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. 1999 Sep 1;342(Pt 2):337–344.

Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes.

G Hansra 1, P Garcia-Paramio 1, C Prevostel 1, R D Whelan 1, F Bornancin 1, P J Parker 1
PMCID: PMC1220470  PMID: 10455020

Abstract

The generation of antisera specific for the priming phosphorylation sites on protein kinase Calpha (PKCalpha) has permitted analysis of the dephosphorylation of these sites in relation to the down-regulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKCalpha is able to stimulate the dephosphorylation of wild-type PKCalpha in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKCalpha on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKCalpha is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKCbeta1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.

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Selected References

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  1. Adams J. A., McGlone M. L., Gibson R., Taylor S. S. Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase. Biochemistry. 1995 Feb 28;34(8):2447–2454. doi: 10.1021/bi00008a007. [DOI] [PubMed] [Google Scholar]
  2. Alessi D. R. The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase-1beta (Rsk-2) and p70 S6 kinase. FEBS Lett. 1997 Feb 3;402(2-3):121–123. doi: 10.1016/s0014-5793(96)01510-4. [DOI] [PubMed] [Google Scholar]
  3. Bornancin F., Parker P. J. 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. 1997 Feb 7;272(6):3544–3549. doi: 10.1074/jbc.272.6.3544. [DOI] [PubMed] [Google Scholar]
  4. Bornancin F., Parker P. J. Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase Calpha. Curr Biol. 1996 Sep 1;6(9):1114–1123. doi: 10.1016/s0960-9822(02)70678-7. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Cazaubon S. M., Parker P. J. Identification of the phosphorylated region responsible for the permissive activation of protein kinase C. J Biol Chem. 1993 Aug 15;268(23):17559–17563. [PubMed] [Google Scholar]
  7. Cazaubon S., Bornancin F., Parker P. J. Threonine-497 is a critical site for permissive activation of protein kinase C alpha. Biochem J. 1994 Jul 15;301(Pt 2):443–448. doi: 10.1042/bj3010443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cazaubon S., Marais R., Parker P., Strosberg A. D. Monoclonal antibodies to protein kinase C gamma. Functional relationship between epitopes and cofactor binding sites. Eur J Biochem. 1989 Jun 15;182(2):401–406. doi: 10.1111/j.1432-1033.1989.tb14845.x. [DOI] [PubMed] [Google Scholar]
  9. Cazaubon S., Webster C., Camoin L., Strosberg A. D., Parker P. Effector-dependent conformational changes in protein kinase C gamma through epitope mapping with inhibitory monoclonal antibodies. Eur J Biochem. 1990 Dec 27;194(3):799–804. doi: 10.1111/j.1432-1033.1990.tb19472.x. [DOI] [PubMed] [Google Scholar]
  10. Chou M. M., Hou W., Johnson J., Graham L. K., Lee M. H., Chen C. S., Newton A. C., Schaffhausen B. S., Toker A. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr Biol. 1998 Sep 24;8(19):1069–1077. doi: 10.1016/s0960-9822(98)70444-0. [DOI] [PubMed] [Google Scholar]
  11. Coghlan V. M., Hausken Z. E., Scott J. D. Subcellular targeting of kinases and phosphatases by association with bifunctional anchoring proteins. Biochem Soc Trans. 1995 Aug;23(3):592–596. doi: 10.1042/bst0230592. [DOI] [PubMed] [Google Scholar]
  12. Coussens L., Parker P. J., Rhee L., Yang-Feng T. L., Chen E., Waterfield M. D., Francke U., Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science. 1986 Aug 22;233(4766):859–866. doi: 10.1126/science.3755548. [DOI] [PubMed] [Google Scholar]
  13. Dekker L. V., Parker P. J. Protein kinase C--a question of specificity. Trends Biochem Sci. 1994 Feb;19(2):73–77. doi: 10.1016/0968-0004(94)90038-8. [DOI] [PubMed] [Google Scholar]
  14. Edwards A. S., Newton A. C. Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C. J Biol Chem. 1997 Jul 18;272(29):18382–18390. doi: 10.1074/jbc.272.29.18382. [DOI] [PubMed] [Google Scholar]
  15. Exton J. H. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990 Jan 5;265(1):1–4. [PubMed] [Google Scholar]
  16. Freisewinkel I., Riethmacher D., Stabel S. Downregulation of protein kinase C-gamma is independent of a functional kinase domain. FEBS Lett. 1991 Mar 25;280(2):262–266. doi: 10.1016/0014-5793(91)80307-o. [DOI] [PubMed] [Google Scholar]
  17. Garcia-Paramio P., Cabrerizo Y., Bornancin F., Parker P. J. The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. Biochem J. 1998 Aug 1;333(Pt 3):631–636. doi: 10.1042/bj3330631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gasson J. C., Ryden T., Bourgeois S. Role of de novo DNA methylation in the glucocorticoid resistance of a T-lymphoid cell line. Nature. 1983 Apr 14;302(5909):621–623. doi: 10.1038/302621a0. [DOI] [PubMed] [Google Scholar]
  19. Goode N. T., Hajibagheri M. A., Parker P. J. Protein kinase C (PKC)-induced PKC down-regulation. Association with up-regulation of vesicle traffic. J Biol Chem. 1995 Feb 10;270(6):2669–2673. doi: 10.1074/jbc.270.6.2669. [DOI] [PubMed] [Google Scholar]
  20. Hanks S. K., Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576–596. [PubMed] [Google Scholar]
  21. Hansra G., Bornancin F., Whelan R., Hemmings B. A., Parker P. J. 12-O-Tetradecanoylphorbol-13-acetate-induced dephosphorylation of protein kinase Calpha correlates with the presence of a membrane-associated protein phosphatase 2A heterotrimer. J Biol Chem. 1996 Dec 20;271(51):32785–32788. doi: 10.1074/jbc.271.51.32785. [DOI] [PubMed] [Google Scholar]
  22. House C., Kemp B. E. Protein kinase C pseudosubstrate prototope: structure-function relationships. Cell Signal. 1990;2(2):187–190. doi: 10.1016/0898-6568(90)90022-3. [DOI] [PubMed] [Google Scholar]
  23. Hug H., Sarre T. F. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 1993 Apr 15;291(Pt 2):329–343. doi: 10.1042/bj2910329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hyatt S. L., Liao L., Chapline C., Jaken S. Identification and characterization of alpha-protein kinase C binding proteins in normal and transformed REF52 cells. Biochemistry. 1994 Feb 8;33(5):1223–1228. doi: 10.1021/bi00171a023. [DOI] [PubMed] [Google Scholar]
  25. Kajikawa N., Kikkawa U., Itoh K., Nishizuka Y. Membrane-permeable diacylglycerol, its application to platelet secretion, and regulation of platelet protein kinase C. Methods Enzymol. 1989;169:430–442. doi: 10.1016/0076-6879(89)69079-9. [DOI] [PubMed] [Google Scholar]
  26. Keranen L. M., Dutil E. M., Newton A. C. Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr Biol. 1995 Dec 1;5(12):1394–1403. doi: 10.1016/s0960-9822(95)00277-6. [DOI] [PubMed] [Google Scholar]
  27. Kiley S. C., Adams P. D., Parker P. J. Cloning and characterization of phorbol ester differentiation-resistant U937 cell variants. Cell Growth Differ. 1997 Feb;8(2):221–230. [PubMed] [Google Scholar]
  28. Kiley S. C., Parker P. J., Fabbro D., Jaken S. Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells. J Biol Chem. 1991 Dec 15;266(35):23761–23768. [PubMed] [Google Scholar]
  29. Krueger K. M., Daaka Y., Pitcher J. A., Lefkowitz R. J. The role of sequestration in G protein-coupled receptor resensitization. Regulation of beta2-adrenergic receptor dephosphorylation by vesicular acidification. J Biol Chem. 1997 Jan 3;272(1):5–8. doi: 10.1074/jbc.272.1.5. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Le Good J. A., Ziegler W. H., Parekh D. B., Alessi D. R., Cohen P., Parker P. J. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science. 1998 Sep 25;281(5385):2042–2045. doi: 10.1126/science.281.5385.2042. [DOI] [PubMed] [Google Scholar]
