Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Nov 1;375(Pt 3):663–671. doi: 10.1042/BJ20030926

Caspase processing activates atypical protein kinase C zeta by relieving autoinhibition and destabilizes the protein.

Lucinda Smith 1, Zhi Wang 1, Jeffrey B Smith 1
PMCID: PMC1223714  PMID: 12887331

Abstract

Treatment of HeLa cells with tumour necrosis factor alpha (TNFalpha) induced caspase processing of ectopic PKC (protein kinase C) zeta, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC zeta. Atypical PKC iota is largely identical with PKC zeta, except for a 60-amino-acid segment that lacks the caspase-processing sites of the zeta isoform. Replacement of this segment of PKC zeta with the corresponding segment of PKC iota prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC zeta by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC zeta in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC zeta, which is required for catalytic competency. The freed kinase domain of PKC zeta had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-alpha shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin-proteasome system.

Full Text

The Full Text of this article is available as a PDF (271.0 KB).

Selected References

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

  1. Balendran A., Biondi R. M., Cheung P. C., Casamayor A., Deak M., Alessi D. R. A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1. J Biol Chem. 2000 Jul 7;275(27):20806–20813. doi: 10.1074/jbc.M000421200. [DOI] [PubMed] [Google Scholar]
  2. Biondi R. M., Cheung P. C., Casamayor A., Deak M., Currie R. A., Alessi D. R. Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA. EMBO J. 2000 Mar 1;19(5):979–988. doi: 10.1093/emboj/19.5.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourbon N. A., Yun J., Kester M. Ceramide directly activates protein kinase C zeta to regulate a stress-activated protein kinase signaling complex. J Biol Chem. 2000 Nov 10;275(45):35617–35623. doi: 10.1074/jbc.M007346200. [DOI] [PubMed] [Google Scholar]
  4. Carlin S., Yang K. X., Donnelly R., Black J. L. Protein kinase C isoforms in human airway smooth muscle cells: activation of PKC-zeta during proliferation. Am J Physiol. 1999 Mar;276(3 Pt 1):L506–L512. doi: 10.1152/ajplung.1999.276.3.L506. [DOI] [PubMed] [Google Scholar]
  5. Cepko C., Ryder E. F., Austin C. P., Walsh C., Fekete D. M. Lineage analysis using retrovirus vectors. Methods Enzymol. 1995;254:387–419. doi: 10.1016/0076-6879(95)54027-x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Earnshaw W. C., Martins L. M., Kaufmann S. H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem. 1999;68:383–424. doi: 10.1146/annurev.biochem.68.1.383. [DOI] [PubMed] [Google Scholar]
  8. Frutos S., Moscat J., Diaz-Meco M. T. Cleavage of zetaPKC but not lambda/iotaPKC by caspase-3 during UV-induced apoptosis. J Biol Chem. 1999 Apr 16;274(16):10765–10770. doi: 10.1074/jbc.274.16.10765. [DOI] [PubMed] [Google Scholar]
  9. Herrera-Velit P., Knutson K. L., Reiner N. E. Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-zeta in bacterial lipopolysaccharide-treated human monocytes. J Biol Chem. 1997 Jun 27;272(26):16445–16452. doi: 10.1074/jbc.272.26.16445. [DOI] [PubMed] [Google Scholar]
  10. Jordan M., Schallhorn A., Wurm F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 1996 Feb 15;24(4):596–601. doi: 10.1093/nar/24.4.596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Leitges M., Sanz L., Martin P., Duran A., Braun U., García J. F., Camacho F., Diaz-Meco M. T., Rennert P. D., Moscat J. Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Mol Cell. 2001 Oct;8(4):771–780. doi: 10.1016/s1097-2765(01)00361-6. [DOI] [PubMed] [Google Scholar]
  15. Mellor H., Parker P. J. The extended protein kinase C superfamily. Biochem J. 1998 Jun 1;332(Pt 2):281–292. doi: 10.1042/bj3320281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Murray N. R., Fields A. P. Atypical protein kinase C iota protects human leukemia cells against drug-induced apoptosis. J Biol Chem. 1997 Oct 31;272(44):27521–27524. doi: 10.1074/jbc.272.44.27521. [DOI] [PubMed] [Google Scholar]
  17. Mutter R., Wills M. Chemistry and clinical biology of the bryostatins. Bioorg Med Chem. 2000 Aug;8(8):1841–1860. doi: 10.1016/s0968-0896(00)00150-4. [DOI] [PubMed] [Google Scholar]
  18. Nakanishi H., Brewer K. A., Exton J. H. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
  19. Nakanishi H., Exton J. H. Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. J Biol Chem. 1992 Aug 15;267(23):16347–16354. [PubMed] [Google Scholar]
  20. Newton A. C., Keranen L. M. Phosphatidyl-L-serine is necessary for protein kinase C's high-affinity interaction with diacylglycerol-containing membranes. Biochemistry. 1994 May 31;33(21):6651–6658. doi: 10.1021/bi00187a035. [DOI] [PubMed] [Google Scholar]
  21. Newton A. C. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev. 2001 Aug;101(8):2353–2364. doi: 10.1021/cr0002801. [DOI] [PubMed] [Google Scholar]
  22. Newton Alexandra C. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J. 2003 Mar 1;370(Pt 2):361–371. doi: 10.1042/BJ20021626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995 Apr;9(7):484–496. [PubMed] [Google Scholar]
  24. Okuda H., Saitoh K., Hirai S., Iwai K., Takaki Y., Baba M., Minato N., Ohno S., Shuin T. The von Hippel-Lindau tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. J Biol Chem. 2001 Sep 26;276(47):43611–43617. doi: 10.1074/jbc.M107880200. [DOI] [PubMed] [Google Scholar]
  25. Omura S., Matsuzaki K., Fujimoto T., Kosuge K., Furuya T., Fujita S., Nakagawa A. Structure of lactacystin, a new microbial metabolite which induces differentiation of neuroblastoma cells. J Antibiot (Tokyo) 1991 Jan;44(1):117–118. doi: 10.7164/antibiotics.44.117. [DOI] [PubMed] [Google Scholar]
  27. Rechsteiner M., Rogers S. W. PEST sequences and regulation by proteolysis. Trends Biochem Sci. 1996 Jul;21(7):267–271. [PubMed] [Google Scholar]
  28. Smith L., Chen L., Reyland M. E., DeVries T. A., Talanian R. V., Omura S., Smith J. B. Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system. J Biol Chem. 2000 Dec 22;275(51):40620–40627. doi: 10.1074/jbc.M908517199. [DOI] [PubMed] [Google Scholar]
  29. Smith Lucinda, Smith Jeffrey B. Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop. J Biol Chem. 2002 Sep 19;277(48):45866–45873. doi: 10.1074/jbc.M206420200. [DOI] [PubMed] [Google Scholar]
  30. Sonnenburg E. D., Gao T., Newton A. C. 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. 2001 Sep 28;276(48):45289–45297. doi: 10.1074/jbc.M107416200. [DOI] [PubMed] [Google Scholar]
  31. Standaert M. L., Bandyopadhyay G., Perez L., Price D., Galloway L., Poklepovic A., Sajan M. P., Cenni V., Sirri A., Moscat J. Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes. J Biol Chem. 1999 Sep 3;274(36):25308–25316. doi: 10.1074/jbc.274.36.25308. [DOI] [PubMed] [Google Scholar]
  32. Talanian R. V., Quinlan C., Trautz S., Hackett M. C., Mankovich J. A., Banach D., Ghayur T., Brady K. D., Wong W. W. Substrate specificities of caspase family proteases. J Biol Chem. 1997 Apr 11;272(15):9677–9682. doi: 10.1074/jbc.272.15.9677. [DOI] [PubMed] [Google Scholar]
  33. Yang H., Kaelin W. G., Jr Molecular pathogenesis of the von Hippel-Lindau hereditary cancer syndrome: implications for oxygen sensing. Cell Growth Differ. 2001 Sep;12(9):447–455. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES