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. 1987 Mar;84(6):1502–1506. doi: 10.1073/pnas.84.6.1502

Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro.

L B Ray, T W Sturgill
PMCID: PMC304462  PMID: 2951732

Abstract

Insulin treatment (Kact, 5 X 10(-9) M) of serum-starved 3T3-L1 adipocytes stimulates a soluble serine/threonine kinase that catalyzes phosphorylation of microtubule-associated protein 2 (MAP-2) in vitro. Maximal activation of MAP-2 kinase activity by 80 nM insulin was observed after 10 min of hormonal stimulation, prior to maximal stimulation of S6 kinase activity (20 min). The insulin-stimulatable MAP-2 kinase activity is not adsorbed to phosphocellulose, whereas the principal S6 kinase activity is retained and elutes at approximately 0.5 M NaCl. The insulin-stimulatable MAP-2 kinase is less stable during incubation at 30 degrees C than S6 kinase activity. Inclusion of phosphatase inhibitors decreases the rate at which the stimulated MAP-2 kinase activity is lost from extract supernatants incubated at 30 degrees C. p-Nitrophenyl phosphate is more effective than DL-phosphotyrosine, whereas DL-phosphoserine is without effect at the concentration used (40 mM). The difference in MAP-2 kinase activity in extract supernatants from control and insulin-treated cells is also preserved after rapid chromatography on Sephadex G-25. These results show that a soluble serine/threonine kinase is rapidly activated by insulin, possibly by phosphorylation of either the kinase itself or an interacting modulator.

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Selected References

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

  1. Beemon K., Hunter T. Characterization of Rous sarcoma virus src gene products synthesized in vitro. J Virol. 1978 Nov;28(2):551–566. doi: 10.1128/jvi.28.2.551-566.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  3. Christopherson R. I., Jones M. E., Finch L. R. A simple centrifuge column for desalting protein solutions. Anal Biochem. 1979 Nov 15;100(1):184–187. doi: 10.1016/0003-2697(79)90130-1. [DOI] [PubMed] [Google Scholar]
  4. Cobb M. H., Rosen O. M. Description of a protein kinase derived from insulin-treated 3T3-L1 cells that catalyzes the phosphorylation of ribosomal protein S6 and casein. J Biol Chem. 1983 Oct 25;258(20):12472–12481. [PubMed] [Google Scholar]
  5. 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]
  6. Cohen S. Purification of the receptor for epidermal growth factor from A-431 cells: its function as a tyrosyl kinase. Methods Enzymol. 1983;99:379–387. doi: 10.1016/0076-6879(83)99074-2. [DOI] [PubMed] [Google Scholar]
  7. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  8. Denton R. M., Brownsey R. W., Hopkirk T. J., Belsham G. J. Protein kinases and insulin action in fat cells. Biochem Soc Trans. 1984 Oct;12(5):768–771. doi: 10.1042/bst0120768. [DOI] [PubMed] [Google Scholar]
  9. Johnson R. A., Walseth T. F. The enzymatic preparation of [alpha-32P]ATP, [alpha-32P]GTP, [32P]cAMP, and [32P]cGMP, and their use in the assay of adenylate and guanylate cyclases and cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1979;10:135–167. [PubMed] [Google Scholar]
  10. Kadowaki T., Fujita-Yamaguchi Y., Nishida E., Takaku F., Akiyama T., Kathuria S., Akanuma Y., Kasuga M. Phosphorylation of tubulin and microtubule-associated proteins by the purified insulin receptor kinase. J Biol Chem. 1985 Apr 10;260(7):4016–4020. [PubMed] [Google Scholar]
  11. Kim H., Binder L. I., Rosenbaum J. L. The periodic association of MAP2 with brain microtubules in vitro. J Cell Biol. 1979 Feb;80(2):266–276. doi: 10.1083/jcb.80.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Novak-Hofer I., Thomas G. Epidermal growth factor-mediated activation of an S6 kinase in Swiss mouse 3T3 cells. J Biol Chem. 1985 Aug 25;260(18):10314–10319. [PubMed] [Google Scholar]
  14. Petruzzelli L. M., Ganguly S., Smith C. J., Cobb M. H., Rubin C. S., Rosen O. M. Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6792–6796. doi: 10.1073/pnas.79.22.6792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rubin C. S., Hirsch A., Fung C., Rosen O. M. Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem. 1978 Oct 25;253(20):7570–7578. [PubMed] [Google Scholar]
  16. Sparks J. W., Brautigan D. L. Molecular basis for substrate specificity of protein kinases and phosphatases. Int J Biochem. 1986;18(6):497–504. doi: 10.1016/0020-711x(86)90159-x. [DOI] [PubMed] [Google Scholar]
  17. Stefanovic D., Erikson E., Pike L. J., Maller J. L. Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase. EMBO J. 1986 Jan;5(1):157–160. doi: 10.1002/j.1460-2075.1986.tb04190.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sturgill T. W., Ray L. B. Muscle proteins related to microtubule associated protein-2 are substrates for an insulin-stimulatable kinase. Biochem Biophys Res Commun. 1986 Jan 29;134(2):565–571. doi: 10.1016/s0006-291x(86)80457-0. [DOI] [PubMed] [Google Scholar]
  19. Swarup G., Cohen S., Garbers D. L. Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J Biol Chem. 1981 Aug 10;256(15):8197–8201. [PubMed] [Google Scholar]
  20. Tabarini D., Heinrich J., Rosen O. M. Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4369–4373. doi: 10.1073/pnas.82.13.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zasloff M., Ochoa S. Purification of eukaryotic initiation factor 1 (EIF1) from Artemia salina embryos. Methods Enzymol. 1974;30:197–206. doi: 10.1016/0076-6879(74)30022-5. [DOI] [PubMed] [Google Scholar]

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