In Vitro Autophosphorylation of Protein Kinase C Isozymes

  • Antonio M. Pepio
  • Wayne S. Sossin
Part of the Methods in Molecular Biology™ book series (MIMB, volume 233)

Abstract

Protein kinase C (PKC) autophosphorylates at multiple sites. Two of these sites that are important for PKC activity are usually quantitatively phosphorylated in vivo (1, 2, 3, 4, 5, 6, 7, 8). Therefore, these sites are poorly detected in vitro autophosphorylation studies unless phosphatases are used first to remove endogenous phosphorylation. PKCs also autophosphorylate at other sites that are not quantitatively phosphorylated in cells and are not required for PKC activity (7, 8, 9, 10, 11, 12, 13, 14). These sites may nevertheless be important for modulating the activity of PKC (10,12,13). These sites can be isoform specific and may also be important in determining differences in isoform activation.

Keywords

Cellulose Rubber Electrophoresis Pyridine Lysine 

References

  1. 1.
    Bornancin, F. and Parker, P. J. (1996) Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase c-alpha. Curr. Biol. 6, 1114–1123.PubMedCrossRefGoogle Scholar
  2. 2.
    Bornancin, F. and Parker, P. J. (1997) Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J. Biol. Chem. 272, 3544–3549.PubMedCrossRefGoogle Scholar
  3. 3.
    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
  4. 4.
    Dutil, E. M., Keranen, L. M., DePaoli, R. A., and Newton, A. C. (1994) In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation. J. Biol. Chem. 269, 29,359–29,362.PubMedGoogle Scholar
  5. 5.
    Edwards, A. S. and Newton, A. C. (1997) Phosphorylation at conserved carboxylterminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C. J. Biol. Chem. 272, 18,382–18,390.PubMedCrossRefGoogle Scholar
  6. 6.
    Edwards, A. S., Faux, M. C., Scott, J. D., and Newton, A. C. (1999) Carboxylterminal phosphorylation regulates the function and subcellular localization of protein kinase C beta II. J. Biol. Chem. 274, 6461–6468.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, 1394–1403.PubMedCrossRefGoogle Scholar
  8. 8.
    Tsutakawa, S. E., Medzihradszky, K. F., Flint, A. J., Burlingame, A. L., and Koshland, D. E., Jr. (1995) Determination of in vivo phosphorylation sites in protein kinase C. J. Biol. Chem. 270, 26,807–26,812.PubMedCrossRefGoogle Scholar
  9. 9.
    Flint, A. J., Paladini, R. D., and Koshland, D. E., Jr. (1990) Autophosphorylation of protein kinase C at three separated regions of its primary sequence. Science 249, 408–411.PubMedCrossRefGoogle Scholar
  10. 10.
    Orr, J. W., Keranen, L. M., and Newton, A. C. (1992) Reversible exposure of the pseudosubstrate domain of protein kinase C by phosphatidylserine and diacylglycerol. J. Biol. Chem. 267, 15,263–15,266.PubMedGoogle Scholar
  11. 11.
    Ng, T., Squire, A., Hansra, G., Bornancin, F., Prevostel, C., Hanby, A., et al. (1999) Imaging protein kinase Calpha activation in cells. Science 283, 2085–2089.PubMedCrossRefGoogle Scholar
  12. 12.
    Nakhost, A., Dyer, J. R., Pepio, A. M., Fan, X., and Sossin, W. S. (1999) Protein kinase C phosphorylated at a conserved threonine is retained in the cytoplasm. J. Biol. Chem. 274, 28,944–28,949.PubMedCrossRefGoogle Scholar
  13. 13.
    Pepio, A. M. and Sossin, W. S. (2001) Membrane translocation of nPKCs is regulated by phosphorylation of the C2 domain. J. Biol. Chem. 276, 3846–3855.PubMedCrossRefGoogle Scholar
  14. 14.
    Sweatt, J. D., Atkins, C. M., Johnson, J., English, J. D., Roberson, E. D., Chen, S. J., et al. (1998) Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation. J. Neurochem. 71, 1075–1085.PubMedCrossRefGoogle Scholar
  15. 15.
    Byrne, J. H. and Kandel, E. R. (1996) Presynaptic facilitation revisited: state and time dependence. J. Neurosci. 16, 425–435.PubMedGoogle Scholar
  16. 16.
    Byrne, J. H., Zwartjes, R., Homayouni, R., Critz, S. D., and Eskin, A. (1993) Roles of second messenger pathways in neuronal plasticity and in learning and memory. Insights gained from Aplysia. Adv. Second Messenger Phosphoprotein Res. 27, 47–108.PubMedGoogle Scholar
  17. 17.
    Kruger, K. E., Sossin, W. S., Sacktor, T. C., Bergold, P. J., Beushausen, S., and Schwartz, J. H. (1991) Cloning and characterization of Ca(2+)-dependent and Ca(2+)-independent PKCs expressed in Aplysia sensory cells. J. Neurosci. 11, 2303–2313.PubMedGoogle Scholar
  18. 18.
    Sossin, W. S., Diaz, A. R., and Schwartz, J. H. (1993) Characterization of two isoforms of protein kinase C in the nervous system of Aplysia californica. J. Biol. Chem. 268, 5763–5768.PubMedGoogle Scholar
  19. 19.
    Sossin, W. S. and Schwartz, J. H. (1992) Selective activation of Ca(2+)-activated PKCs in Aplysia neurons by 5-HT. J. Neurosci. 12, 1160–1168.PubMedGoogle Scholar
  20. 20.
    Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine. J. Biol. Chem. 260, 10,039–10,043.PubMedGoogle Scholar
  21. 21.
    Newton, A. C. and Koshland, D. E., Jr. (1990) Phosphatidylserine affects specificity of protein kinase C substrate phosphorylation and autophosphorylation. Biochemistry 29, 6656–6661.PubMedCrossRefGoogle Scholar
  22. 22.
    Newton, A. C. and Koshland, D. E., Jr. (1987) Protein kinase C autophosphorylates by an intrapeptide reaction. J. Biol. Chem. 262, 10,185–10,188.PubMedGoogle Scholar
  23. 23.
    Boyle, W. J., van, der, Geer, P., and Hunter, T. (1991) Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201, 110–149.PubMedCrossRefGoogle Scholar
  24. 24.
    Nagahara, H., Latek, R. R., Ezhevsky, S. A., and Dowdy, S. F. (1999) 2-D phosphopeptide mapping. Methods Mol. Biol. 112, 271–279.PubMedGoogle Scholar
  25. 25.
    Resing, K. A. and Ahn, N. G. (1997) Protein phosphorylation analysis by electrospray ionization-mass spectrometry. Methods Enzymol. 283, 29–44.PubMedCrossRefGoogle Scholar
  26. 26.
    Zhou, H., Watts, J. D., and Aebersold, R. (2001) A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378.PubMedCrossRefGoogle Scholar
  27. 27.
    Shi, S. D., Hemling, M. E., Carr, S. A., Horn, D. M., Lindh, I., & McLafferty, F. W. (2001) Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry. Anal. Chem. 73, 19–22.PubMedCrossRefGoogle Scholar
  28. 28.
    Annan, R. S. and Carr, S. A. (1996) Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry. Anal. Chem. 68, 3413–3421.PubMedCrossRefGoogle Scholar
  29. 29.
    Carr, S. A., Huddleston, M. J., & Annan, R. S. (1996) Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry. Anal. Biochem. 239, 180–192.PubMedCrossRefGoogle Scholar
  30. 30.
    Quadroni, M. and James, P. (2000) Phosphopeptide analysis. Exs 88, 199–213.PubMedGoogle Scholar
  31. 31.
    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
  32. 32.
    Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., & Hannun, Y. A. (1998) Visualization of dynamic trafficking of a protein kinase C betaII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization. J. Biol. Chem. 273, 10,755–10,762.PubMedCrossRefGoogle Scholar
  33. 33.
    Feng, X., Becker, K. P., Stribling, S. D., Peters, K. G., & Hannun, Y. A. (2000). Regulation of receptor-mediated protein kinase C membrane trafficking by autophosphorylation. J. Biol. Chem. 275, 17,024–17,034.PubMedCrossRefGoogle Scholar
  34. 34.
    Oancea, E. and Meyer, T. (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307–318.PubMedCrossRefGoogle Scholar
  35. 35.
    Pepio, A. M., Fan, X., and Sossin, W. S. (1998) The role of C2 domains in Ca2+-activated and Ca2+-independent protein kinase Cs in Aplysia J. Biol. Chem. 273, 19,040–19,048.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Antonio M. Pepio
    • 1
  • Wayne S. Sossin
    • 2
  1. 1.Elan PharmaceuticalsSan Diego
  2. 2.Department of Neurology and NeurosurgeryMcGill UniversityMontrealCanada

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