm A., Meulia T., Groudine M. Common mechanisms for the control of eukaryotic transcriptional elongation. Bioessays. 1993 Oct;15(10):659–665. doi: 10.1002/bies.950151005. [DOI] [PubMed] [Google Scholar]
  34. Krämer A., Haars R., Kabisch R., Will H., Bautz F. A., Bautz E. K. Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster. Mol Gen Genet. 1980;180(1):193–199. doi: 10.1007/BF00267369. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Lee J. M., Greenleaf A. L. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II. Proc Natl Acad Sci U S A. 1989 May;86(10):3624–3628. doi: 10.1073/pnas.86.10.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lee J. M., Greenleaf A. L. CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae. Gene Expr. 1991 May;1(2):149–167. [PMC free article] [PubMed] [Google Scholar]
  38. Legagneux V., Morange M., Bensaude O. Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. Eur J Biochem. 1990 Oct 5;193(1):121–126. doi: 10.1111/j.1432-1033.1990.tb19312.x. [DOI] [PubMed] [Google Scholar]
  39. Lu H., Flores O., Weinmann R., Reinberg D. The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10004–10008. doi: 10.1073/pnas.88.22.10004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lu H., Zawel L., Fisher L., Egly J. M., Reinberg D. Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II. Nature. 1992 Aug 20;358(6388):641–645. doi: 10.1038/358641a0. [DOI] [PubMed] [Google Scholar]
  41. Maller J. MAP kinase activation. Curr Biol. 1991 Oct;1(5):334–335. doi: 10.1016/0960-9822(91)90104-5. [DOI] [PubMed] [Google Scholar]
  42. Meloche S., Pagès G., Pouysségur J. Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk. Mol Biol Cell. 1992 Jan;3(1):63–71. doi: 10.1091/mbc.3.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Meulia T., Krumm A., Groudine M. Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis. Mol Cell Biol. 1993 Sep;13(9):5647–5658. doi: 10.1128/mcb.13.9.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Neiman A. M. Conservation and reiteration of a kinase cascade. Trends Genet. 1993 Nov;9(11):390–394. doi: 10.1016/0168-9525(93)90139-9. [DOI] [PubMed] [Google Scholar]
  45. Nishida E., Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. doi: 10.1016/0968-0004(93)90019-j. [DOI] [PubMed] [Google Scholar]
  46. Nolan G. P., Fiering S., Nicolas J. F., Herzenberg L. A. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2603–2607. doi: 10.1073/pnas.85.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. O'Brien T., Hardin S., Greenleaf A., Lis J. T. Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature. 1994 Jul 7;370(6484):75–77. doi: 10.1038/370075a0. [DOI] [PubMed] [Google Scholar]
  48. O'Brien T., Lis J. T. RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene. Mol Cell Biol. 1991 Oct;11(10):5285–5290. doi: 10.1128/mcb.11.10.5285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Peterson M. G., Tjian R. Transcription. The tell-tail trigger. Nature. 1992 Aug 20;358(6388):620–621. doi: 10.1038/358620a0. [DOI] [PubMed] [Google Scholar]
  51. Rougvie A. E., Lis J. T. Postinitiation transcriptional control in Drosophila melanogaster. Mol Cell Biol. 1990 Nov;10(11):6041–6045. doi: 10.1128/mcb.10.11.6041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Ruderman J. V. MAP kinase and the activation of quiescent cells. Curr Opin Cell Biol. 1993 Apr;5(2):207–213. doi: 10.1016/0955-0674(93)90104-x. [DOI] [PubMed] [Google Scholar]
  53. Sanes J. R., Rubenstein J. L., Nicolas J. F. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. doi: 10.1002/j.1460-2075.1986.tb04620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sawadogo M., Sentenac A. RNA polymerase B (II) and general transcription factors. Annu Rev Biochem. 1990;59:711–754. doi: 10.1146/annurev.bi.59.070190.003431. [DOI] [PubMed] [Google Scholar]
  55. Scafe C., Chao D., Lopes J., Hirsch J. P., Henry S., Young R. A. RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals. Nature. 1990 Oct 4;347(6292):491–494. doi: 10.1038/347491a0. [DOI] [PubMed] [Google Scholar]
  56. Seipel K., Georgiev O., Gerber H. P., Schaffner W. C-terminal domain (CTD) of RNA-polymerase II and N-terminal segment of the human TATA binding protein (TBP) can mediate remote and proximal transcriptional activation, respectively. Nucleic Acids Res. 1993 Dec 11;21(24):5609–5615. doi: 10.1093/nar/21.24.5609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Serizawa H., Conaway R. C., Conaway J. W. A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7476–7480. doi: 10.1073/pnas.89.16.7476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tamm I., Hand R., Caliguiri L. A. Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells. J Cell Biol. 1976 May;69(2):229–240. doi: 10.1083/jcb.69.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Thompson N. E., Steinberg T. H., Aronson D. B., Burgess R. R. Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J Biol Chem. 1989 Jul 5;264(19):11511–11520. [PubMed] [Google Scholar]
  60. 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]
  61. Ullmann A. One-step purification of hybrid proteins which have beta-galactosidase activity. Gene. 1984 Jul-Aug;29(1-2):27–31. doi: 10.1016/0378-1119(84)90162-8. [DOI] [PubMed] [Google Scholar]
  62. Wang Y., Simonson M. S., Pouysségur J., Dunn M. J. Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells. Biochem J. 1992 Oct 15;287(Pt 2):589–594. doi: 10.1042/bj2870589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weeks J. R., Hardin S. E., Shen J., Lee J. M., Greenleaf A. L. Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev. 1993 Dec;7(12A):2329–2344. doi: 10.1101/gad.7.12a.2329. [DOI] [PubMed] [Google Scholar]
  64. Wick M., Bürger C., Brüsselbach S., Lucibello F. C., Müller R. Identification of serum-inducible genes: different patterns of gene regulation during G0-->S and G1-->S progression. J Cell Sci. 1994 Jan;107(Pt 1):227–239. doi: 10.1242/jcs.107.1.227. [DOI] [PubMed] [Google Scholar]
  65. Woodgett J. R. A common denominator linking glycogen metabolism, nuclear oncogenes and development. Trends Biochem Sci. 1991 May;16(5):177–181. doi: 10.1016/0968-0004(91)90071-3. [DOI] [PubMed] [Google Scholar]
  66. Young R. A. RNA polymerase II. Annu Rev Biochem. 1991;60:689–715. doi: 10.1146/annurev.bi.60.070191.003353. [DOI] [PubMed] [Google Scholar]
  67. Zandomeni R., Zandomeni M. C., Shugar D., Weinmann R. Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription. J Biol Chem. 1986 Mar 5;261(7):3414–3419. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES