Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 May 1;323(Pt 3):621–627. doi: 10.1042/bj3230621

Insulin regulation of mitogen-activated protein kinase kinase (MEK), mitogen-activated protein kinase and casein kinase in the cell nucleus: a possible role in the regulation of gene expression.

S J Kim 1, C R Kahn 1
PMCID: PMC1218363  PMID: 9169593

Abstract

After insulin receptor activation, many cytoplasmic enzymes, including mitogen-activated protein (MAP) kinase, MAP kinase kinase (MEK) and casein kinase II (CKII) are activated, but exactly how insulin signalling progresses to the nucleus remains poorly understood. In Chinese hamster ovary cells overexpressing human insulin receptors [CHO(Hirc)], MEK, CKII and the MAP kinases ERK I and ERK II can be detected by immunoblotting in the nucleus, as well as in the cytoplasm, in the unstimulated state. Nuclear localization of MAP kinase is also observed in 3T3-F442A adipocytes, NIH-3T3 cells and Fao hepatoma cells, whereas MEK is found in the nucleus only in Fao and CHO cells. Insulin treatment for 5-30 min induces a translocation of MEK from the cytoplasm to the nucleus, whereas the MAP kinases and CKII are not translocated into the nucleus in response to insulin during this period. However, nuclear MAP kinase and CKII activities increase by 2-3-fold within 1-10 min after stimulation with insulin. By using gel-shift assays, it has been shown that insulin also stimulates nuclear protein binding to an AP-1 site with kinetics similar to MEK translocation and MAP kinase and CKII activation. Treatment of the extracts in vitro with protein phosphatase 2A or treatment of the intact cells with 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole, a cell-permeable inhibitor of CKII, almost completely blocks the insulin-induced DNA-binding activity, whereas incubation of cells with a MEK inhibitor produces only a slight decrease. These results suggest that insulin signalling results in the activation of serine kinases in the nucleus via two pathways: (1) insulin stimulates the nuclear translocation of some kinases, such as MEK, which might directly phosphorylate nuclear protein substrates or activate other nuclear kinases, and (2) insulin activates nuclear kinases without translocation. The latter is true of CKII, which seems to regulate the binding of nuclear proteins to the AP-1 site, possibly by phosphorylation of AP-1 transcription factors.

Full Text

The Full Text of this article is available as a PDF (370.9 KB).

Selected References

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

  1. Abdel-Hafiz H. A., Heasley L. E., Kyriakis J. M., Avruch J., Kroll D. J., Johnson G. L., Hoeffler J. P. Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases. Mol Endocrinol. 1992 Dec;6(12):2079–2089. doi: 10.1210/mend.6.12.1337144. [DOI] [PubMed] [Google Scholar]
  2. Ahmed K. Significance of the casein kinase system in cell growth and proliferation with emphasis on studies of the androgenic regulation of the prostate. Cell Mol Biol Res. 1994;40(1):1–11. [PubMed] [Google Scholar]
  3. Alvarez E., Northwood I. C., Gonzalez F. A., Latour D. A., Seth A., Abate C., Curran T., Davis R. J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J Biol Chem. 1991 Aug 15;266(23):15277–15285. [PubMed] [Google Scholar]
  4. Baker S. J., Kerppola T. K., Luk D., Vandenberg M. T., Marshak D. R., Curran T., Abate C. Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts. Mol Cell Biol. 1992 Oct;12(10):4694–4705. doi: 10.1128/mcb.12.10.4694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernstein L. R., Ferris D. K., Colburn N. H., Sobel M. E. A family of mitogen-activated protein kinase-related proteins interacts in vivo with activator protein-1 transcription factor. J Biol Chem. 1994 Apr 1;269(13):9401–9404. [PubMed] [Google Scholar]
  6. Blobel G., Potter V. R. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 1966 Dec 30;154(3757):1662–1665. doi: 10.1126/science.154.3757.1662. [DOI] [PubMed] [Google Scholar]
  7. Chen R. H., Sarnecki C., Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol. 1992 Mar;12(3):915–927. doi: 10.1128/mcb.12.3.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng J. T., Cobb M. H., Baer R. Phosphorylation of the TAL1 oncoprotein by the extracellular-signal-regulated protein kinase ERK1. Mol Cell Biol. 1993 Feb;13(2):801–808. doi: 10.1128/mcb.13.2.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Csermely P., Kahn C. R. Insulin induces the phosphorylation of DNA-binding nuclear proteins including lamins in 3T3-F442A. Biochemistry. 1992 Oct 20;31(41):9940–9946. doi: 10.1021/bi00156a012. [DOI] [PubMed] [Google Scholar]
  10. Csermely P., Schnaider T., Cheatham B., Olson M. O., Kahn C. R. Insulin induces the phosphorylation of nucleolin. A possible mechanism of insulin-induced RNA efflux from nuclei. J Biol Chem. 1993 May 5;268(13):9747–9752. [PubMed] [Google Scholar]
  11. 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]
  12. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  13. Erikson E., Maller J. L. In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocyte maturation. J Biol Chem. 1989 Aug 15;264(23):13711–13717. [PubMed] [Google Scholar]
  14. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  15. Getzenberg R. H., Pienta K. J., Coffey D. S. The tissue matrix: cell dynamics and hormone action. Endocr Rev. 1990 Aug;11(3):399–417. doi: 10.1210/edrv-11-3-399. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Haystead T. A., Weiel J. E., Litchfield D. W., Tsukitani Y., Fischer E. H., Krebs E. G. Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal. J Biol Chem. 1990 Sep 25;265(27):16571–16580. [PubMed] [Google Scholar]
  19. Hopcroft D. W., Mason D. R., Scott R. S. Standardization of insulin secretion from pancreatic islets: validation of a DNA assay. Horm Metab Res. 1985 Nov;17(11):559–561. doi: 10.1055/s-2007-1013606. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Huang W., Erikson R. L. Constitutive activation of Mek1 by mutation of serine phosphorylation sites. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8960–8963. doi: 10.1073/pnas.91.19.8960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kameshita I., Fujisawa H. A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem. 1989 Nov 15;183(1):139–143. doi: 10.1016/0003-2697(89)90181-4. [DOI] [PubMed] [Google Scholar]
  24. Kim S. J., Kahn C. R. Insulin induces rapid accumulation of insulin receptors and increases tyrosine kinase activity in the nucleus of cultured adipocytes. J Cell Physiol. 1993 Nov;157(2):217–228. doi: 10.1002/jcp.1041570203. [DOI] [PubMed] [Google Scholar]
  25. Kim S. J., Kahn C. R. Insulin stimulates phosphorylation of c-Jun, c-Fos, and Fos-related proteins in cultured adipocytes. J Biol Chem. 1994 Apr 22;269(16):11887–11892. [PubMed] [Google Scholar]
  26. Kuo C. J., Chung J., Fiorentino D. F., Flanagan W. M., Blenis J., Crabtree G. R. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature. 1992 Jul 2;358(6381):70–73. doi: 10.1038/358070a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Lee K. A., Bindereif A., Green M. R. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech. 1988 Mar-Apr;5(2):22–31. doi: 10.1016/0735-0651(88)90023-4. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lin A., Frost J., Deng T., Smeal T., al-Alawi N., Kikkawa U., Hunter T., Brenner D., Karin M. Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell. 1992 Sep 4;70(5):777–789. doi: 10.1016/0092-8674(92)90311-y. [DOI] [PubMed] [Google Scholar]
  32. Mansour S. J., Matten W. T., Hermann A. S., Candia J. M., Rong S., Fukasawa K., Vande Woude G. F., Ahn N. G. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994 Aug 12;265(5174):966–970. doi: 10.1126/science.8052857. [DOI] [PubMed] [Google Scholar]
  33. Miyata Y., Yahara I. The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity. J Biol Chem. 1992 Apr 5;267(10):7042–7047. [PubMed] [Google Scholar]
  34. Nakajima T., Kinoshita S., Sasagawa T., Sasaki K., Naruto M., Kishimoto T., Akira S. Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2207–2211. doi: 10.1073/pnas.90.6.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. O'Brien R. M., Lucas P. C., Forest C. D., Magnuson M. A., Granner D. K. Identification of a sequence in the PEPCK gene that mediates a negative effect of insulin on transcription. Science. 1990 Aug 3;249(4968):533–537. doi: 10.1126/science.2166335. [DOI] [PubMed] [Google Scholar]
  36. Pinna L. A. Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys Acta. 1990 Sep 24;1054(3):267–284. doi: 10.1016/0167-4889(90)90098-x. [DOI] [PubMed] [Google Scholar]
  37. Podlecki D. A., Smith R. M., Kao M., Tsai P., Huecksteadt T., Brandenburg D., Lasher R. S., Jarett L., Olefsky J. M. Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects. J Biol Chem. 1987 Mar 5;262(7):3362–3368. [PubMed] [Google Scholar]
  38. Posada J., Yew N., Ahn N. G., Vande Woude G. F., Cooper J. A. Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol Cell Biol. 1993 Apr;13(4):2546–2553. doi: 10.1128/mcb.13.4.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  40. Ray L. B., Sturgill T. W. Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells. J Biol Chem. 1988 Sep 5;263(25):12721–12727. [PubMed] [Google Scholar]
  41. Reusch J. E., Hsieh P., Klemm D., Hoeffler J., Draznin B. Insulin inhibits dephosphorylation of adenosine 3',5'-monophosphate response element-binding protein/activating transcription factor-1: effect on nuclear phosphoserine phosphatase-2a. Endocrinology. 1994 Dec;135(6):2418–2422. doi: 10.1210/endo.135.6.7988426. [DOI] [PubMed] [Google Scholar]
  42. Sanghera J. S., Peter M., Nigg E. A., Pelech S. L. Immunological characterization of avian MAP kinases: evidence for nuclear localization. Mol Biol Cell. 1992 Jul;3(7):775–787. doi: 10.1091/mbc.3.7.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Sommercorn J., Mulligan J. A., Lozeman F. J., Krebs E. G. Activation of casein kinase II in response to insulin and to epidermal growth factor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8834–8838. doi: 10.1073/pnas.84.24.8834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sun H., Tonks N. K. The coordinated action of protein tyrosine phosphatases and kinases in cell signaling. Trends Biochem Sci. 1994 Nov;19(11):480–485. doi: 10.1016/0968-0004(94)90134-1. [DOI] [PubMed] [Google Scholar]
  47. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zheng C. F., Guan K. L. Cytoplasmic localization of the mitogen-activated protein kinase activator MEK. J Biol Chem. 1994 Aug 5;269(31):19947–19952. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES