Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Sep 17;93(19):10228–10233. doi: 10.1073/pnas.93.19.10228

Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

H Eldar-Finkelman 1, G M Argast 1, O Foord 1, E H Fischer 1, E G Krebs 1
PMCID: PMC38366  PMID: 8816781

Abstract

In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism.

Full text

PDF
10228

Images in this article

10230Fig. 1
on p.10230
10230Fig. 2
on p.10230
10231Fig. 3
on p.10231
10231Fig. 4
on p.10231

Selected References

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

  1. Andersson S., Davis D. L., Dahlbäck H., Jörnvall H., Russell D. W. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed] [Google Scholar]
  2. Bellacosa A., Testa J. R., Staal S. P., Tsichlis P. N. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science. 1991 Oct 11;254(5029):274–277. doi: 10.1126/science.254.5029.274. [DOI] [PubMed] [Google Scholar]
  3. Benjamin W. B., Pentyala S. N., Woodgett J. R., Hod Y., Marshak D. ATP citrate-lyase and glycogen synthase kinase-3 beta in 3T3-L1 cells during differentiation into adipocytes. Biochem J. 1994 Jun 1;300(Pt 2):477–482. doi: 10.1042/bj3000477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyle W. J., Smeal T., Defize L. H., Angel P., Woodgett J. R., Karin M., Hunter T. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell. 1991 Feb 8;64(3):573–584. doi: 10.1016/0092-8674(91)90241-p. [DOI] [PubMed] [Google Scholar]
  5. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coffer P. J., Woodgett J. R. Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem. 1991 Oct 15;201(2):475–481. doi: 10.1111/j.1432-1033.1991.tb16305.x. [DOI] [PubMed] [Google Scholar]
  7. Cohen P. The role of protein phosphorylation in the hormonal control of enzyme activity. Eur J Biochem. 1985 Sep 16;151(3):439–448. doi: 10.1111/j.1432-1033.1985.tb09121.x. [DOI] [PubMed] [Google Scholar]
  8. Cross D. A., Alessi D. R., Cohen P., Andjelkovich M., Hemmings B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995 Dec 21;378(6559):785–789. doi: 10.1038/378785a0. [DOI] [PubMed] [Google Scholar]
  9. Cross D. A., Alessi D. R., Vandenheede J. R., McDowell H. E., Hundal H. S., Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J. 1994 Oct 1;303(Pt 1):21–26. doi: 10.1042/bj3030021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dominguez I., Itoh K., Sokol S. Y. Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8498–8502. doi: 10.1073/pnas.92.18.8498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eldar-Finkelman H., Seger R., Vandenheede J. R., Krebs E. G. Inactivation of glycogen synthase kinase-3 by epidermal growth factor is mediated by mitogen-activated protein kinase/p90 ribosomal protein S6 kinase signaling pathway in NIH/3T3 cells. J Biol Chem. 1995 Jan 20;270(3):987–990. doi: 10.1074/jbc.270.3.987. [DOI] [PubMed] [Google Scholar]
  12. Fiol C. J., Mahrenholz A. M., Wang Y., Roeske R. W., Roach P. J. Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J Biol Chem. 1987 Oct 15;262(29):14042–14048. [PubMed] [Google Scholar]
  13. Fiol C. J., Williams J. S., Chou C. H., Wang Q. M., Roach P. J., Andrisani O. M. A secondary phosphorylation of CREB341 at Ser129 is required for the cAMP-mediated control of gene expression. A role for glycogen synthase kinase-3 in the control of gene expression. J Biol Chem. 1994 Dec 23;269(51):32187–32193. [PubMed] [Google Scholar]
  14. Flotow H., Roach P. J. Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase. J Biol Chem. 1989 Jun 5;264(16):9126–9128. [PubMed] [Google Scholar]
  15. Franke T. F., Yang S. I., Chan T. O., Datta K., Kazlauskas A., Morrison D. K., Kaplan D. R., Tsichlis P. N. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995 Jun 2;81(5):727–736. doi: 10.1016/0092-8674(95)90534-0. [DOI] [PubMed] [Google Scholar]
  16. He X., Saint-Jeannet J. P., Woodgett J. R., Varmus H. E., Dawid I. B. Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature. 1995 Apr 13;374(6523):617–622. doi: 10.1038/374617a0. [DOI] [PubMed] [Google Scholar]
  17. Hemmings B. A., Aitken A., Cohen P., Rymond M., Hofmann F. Phosphorylation of the type-II regulatory subunit of cyclic-AMP-dependent protein kinase by glycogen synthase kinase 3 and glycogen synthase kinase 5. Eur J Biochem. 1982 Oct;127(3):473–481. doi: 10.1111/j.1432-1033.1982.tb06896.x. [DOI] [PubMed] [Google Scholar]
  18. Hemmings B. A., Yellowlees D., Kernohan J. C., Cohen P. Purification of glycogen synthase kinase 3 from rabbit skeletal muscle. Copurification with the activating factor (FA) of the (Mg-ATP) dependent protein phosphatase. Eur J Biochem. 1981 Oct;119(3):443–451. doi: 10.1111/j.1432-1033.1981.tb05628.x. [DOI] [PubMed] [Google Scholar]
  19. Hughes K., Nikolakaki E., Plyte S. E., Totty N. F., Woodgett J. R. Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation. EMBO J. 1993 Feb;12(2):803–808. doi: 10.1002/j.1460-2075.1993.tb05715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hughes K., Ramakrishna S., Benjamin W. B., Woodgett J. R. Identification of multifunctional ATP-citrate lyase kinase as the alpha-isoform of glycogen synthase kinase-3. Biochem J. 1992 Nov 15;288(Pt 1):309–314. doi: 10.1042/bj2880309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lawrence J. C., Jr, Zhang J. N. Control of glycogen synthase and phosphorylase by amylin in rat skeletal muscle. Hormonal effects on the phosphorylation of phosphorylase and on the distribution of phosphate in the synthase subunit. J Biol Chem. 1994 Apr 15;269(15):11595–11600. [PubMed] [Google Scholar]
  23. Mandelkow E. M., Drewes G., Biernat J., Gustke N., Van Lint J., Vandenheede J. R., Mandelkow E. Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. FEBS Lett. 1992 Dec 21;314(3):315–321. doi: 10.1016/0014-5793(92)81496-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Nakielny S., Campbell D. G., Cohen P. The molecular mechanism by which adrenalin inhibits glycogen synthesis. Eur J Biochem. 1991 Aug 1;199(3):713–722. doi: 10.1111/j.1432-1033.1991.tb16175.x. [DOI] [PubMed] [Google Scholar]
  26. Nikolakaki E., Coffer P. J., Hemelsoet R., Woodgett J. R., Defize L. H. Glycogen synthase kinase 3 phosphorylates Jun family members in vitro and negatively regulates their transactivating potential in intact cells. Oncogene. 1993 Apr;8(4):833–840. [PubMed] [Google Scholar]
  27. Parker P. J., Caudwell F. B., Cohen P. Glycogen synthase from rabbit skeletal muscle; effect of insulin on the state of phosphorylation of the seven phosphoserine residues in vivo. Eur J Biochem. 1983 Jan 17;130(1):227–234. doi: 10.1111/j.1432-1033.1983.tb07140.x. [DOI] [PubMed] [Google Scholar]
  28. Peifer M., Pai L. M., Casey M. Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. Dev Biol. 1994 Dec;166(2):543–556. doi: 10.1006/dbio.1994.1336. [DOI] [PubMed] [Google Scholar]
  29. Pierce S. B., Kimelman D. Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development. 1995 Mar;121(3):755–765. doi: 10.1242/dev.121.3.755. [DOI] [PubMed] [Google Scholar]
  30. Plyte S. E., Hughes K., Nikolakaki E., Pulverer B. J., Woodgett J. R. Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim Biophys Acta. 1992 Dec 16;1114(2-3):147–162. doi: 10.1016/0304-419x(92)90012-n. [DOI] [PubMed] [Google Scholar]
  31. Puziss J. W., Hardy T. A., Johnson R. B., Roach P. J., Hieter P. MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3. Mol Cell Biol. 1994 Jan;14(1):831–839. doi: 10.1128/mcb.14.1.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Robbins D. J., Zhen E., Owaki H., Vanderbilt C. A., Ebert D., Geppert T. D., Cobb M. H. Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J Biol Chem. 1993 Mar 5;268(7):5097–5106. [PubMed] [Google Scholar]
  33. Rylatt D. B., Aitken A., Bilham T., Condon G. D., Embi N., Cohen P. Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase. Eur J Biochem. 1980 Jun;107(2):529–537. [PubMed] [Google Scholar]
  34. Saito Y., Vandenheede J. R., Cohen P. The mechanism by which epidermal growth factor inhibits glycogen synthase kinase 3 in A431 cells. Biochem J. 1994 Oct 1;303(Pt 1):27–31. doi: 10.1042/bj3030027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Saksela K., Mäkelä T. P., Hughes K., Woodgett J. R., Alitalo K. Activation of protein kinase C increases phosphorylation of the L-myc trans-activator domain at a GSK-3 target site. Oncogene. 1992 Feb;7(2):347–353. [PubMed] [Google Scholar]
  36. Siegfried E., Chou T. B., Perrimon N. wingless signaling acts through zeste-white 3, the Drosophila homolog of glycogen synthase kinase-3, to regulate engrailed and establish cell fate. Cell. 1992 Dec 24;71(7):1167–1179. doi: 10.1016/s0092-8674(05)80065-0. [DOI] [PubMed] [Google Scholar]
  37. Skurat A. V., Wang Y., Roach P. J. Rabbit skeletal muscle glycogen synthase expressed in COS cells. Identification of regulatory phosphorylation sites. J Biol Chem. 1994 Oct 14;269(41):25534–25542. [PubMed] [Google Scholar]
  38. Stambolic V., Woodgett J. R. Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation. Biochem J. 1994 Nov 1;303(Pt 3):701–704. doi: 10.1042/bj3030701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sutherland C., Leighton I. A., Cohen P. Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem J. 1993 Nov 15;296(Pt 1):15–19. doi: 10.1042/bj2960015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Thomas J. A., Schlender K. K., Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem. 1968 Oct 24;25(1):486–499. doi: 10.1016/0003-2697(68)90127-9. [DOI] [PubMed] [Google Scholar]
  41. Vandenheede J. R., Yang S. D., Goris J., Merlevede W. ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. II. Purification of the activating factor and its characterization as a bifunctional protein also displaying synthase kinase activity. J Biol Chem. 1980 Dec 25;255(24):11768–11774. [PubMed] [Google Scholar]
  42. Wang Q. M., Fiol C. J., DePaoli-Roach A. A., Roach P. J. Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. J Biol Chem. 1994 May 20;269(20):14566–14574. [PubMed] [Google Scholar]
  43. Wang Q. M., Roach P. J., Fiol C. J. Use of a synthetic peptide as a selective substrate for glycogen synthase kinase 3. Anal Biochem. 1994 Aug 1;220(2):397–402. doi: 10.1006/abio.1994.1356. [DOI] [PubMed] [Google Scholar]
  44. Wang Y., Roach P. J. Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a). J Biol Chem. 1993 Nov 15;268(32):23876–23880. [PubMed] [Google Scholar]
  45. Welsh G. I., Foulstone E. J., Young S. W., Tavaré J. M., Proud C. G. Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase. Biochem J. 1994 Oct 1;303(Pt 1):15–20. doi: 10.1042/bj3030015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Welsh G. I., Proud C. G. Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B. Biochem J. 1993 Sep 15;294(Pt 3):625–629. doi: 10.1042/bj2940625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Woodgett J. R., Cohen P. Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5). Biochim Biophys Acta. 1984 Aug 14;788(3):339–347. doi: 10.1016/0167-4838(84)90047-5. [DOI] [PubMed] [Google Scholar]
  49. Woodgett J. R. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J. 1990 Aug;9(8):2431–2438. doi: 10.1002/j.1460-2075.1990.tb07419.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES