Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 May 15;372(Pt 1):137–143. doi: 10.1042/BJ20021901

Effect of caspase cleavage-site phosphorylation on proteolysis.

József Tözsér 1, Péter Bagossi 1, Gábor Zahuczky 1, Suzanne I Specht 1, Eva Majerova 1, Terry D Copeland 1
PMCID: PMC1223375  PMID: 12589706

Abstract

Caspases are important mediators of apoptotic cell death. Several cellular protein substrates of caspases contain potential phosphorylation site(s) at the cleavage-site region, and some of these sites have been verified to be phosphorylated. Since phosphorylation may affect substantially the substrate susceptibility towards proteolysis, phosphorylated, non-phosphorylated and substituted oligopeptides representing such cleavage sites were studied as substrates of apoptotic caspases 3, 7 and 8. Peptides containing phosphorylated serine residues at P4 and P1' positions were found to be substantially less susceptible towards proteolysis as compared with the serine-containing analogues, while phosphoserine at P3 did not have a substantial effect. P1 serine as well as P1-phosphorylated, serine-containing analogues of an oligopeptide representing the poly(ADP-ribose) polymerase cleavage site of caspase-3 were not hydrolysed by any of these enzymes, whereas the P1 aspartate-containing peptides were efficiently hydrolysed. These findings were interpreted with the aid of molecular modelling. Our results suggest that cleavage-site phosphorylation in certain positions could be disadvantageous or detrimental with respect to cleavability by caspases. Cleavage-site phosphorylation may therefore provide a regulatory mechanism to protect substrates from caspase-mediated degradation.

Full Text

The Full Text of this article is available as a PDF (151.2 KB).

Selected References

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

  1. Barkett M., Xue D., Horvitz H. R., Gilmore T. D. Phosphorylation of IkappaB-alpha inhibits its cleavage by caspase CPP32 in vitro. J Biol Chem. 1997 Nov 21;272(47):29419–29422. doi: 10.1074/jbc.272.47.29419. [DOI] [PubMed] [Google Scholar]
  2. Berti P. J., Faerman C. H., Storer A. C. Cooperativity of papain-substrate interaction energies in the S2 to S2' subsites. Biochemistry. 1991 Feb 5;30(5):1394–1402. doi: 10.1021/bi00219a033. [DOI] [PubMed] [Google Scholar]
  3. Blanchard H., Donepudi M., Tschopp M., Kodandapani L., Wu J. C., Grütter M. G. Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase-8-Z-DEVD-cho complex. J Mol Biol. 2000 Sep 8;302(1):9–16. doi: 10.1006/jmbi.2000.4041. [DOI] [PubMed] [Google Scholar]
  4. Bone R., Sampson N. S., Bartlett P. A., Agard D. A. Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters. Biochemistry. 1991 Feb 26;30(8):2263–2272. doi: 10.1021/bi00222a032. [DOI] [PubMed] [Google Scholar]
  5. Brancolini C., Benedetti M., Schneider C. Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J. 1995 Nov 1;14(21):5179–5190. doi: 10.1002/j.1460-2075.1995.tb00202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brancolini C., Schneider C. Phosphorylation of the growth arrest-specific protein Gas2 is coupled to actin rearrangements during Go-->G1 transition in NIH 3T3 cells. J Cell Biol. 1994 Mar;124(5):743–756. doi: 10.1083/jcb.124.5.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davies D. R. The structure and function of the aspartic proteinases. Annu Rev Biophys Biophys Chem. 1990;19:189–215. doi: 10.1146/annurev.bb.19.060190.001201. [DOI] [PubMed] [Google Scholar]
  8. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  9. Desagher S., Osen-Sand A., Montessuit S., Magnenat E., Vilbois F., Hochmann A., Journot L., Antonsson B., Martinou J. C. Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell. 2001 Sep;8(3):601–611. doi: 10.1016/s1097-2765(01)00335-5. [DOI] [PubMed] [Google Scholar]
  10. Egelhoff T. T., Lee R. J., Spudich J. A. Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo. Cell. 1993 Oct 22;75(2):363–371. doi: 10.1016/0092-8674(93)80077-r. [DOI] [PubMed] [Google Scholar]
  11. Gechtman Z., Shaltiel S. Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin. Eur J Biochem. 1997 Jan 15;243(1-2):493–501. doi: 10.1111/j.1432-1033.1997.0493a.x. [DOI] [PubMed] [Google Scholar]
  12. Graves J. D., Draves K. E., Gotoh Y., Krebs E. G., Clark E. A. Both phosphorylation and caspase-mediated cleavage contribute to regulation of the Ste20-like protein kinase Mst1 during CD95/Fas-induced apoptosis. J Biol Chem. 2001 Feb 13;276(18):14909–14915. doi: 10.1074/jbc.M010905200. [DOI] [PubMed] [Google Scholar]
  13. Grunberg J., Walter J., Loetscher H., Deuschle U., Jacobsen H., Haass C. Alzheimer's disease associated presenilin-1 holoprotein and its 18-20 kDa C-terminal fragment are death substrates for proteases of the caspase family. Biochemistry. 1998 Feb 24;37(8):2263–2270. doi: 10.1021/bi972106l. [DOI] [PubMed] [Google Scholar]
  14. Huang W., Erikson R. L. Constitutive activation of Mek1 by mutation of serine phosphorylation sites. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8960–8963. doi: 10.1073/pnas.91.19.8960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaspari A., Diefenthal T., Grosche G., Schierhorn A., Demuth H. U. Substrates containing phosphorylated residues adjacent to proline decrease the cleavage by proline-specific peptidases. Biochim Biophys Acta. 1996 Mar 7;1293(1):147–153. doi: 10.1016/0167-4838(95)00238-3. [DOI] [PubMed] [Google Scholar]
  16. Kim T. W., Pettingell W. H., Jung Y. K., Kovacs D. M., Tanzi R. E. Alternative cleavage of Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease. Science. 1997 Jul 18;277(5324):373–376. doi: 10.1126/science.277.5324.373. [DOI] [PubMed] [Google Scholar]
  17. Krippner-Heidenreich A., Talanian R. V., Sekul R., Kraft R., Thole H., Ottleben H., Lüscher B. Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1. Biochem J. 2001 Sep 15;358(Pt 3):705–715. doi: 10.1042/0264-6021:3580705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee F. S., Peters R. T., Dang L. C., Maniatis T. MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9319–9324. doi: 10.1073/pnas.95.16.9319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee K. K., Tang M. K., Yew D. T., Chow P. H., Yee S. P., Schneider C., Brancolini C. gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb. Dev Biol. 1999 Mar 1;207(1):14–25. doi: 10.1006/dbio.1998.9086. [DOI] [PubMed] [Google Scholar]
  20. Mittl P. R., Di Marco S., Krebs J. F., Bai X., Karanewsky D. S., Priestle J. P., Tomaselli K. J., Grütter M. G. Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone. J Biol Chem. 1997 Mar 7;272(10):6539–6547. doi: 10.1074/jbc.272.10.6539. [DOI] [PubMed] [Google Scholar]
  21. Nicholson D. W. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 1999 Nov;6(11):1028–1042. doi: 10.1038/sj.cdd.4400598. [DOI] [PubMed] [Google Scholar]
  22. Park J. A., Kim K. W., Kim S. I., Lee S. K. Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells. Eur J Biochem. 1998 Oct 1;257(1):242–248. doi: 10.1046/j.1432-1327.1998.2570242.x. [DOI] [PubMed] [Google Scholar]
  23. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  24. Scheuner D., Eckman C., Jensen M., Song X., Citron M., Suzuki N., Bird T. D., Hardy J., Hutton M., Kukull W. Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease. Nat Med. 1996 Aug;2(8):864–870. doi: 10.1038/nm0896-864. [DOI] [PubMed] [Google Scholar]
  25. Schneider C., King R. M., Philipson L. Genes specifically expressed at growth arrest of mammalian cells. Cell. 1988 Sep 9;54(6):787–793. doi: 10.1016/s0092-8674(88)91065-3. [DOI] [PubMed] [Google Scholar]
  26. Scott M. T., Morrice N., Ball K. L. Reversible phosphorylation at the C-terminal regulatory domain of p21(Waf1/Cip1) modulates proliferating cell nuclear antigen binding. J Biol Chem. 2000 Apr 14;275(15):11529–11537. doi: 10.1074/jbc.275.15.11529. [DOI] [PubMed] [Google Scholar]
  27. Slee E. A., Adrain C., Martin S. J. Serial killers: ordering caspase activation events in apoptosis. Cell Death Differ. 1999 Nov;6(11):1067–1074. doi: 10.1038/sj.cdd.4400601. [DOI] [PubMed] [Google Scholar]
  28. Stennicke H. R., Renatus M., Meldal M., Salvesen G. S. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem J. 2000 Sep 1;350(Pt 2):563–568. [PMC free article] [PubMed] [Google Scholar]
  29. Stennicke H. R., Salvesen G. S. Caspases: preparation and characterization. Methods. 1999 Apr;17(4):313–319. doi: 10.1006/meth.1999.0745. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Thornberry N. A. Caspases: a decade of death research. Cell Death Differ. 1999 Nov;6(11):1023–1027. doi: 10.1038/sj.cdd.4400607. [DOI] [PubMed] [Google Scholar]
  32. Thornberry N. A., Lazebnik Y. Caspases: enemies within. Science. 1998 Aug 28;281(5381):1312–1316. doi: 10.1126/science.281.5381.1312. [DOI] [PubMed] [Google Scholar]
  33. Tözsér J., Bagossi P., Boross P., Louis J. M., Majerova E., Oroszlan S., Copeland T. D. Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates. Eur J Biochem. 1999 Oct 1;265(1):423–429. doi: 10.1046/j.1432-1327.1999.00756.x. [DOI] [PubMed] [Google Scholar]
  34. Tözsér J., Bagossi P., Weber I. T., Louis J. M., Copeland T. D., Oroszlan S. Studies on the symmetry and sequence context dependence of the HIV-1 proteinase specificity. J Biol Chem. 1997 Jul 4;272(27):16807–16814. doi: 10.1074/jbc.272.27.16807. [DOI] [PubMed] [Google Scholar]
  35. Ura S., Masuyama N., Graves J. D., Gotoh Y. Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation. Proc Natl Acad Sci U S A. 2001 Aug 21;98(18):10148–10153. doi: 10.1073/pnas.181161698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walter B. N., Huang Z., Jakobi R., Tuazon P. T., Alnemri E. S., Litwack G., Traugh J. A. Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity. J Biol Chem. 1998 Oct 30;273(44):28733–28739. doi: 10.1074/jbc.273.44.28733. [DOI] [PubMed] [Google Scholar]
  37. Walter J., Grünberg J., Capell A., Pesold B., Schindzielorz A., Citron M., Mendla K., George-Hyslop P. S., Multhaup G., Selkoe D. J. Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5349–5354. doi: 10.1073/pnas.94.10.5349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Walter J., Grünberg J., Schindzielorz A., Haass C. Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms. Biochemistry. 1998 Apr 28;37(17):5961–5967. doi: 10.1021/bi971763a. [DOI] [PubMed] [Google Scholar]
  39. Walter J., Schindzielorz A., Grünberg J., Haass C. Phosphorylation of presenilin-2 regulates its cleavage by caspases and retards progression of apoptosis. Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1391–1396. doi: 10.1073/pnas.96.4.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang X., Pai J. T., Wiedenfeld E. A., Medina J. C., Slaughter C. A., Goldstein J. L., Brown M. S. Purification of an interleukin-1 beta converting enzyme-related cysteine protease that cleaves sterol regulatory element-binding proteins between the leucine zipper and transmembrane domains. J Biol Chem. 1995 Jul 28;270(30):18044–18050. doi: 10.1074/jbc.270.30.18044. [DOI] [PubMed] [Google Scholar]
  41. Wei Y., Fox T., Chambers S. P., Sintchak J., Coll J. T., Golec J. M., Swenson L., Wilson K. P., Charifson P. S. The structures of caspases-1, -3, -7 and -8 reveal the basis for substrate and inhibitor selectivity. Chem Biol. 2000 Jun;7(6):423–432. doi: 10.1016/s1074-5521(00)00123-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES