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PDK-1 and Protein Kinase C Phosphorylation

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 233)

Abstract

The discovery of the phosphoinositide-dependent kinase-1 (PDK-1) as the upstream kinase for protein kinase C (PKC) represented an important step in the understanding of the regulation of this crucial lipid-signaling enzyme. Three laboratories simultaneously described PDK-1 as the activation loop upstream kinase for conventional (PKCα and PKCβII; ref. 1), novel (PKCδ and PKCε ref. 2), and atypical (PKCζ refs. 2 and 3) isozymes. It is now well established that PDK-1 is the upstream kinase for all PKC family members, and numerous studies have addressed the detailed biochemical mechanisms by which PDK-1 phosphorylates PKC thereby regulating its function in cells (for reviews, see refs. 4, 5, 6). PDK-1 phosphorylates PKCs at a critical Thr residue in the so-called activation loop sequence of the highly conserved catalytic kinase domain (see  Chapter 13), and this event is required for PKC to gain catalytic competency. Phosphorylation of the activation loop Thr correctly aligns residues within the active site and this permits transfer of the gamma phosphate of ATP to an exogenous substrate. This phosphorylation triggers two phosphorylations at the carboxyl-terminus required to stabilize the catalytically competent species of PKC. Therefore, phosphorylation is a rate-limiting step in the regulation of PKC and precedes other regulatory events, including binding of lipid activators (7).

Keywords

Invitrogen Life Technology Activation Loop Mammalian Expression Vector Spinner Flask Phosphospecific Antibody 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References

  1. 1.
    Dutil, E. M., Toker, A., and Newton, A. C. (1998) Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1). Curr. Biol. 8, 1366–1375.PubMedCrossRefGoogle Scholar
  2. 2.
    Le Good, J. A., Ziegler, W. H., Parekh, D. B., Alessi, D. R., Cohen, P., and Parker, P. J. (1998) Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281, 2042–2045.PubMedCrossRefGoogle Scholar
  3. 3.
    Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., et al. (1998) Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr. Biol. 8, 1069–1077.PubMedCrossRefGoogle Scholar
  4. 4.
    Toker, A. and Newton, A. C. (2000) Cellular signaling: pivoting around PDK-1. Cell 103(2), 185–188.PubMedCrossRefGoogle Scholar
  5. 5.
    Storz, P. and Toker, A. (2002) 3′-phosphoinositide-dependent kinase-1 (PDK-1) in PI 3-kinase signaling. Front. Biosci. 7, d886–d902.PubMedCrossRefGoogle Scholar
  6. 6.
    Parekh, D. B., Ziegler, W., and Parker, P. J. (2000) Multiple pathways control protein kinase C phosphorylation. EMBO J. 19(4), 496–503.PubMedCrossRefGoogle Scholar
  7. 7.
    Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr. Biol. 5(12), 1394–1403.PubMedCrossRefGoogle Scholar
  8. 8.
    Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., et al. (1997) Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr. Biol. 7, 261–269.PubMedCrossRefGoogle Scholar
  9. 9.
    Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese, C. B., Painter, G. F., et al. (1997) Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277, 567–570.PubMedCrossRefGoogle Scholar
  10. 10.
    Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., et al. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15, 6541–6551.PubMedGoogle Scholar
  11. 11.
    Moriya, S., Kazlauskas, A., Akimoto, K., Hirai, S., Mizuno, K., Takenawa, T., et al. (1996) Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA 93(1), 151–155.PubMedCrossRefGoogle Scholar
  12. 12.
    Parekh, D., Ziegler, W., Yonezawa, K., Hara, K., and Parker, P. J. (1999) Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon. J. Biol. Chem. 274, 34,758–34,764.PubMedCrossRefGoogle Scholar
  13. 13.
    Cenni, V., Doppler, H., Sonnenburg, E. D., Maraldi, N., Newton, A. C., and Toker, A. (2002) Regulation of novel protein kinase C epsilon by phosphorylation. Biochem. J. 363, 537–545.PubMedCrossRefGoogle Scholar
  14. 14.
    Sonnenburg, E. D., Gao, T., and Newton, A. C. (2001) The phosphoinositide-dependent kinase, PDK-1, phosphorylates conventional protein kinase C isozymes by a mechanism that is independent of phosphoinositide 3-kinase. J. Biol. Chem. 276, 45,289–45,297.PubMedCrossRefGoogle Scholar
  15. 15.
    Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., et al. (1997). 3-phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr. Biol. 7, 776–789.PubMedCrossRefGoogle Scholar
  16. 16.
    Anderson, K. E., Coadwell, J., Stephens, L. R., and Hawkins, P. T. (1998) Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B. Curr. Biol. 8, 684–691.PubMedCrossRefGoogle Scholar
  17. 17.
    Behn-Krappa, A. and Newton, A. C. (1999) The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation. Curr. Biol. 9, 728–737.PubMedCrossRefGoogle Scholar
  18. 18.
    Ohno, S., Konno, Y., Akita, Y., Yano, A., and Suzuki, K. (1990) A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation. J. Biol. Chem. 265, 6296–6300.PubMedGoogle Scholar
  19. 19.
    Zhang, J., Wang, L., Petrin, J., Bishop, W. R., and Bond, R. W. (1993) Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites. Proc. Natl. Acad. Sci. USA 90, 6130–6134.PubMedCrossRefGoogle Scholar
  20. 20.
    Feng, X. and Hannun, Y. A. (1998) An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane. J. Biol. Chem. 273, 26,870–26,874.PubMedCrossRefGoogle Scholar
  21. 21.
    Hata, A., Akita, Y., Suzuki, K., and Ohno, S. (1993) Functional divergence of protein kinase C (PKC) family members. PKC gamma differs from PKC alpha and-beta II and nPKC epsilon in its competence to mediate-12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive transcriptional activation through a TPA-response element. J. Biol. Chem. 268, 9122–9129.PubMedGoogle Scholar
  22. 22.
    Li, W., Yu, J. C., Shin, D. Y., and Pierce, J. H. (1995) Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J. Biol. Chem. 270, 8311–8318.PubMedCrossRefGoogle Scholar
  23. 23.
    Ueda, E., Ohno, S., Kuroki, T., Livneh, E., Yamada, K., Yamanishi, K., et al. (1996) The eta isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene. J. Biol. Chem. 271, 9790–9794.PubMedCrossRefGoogle Scholar
  24. 24.
    Chang, J. D., Xu, Y., Raychowdhury, M. K., and Ware, J. A. (1993) Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC). A new member of the nPKC family expressed in skeletal muscle, megakaryoblastic cells, and platelets. J. Biol. Chem. 268, 14,208–14,214.PubMedGoogle Scholar
  25. 25.
    Murray, N. R. and Fields, A. P. (1997) Atypical protein kinase C iota protects human leukemia cells against drug-induced apoptosis. J. Biol. Chem. 272, 27,521–27,524.PubMedCrossRefGoogle Scholar
  26. 26.
    Kotani, K., Ogawa, W., Matsumoto, M., Kitamura, T., Sakaue, H., Hino, Y., et al. (1998) Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes. Mol. Cell. Biol. 18, 6971–6982.PubMedGoogle Scholar
  27. 27.
    Newton, A. C. (1997) Regulation of protein kinase C. Curr. Opin. Cell. Biol. 9(2), 161–167.PubMedCrossRefGoogle Scholar
  28. 28.
    Park, J., Hill, M. M., Hess, D., Brazil, D. P., Hofsteenge, J., and Hemmings, B. A. (2001) Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity. J. Biol. Chem. 276(40), 37,459–37,471.PubMedCrossRefGoogle Scholar
  29. 29.
    Toker, A. (1998) Signaling through protein kinase C. Front Biosci 3, D1134–D1147.PubMedGoogle Scholar
  30. 30.
    Edwards, A. S., Faux, M. C., Scott, J. D., and Newton, A. C. (1999) Carboxyl-terminal phosphorylation regulates the function and subcellular localization of protein kinase C betaII. J. Biol. Chem. 274, 6461–6468.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  1. 1.Department of Pathology, Beth Israel Deaconess Medical SchoolHarvard Medical SchoolBoston

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