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Direct control of the Forkhead transcription factor AFX by protein kinase B

Nature volume 398, pages 630–634 (1999)Cite this article

Abstract

The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B (refs 1, 2) regulates certain insulin-responsive genes3,4, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf -16 is regulated by a pathway consisting of insulin-receptor-like daf- 2 and PI(3)K-like age -1 (5–8). Here we show that protein kinase B phosphorylates AFX, a human orthologue of daf -16 (refs 5, 6, 9), both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus.

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Figure 1: Insulin treatment and active forms of PI(3)K and PKB increase phosphorylation of HA–AFX in vivo.
Figure 2: PKB phosphorylates HA–AFX in vitro and in vivo.
Figure 3: PKB cooperates with Ras in the phosphorylation of HA–AFX by insulin.
Figure 4: Phosphorylation of HA–AFX by PKB regulates transcriptional activity.

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References

  1. Burgering, B. M. Th. & Coffer, P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599–602 (1995).

    Article  ADS  CAS  Google Scholar 

  2. Franke, T. F. et al. The protein kinase encoded by the akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81, 727–736 (1995).

    Article  CAS  Google Scholar 

  3. Cichy, S. B. et al. Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence. J. Biol. Chem. 273, 6482–6487 (1998).

    Article  CAS  Google Scholar 

  4. Wang, D. & Sul, H. S. Insulin stimulation of the fatty acid synthase promoter is mediated by the phosphatidylinositol 3-kinase pathway. Involvement of protein kinase B/Akt. J. Biol. Chem. 273, 25420–25426 (1998).

    Article  CAS  Google Scholar 

  5. Ogg, S. et al. The forkhead transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999 (1997).

    Article  ADS  CAS  Google Scholar 

  6. Lin, K., Dorman, J. B., Rodan, A. & Kenyon, C. daf- 16: and HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322 (1997).

    Article  ADS  CAS  Google Scholar 

  7. Kimura, K. D., Tissenbaum, H. A., Liu, Y. & Ruvkun, G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabiditis elegans. Science 277, 942–946 (1997).

    Article  CAS  Google Scholar 

  8. Morris, J. Z., Tissenbaum, H. A. & Ruvkun, G. A. Phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536–539 (1996).

    Article  ADS  CAS  Google Scholar 

  9. Borkhardt, A. et al. Cloning and characterization of AFX, the gene that fuses to MLL in acute leukaemias with a t(X;11)(q13;q23). Oncogene 14, 195–202 (1997).

    Article  CAS  Google Scholar 

  10. Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A. & Cohen, P. Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett. 399, 333–338 (1996).

    Article  CAS  Google Scholar 

  11. Van Weeren, P. C., de Bruyn, K. M., de Vries-Smits, A. M., van Lint, J. & Burgering, B. M. Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. J. Biol. Chem. 273, 13150–13156 (1998).

    Article  CAS  Google Scholar 

  12. Cheatham, B. et al. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell Biol. 14, 4902–4911 (1994).

    Article  CAS  Google Scholar 

  13. Pronk, G. J. & Bos, J. L. The role of p21ras in receptor tyrosine kinase signalling. Biochim. Biophys. Acta 1198, 131–147 (1994).

    PubMed  Google Scholar 

  14. Medema, R. H., Wubbolts, R. & Bos, J. L. Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression. Mol. Cell Biol. 11, 5963–5967 (1991).

    Article  CAS  Google Scholar 

  15. Kauffmann-Zeh, A. et al. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385, 544–548 (1997).

    Article  ADS  CAS  Google Scholar 

  16. Urano, T., Emkey, R. & Feig, L. A. Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. EMBO J. 15, 810–816 (1996).

    Article  CAS  Google Scholar 

  17. Burgering, B. M. & Bos, J. L. Regulation of Ras-mediated signalling: more than one way to skin a cat. Trends Biochem. Sci. 20, 18–22 (1995).

    Article  CAS  Google Scholar 

  18. Wolthuis, R. M. F., de Ruiter, N. D., Cool, R. H. & Bos, J. L. Stimulation of gene induction and cell growth by the ras effector Rlf. EMBO J. 16, 6748–6761 (1997).

    Article  CAS  Google Scholar 

  19. Leevers, S. J., Paterson, H. F. & Marshall, C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369, 411–414 (1994).

    Article  ADS  CAS  Google Scholar 

  20. Wolthuis, R. M. F. et al. RalGDS-like factor (Rlf) is a novel Ras and Rap1A-associating protein. Oncogene 13, 353–362 (1996).

    CAS  PubMed  Google Scholar 

  21. Unterman, T. G. et al. Hepatocyte nuclear factor-3 (HNF-3) binds to the insulin response sequence in the IGF binding protein-1 (IGFBP-1) promoter and enhances promoter function. Biochem. Biophys. Res. Commun. 203, 1835–1841 (1994).

    Article  CAS  Google Scholar 

  22. O'Brien, R. M. et al. Hepatic nuclear factor-3 and hormone regulated expression of the PEPCK and IGFBP-1 genes. Mol. Cell Biol. 15, 1747–1758 (1995).

    Article  CAS  Google Scholar 

  23. Duyndam, M. C., van Dam, H., van der Eb, A. J. & Zantema, A. The CR1 and CR3 domains of the adenovirus type 5 E1A proteins can independently mediate activation of ATF-2. J. Virol. 70, 5852–5859 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Andjelkovic, M. et al. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272, 31515–31524 (1997).

    Article  CAS  Google Scholar 

  25. Paradis, S. & Ruvkun, G. Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 12, 2488–2498 (1998).

    Article  CAS  Google Scholar 

  26. Peters, U., Haberhausen, G., Kostrzewa, M., Nolte, D. & Müller, U. AFX1 and p54nrb: fine mapping, genomic structure, and exclusion as candidate genes of X-linked dystonia parkinsonism. Hum. Genet. 100, 569–572 (1997).

    Article  CAS  Google Scholar 

  27. Didichenko, S. A., Tilton, B., Hemmings, B. A., Ballmer-Hofer, K. & Thelen, M. Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase. Curr. Biol. 6, 1271–1278 (1996).

    Article  CAS  Google Scholar 

  28. Boyle, W. J., Van der Geer, P. & Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201, 110–149 (1991).

    Article  CAS  Google Scholar 

  29. Luo, K. X., Hurley, T. R. & Sefton, B. M. Cyanogen bromide cleavage and proteolytic peptide mapping of proteins immobilized to membranes. Methods Enzymol. 201, 149–152 (1991).

    Article  CAS  Google Scholar 

  30. De Groot, R. P. & Kruijer, W. Transcriptional activation by TGF beta 1 is mediated by the dyad symmetry element (DSE) and the TPA responsive element (TRE). Biochem. Biophys. Res. Commun. 168, 1074–1081 (1990).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. Reedquist, F. Zwartkruis and P. van der Vliet for critically reading the manuscript and D. Nolte for the pUC-AFX1#3 plasmid. This work was supported by grants from NWO, KWF and CBG, and by the NIH (D.R.P.).

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Authors and Affiliations

  1. Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, University of Utrecht, Universiteitsweg 100, Utrecht, 3584, CG, The Netherlands

    Geert J. P. L. Kops, Nancy D. de Ruiter, Alida M. M. De Vries-Smits, Johannes L. Bos & Boudewijn M. Th. Burgering

  2. Department of Pediatrics, Baylor College of Medicine, Houston, 77030, Texas, USA

    David R. Powell

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  1. Geert J. P. L. Kops
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Correspondence to Boudewijn M. Th. Burgering.

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Kops, G., Ruiter, N., De Vries-Smits, A. et al. Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398, 630–634 (1999). https://doi.org/10.1038/19328

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