  32. Lee H. W., Smith L., Pettit G. R., Bingham Smith J. Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin. Am J Physiol. 1996 Jul;271(1 Pt 1):C304–C311. doi: 10.1152/ajpcell.1996.271.1.C304. [DOI] [PubMed] [Google Scholar]
  33. Lee H. W., Smith L., Pettit G. R., Smith J. B. Bryostatin 1 and phorbol ester down-modulate protein kinase C-alpha and -epsilon via the ubiquitin/proteasome pathway in human fibroblasts. Mol Pharmacol. 1997 Mar;51(3):439–447. [PubMed] [Google Scholar]
  34. Lee H. W., Smith L., Pettit G. R., Vinitsky A., Smith J. B. Ubiquitination of protein kinase C-alpha and degradation by the proteasome. J Biol Chem. 1996 Aug 30;271(35):20973–20976. [PubMed] [Google Scholar]
  35. Marais R. M., Parker P. J. Purification and characterisation of bovine brain protein kinase C isotypes alpha, beta and gamma. Eur J Biochem. 1989 Jun 1;182(1):129–137. doi: 10.1111/j.1432-1033.1989.tb14809.x. [DOI] [PubMed] [Google Scholar]
  36. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science. 1995 Apr 14;268(5208):247–251. doi: 10.1126/science.7716516. [DOI] [PubMed] [Google Scholar]
  37. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995 Apr;9(7):484–496. [PubMed] [Google Scholar]
  38. Noh D. Y., Shin S. H., Rhee S. G. Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim Biophys Acta. 1995 Dec 18;1242(2):99–113. doi: 10.1016/0304-419x(95)00006-0. [DOI] [PubMed] [Google Scholar]
  39. Ohno S., Konno Y., Akita Y., Yano A., Suzuki K. 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. 1990 Apr 15;265(11):6296–6300. [PubMed] [Google Scholar]
  40. Olivier A. R., Parker P. J. Expression and characterization of protein kinase C-delta. Eur J Biochem. 1991 Sep 15;200(3):805–810. doi: 10.1111/j.1432-1033.1991.tb16248.x. [DOI] [PubMed] [Google Scholar]
  41. Orr J. W., Newton A. C. Requirement for negative charge on "activation loop" of protein kinase C. J Biol Chem. 1994 Nov 4;269(44):27715–27718. [PubMed] [Google Scholar]
  42. Pears C. J., Kour G., House C., Kemp B. E., Parker P. J. Mutagenesis of the pseudosubstrate site of protein kinase C leads to activation. Eur J Biochem. 1990 Nov 26;194(1):89–94. doi: 10.1111/j.1432-1033.1990.tb19431.x. [DOI] [PubMed] [Google Scholar]
  43. Pears C., Parker P. J. Down-regulation of a kinase defective PKC-alpha. FEBS Lett. 1991 Jun 17;284(1):120–122. doi: 10.1016/0014-5793(91)80776-y. [DOI] [PubMed] [Google Scholar]
  44. Stabel S., Schaap D., Parker P. J. Expression of protein kinase C isotypes using baculovirus vectors. Methods Enzymol. 1991;200:670–673. doi: 10.1016/0076-6879(91)00179-z. [DOI] [PubMed] [Google Scholar]
  45. Tsutakawa S. E., Medzihradszky K. F., Flint A. J., Burlingame A. L., Koshland D. E., Jr Determination of in vivo phosphorylation sites in protein kinase C. J Biol Chem. 1995 Nov 10;270(45):26807–26812. doi: 10.1074/jbc.270.45.26807. [DOI] [PubMed] [Google Scholar]
  46. Whelan R. D., Parker P. J. Loss of protein kinase C function induces an apoptotic response. Oncogene. 1998 Apr 16;16(15):1939–1944. doi: 10.1038/sj.onc.1201725. [DOI] [PubMed] [Google Scholar]
  47. Wilkinson S. E., Parker P. J., Nixon J. S. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993 Sep 1;294(Pt 2):335–337. doi: 10.1042/bj2940335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yedovitzky M., Mochly-Rosen D., Johnson J. A., Gray M. O., Ron D., Abramovitch E., Cerasi E., Nesher R. Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells. J Biol Chem. 1997 Jan 17;272(3):1417–1420. doi: 10.1074/jbc.272.3.1417. [DOI] [PubMed] [Google Scholar]

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