Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Feb 15;378(Pt 1):177–184. doi: 10.1042/BJ20031122

Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon.

Suchira Bose 1, Fiona L L Stratford 1, Kerry I Broadfoot 1, Grant G F Mason 1, A Jennifer Rivett 1
PMCID: PMC1223927  PMID: 14583091

Abstract

In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit.

Full Text

The Full Text of this article is available as a PDF (224.3 KB).

Selected References

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

  1. Bose S., Brooks P., Mason G. G., Rivett A. J. gamma-Interferon decreases the level of 26 S proteasomes and changes the pattern of phosphorylation. Biochem J. 2001 Jan 15;353(Pt 2):291–297. doi: 10.1042/0264-6021:3530291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brophy Victoria A., Tavaré Jeremy M., Rivett A. Jennifer. Treatment of COS-7 cells with proteasome inhibitors or gamma-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis. Arch Biochem Biophys. 2002 Jan 15;397(2):199–205. doi: 10.1006/abbi.2001.2679. [DOI] [PubMed] [Google Scholar]
  3. Cascio Paolo, Call Matthew, Petre Benjamin M., Walz Thomas, Goldberg Alfred L. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes. EMBO J. 2002 Jun 3;21(11):2636–2645. doi: 10.1093/emboj/21.11.2636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Castaño J. G., Mahillo E., Arizti P., Arribas J. Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis. Biochemistry. 1996 Mar 26;35(12):3782–3789. doi: 10.1021/bi952540s. [DOI] [PubMed] [Google Scholar]
  5. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 1998 Dec 15;17(24):7151–7160. doi: 10.1093/emboj/17.24.7151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coffino P. Regulation of cellular polyamines by antizyme. Nat Rev Mol Cell Biol. 2001 Mar;2(3):188–194. doi: 10.1038/35056508. [DOI] [PubMed] [Google Scholar]
  7. Coux O., Tanaka K., Goldberg A. L. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem. 1996;65:801–847. doi: 10.1146/annurev.bi.65.070196.004101. [DOI] [PubMed] [Google Scholar]
  8. DeMartino G. N., Slaughter C. A. The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem. 1999 Aug 6;274(32):22123–22126. doi: 10.1074/jbc.274.32.22123. [DOI] [PubMed] [Google Scholar]
  9. Eytan E., Ganoth D., Armon T., Hershko A. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7751–7755. doi: 10.1073/pnas.86.20.7751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fabunmi R. P., Wigley W. C., Thomas P. J., DeMartino G. N. Interferon gamma regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies. J Cell Sci. 2001 Jan;114(Pt 1):29–36. doi: 10.1242/jcs.114.1.29. [DOI] [PubMed] [Google Scholar]
  11. Feng Y., Longo D. L., Ferris D. K. Polo-like kinase interacts with proteasomes and regulates their activity. Cell Growth Differ. 2001 Jan;12(1):29–37. [PubMed] [Google Scholar]
  12. Fernández Murray Pedro, Pardo Patricia S., Zelada Alicia M., Passeron Susana. In vivo and in vitro phosphorylation of Candida albicans 20S proteasome. Arch Biochem Biophys. 2002 Aug 1;404(1):116–125. doi: 10.1016/s0003-9861(02)00248-5. [DOI] [PubMed] [Google Scholar]
  13. Gerards W. L., de Jong W. W., Bloemendal H., Boelens W. The human proteasomal subunit HsC8 induces ring formation of other alpha-type subunits. J Mol Biol. 1998 Jan 9;275(1):113–121. doi: 10.1006/jmbi.1997.1429. [DOI] [PubMed] [Google Scholar]
  14. Glickman Michael H., Ciechanover Aaron. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002 Apr;82(2):373–428. doi: 10.1152/physrev.00027.2001. [DOI] [PubMed] [Google Scholar]
  15. Goldberg Alfred L., Cascio Paolo, Saric Tomo, Rock Kenneth L. The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides. Mol Immunol. 2002 Oct;39(3-4):147–164. doi: 10.1016/s0161-5890(02)00098-6. [DOI] [PubMed] [Google Scholar]
  16. Gordon C. The intracellular localization of the proteasome. Curr Top Microbiol Immunol. 2002;268:175–184. doi: 10.1007/978-3-642-59414-4_7. [DOI] [PubMed] [Google Scholar]
  17. Groettrup M., van den Broek M., Schwarz K., Macagno A., Khan S., de Giuli R., Schmidtke G. Structural plasticity of the proteasome and its function in antigen processing. Crit Rev Immunol. 2001;21(4):339–358. [PubMed] [Google Scholar]
  18. Groll M., Ditzel L., Löwe J., Stock D., Bochtler M., Bartunik H. D., Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997 Apr 3;386(6624):463–471. doi: 10.1038/386463a0. [DOI] [PubMed] [Google Scholar]
  19. Hilt W., Wolf D. H. Proteasomes: destruction as a programme. Trends Biochem Sci. 1996 Mar;21(3):96–102. [PubMed] [Google Scholar]
  20. Hoffman L., Pratt G., Rechsteiner M. Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate. J Biol Chem. 1992 Nov 5;267(31):22362–22368. [PubMed] [Google Scholar]
  21. Kaltoft M. B., Koch C., Uerkvitz W., Hendil K. B. Monoclonal antibodies to the human multicatalytic proteinase (proteasome). Hybridoma. 1992 Aug;11(4):507–517. doi: 10.1089/hyb.1992.11.507. [DOI] [PubMed] [Google Scholar]
  22. Litchfield David W. Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J. 2003 Jan 1;369(Pt 1):1–15. doi: 10.1042/BJ20021469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ludemann R., Lerea K. M., Etlinger J. D. Copurification of casein kinase II with 20 S proteasomes and phosphorylation of a 30-kDa proteasome subunit. J Biol Chem. 1993 Aug 15;268(23):17413–17417. [PubMed] [Google Scholar]
  24. Löwe J., Stock D., Jap B., Zwickl P., Baumeister W., Huber R. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science. 1995 Apr 28;268(5210):533–539. doi: 10.1126/science.7725097. [DOI] [PubMed] [Google Scholar]
  25. Mason G. G., Hendil K. B., Rivett A. J. Phosphorylation of proteasomes in mammalian cells. Identification of two phosphorylated subunits and the effect of phosphorylation on activity. Eur J Biochem. 1996 Jun 1;238(2):453–462. doi: 10.1111/j.1432-1033.1996.0453z.x. [DOI] [PubMed] [Google Scholar]
  26. Mason G. G., Murray R. Z., Pappin D., Rivett A. J. Phosphorylation of ATPase subunits of the 26S proteasome. FEBS Lett. 1998 Jul 3;430(3):269–274. doi: 10.1016/s0014-5793(98)00676-0. [DOI] [PubMed] [Google Scholar]
  27. Meggio Flavio, Pinna Lorenzo A. One-thousand-and-one substrates of protein kinase CK2? FASEB J. 2003 Mar;17(3):349–368. doi: 10.1096/fj.02-0473rev. [DOI] [PubMed] [Google Scholar]
  28. Niedermann G. Immunological functions of the proteasome. Curr Top Microbiol Immunol. 2002;268:91–136. doi: 10.1007/978-3-642-59414-4_5. [DOI] [PubMed] [Google Scholar]
  29. Pardo P. S., Murray P. F., Walz K., Franco L., Passeron S. In vivo and in vitro phosphorylation of the alpha 7/PRS1 subunit of Saccharomyces cerevisiae 20 S proteasome: in vitro phosphorylation by protein kinase CK2 is absolutely dependent on polylysine. Arch Biochem Biophys. 1998 Jan 15;349(2):397–401. doi: 10.1006/abbi.1997.0466. [DOI] [PubMed] [Google Scholar]
  30. Pereira M. E., Wilk S. Phosphorylation of the multicatalytic proteinase complex from bovine pituitaries by a copurifying cAMP-dependent protein kinase. Arch Biochem Biophys. 1990 Nov 15;283(1):68–74. doi: 10.1016/0003-9861(90)90613-4. [DOI] [PubMed] [Google Scholar]
  31. Rechsteiner M., Hoffman L., Dubiel W. The multicatalytic and 26 S proteases. J Biol Chem. 1993 Mar 25;268(9):6065–6068. [PubMed] [Google Scholar]
  32. Rechsteiner M., Realini C., Ustrell V. The proteasome activator 11 S REG (PA28) and class I antigen presentation. Biochem J. 2000 Jan 1;345(Pt 1):1–15. [PMC free article] [PubMed] [Google Scholar]
  33. Reidlinger J., Pike A. M., Savory P. J., Murray R. Z., Rivett A. J. Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. J Biol Chem. 1997 Oct 3;272(40):24899–24905. doi: 10.1074/jbc.272.40.24899. [DOI] [PubMed] [Google Scholar]
  34. Rivett A. J. Intracellular distribution of proteasomes. Curr Opin Immunol. 1998 Feb;10(1):110–114. doi: 10.1016/s0952-7915(98)80040-x. [DOI] [PubMed] [Google Scholar]
  35. Rivett A. J., Savory P. J., Djaballah H. Multicatalytic endopeptidase complex: proteasome. Methods Enzymol. 1994;244:331–350. doi: 10.1016/0076-6879(94)44026-3. [DOI] [PubMed] [Google Scholar]
  36. Rivett A. Jennifer, Bose Suchira, Pemberton Alexander J., Brooks Paul, Onion David, Shirley David, Stratford Fiona L. L., Forti Katia. Assays of proteasome activity in relation to aging. Exp Gerontol. 2002 Oct-Nov;37(10-11):1217–1222. doi: 10.1016/s0531-5565(02)00127-4. [DOI] [PubMed] [Google Scholar]
  37. Satoh K., Sasajima H., Nyoumura K. I., Yokosawa H., Sawada H. Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit. Biochemistry. 2001 Jan 16;40(2):314–319. doi: 10.1021/bi001815n. [DOI] [PubMed] [Google Scholar]
  38. Sijts Alice, Sun Yuancheng, Janek Katarina, Kral Sylvie, Paschen Annettte, Schadendorf Dirk, Kloetzel Peter-M. The role of the proteasome activator PA28 in MHC class I antigen processing. Mol Immunol. 2002 Oct;39(3-4):165–169. doi: 10.1016/s0161-5890(02)00099-8. [DOI] [PubMed] [Google Scholar]
  39. Tanahashi N., Murakami Y., Minami Y., Shimbara N., Hendil K. B., Tanaka K. Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J Biol Chem. 2000 May 12;275(19):14336–14345. doi: 10.1074/jbc.275.19.14336. [DOI] [PubMed] [Google Scholar]
  40. Tanaka K., Kanayama H., Tamura T., Lee D. H., Kumatori A., Fujiwara T., Ichihara A., Tokunaga F., Aruga R., Iwanaga S. cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells. Biochem Biophys Res Commun. 1990 Sep 14;171(2):676–683. doi: 10.1016/0006-291x(90)91199-3. [DOI] [PubMed] [Google Scholar]
  41. Torres J., Pulido R. The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J Biol Chem. 2001 Jan 12;276(2):993–998. doi: 10.1074/jbc.M009134200. [DOI] [PubMed] [Google Scholar]
  42. Touitou R., Richardson J., Bose S., Nakanishi M., Rivett J., Allday M. J. A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome. EMBO J. 2001 May 15;20(10):2367–2375. doi: 10.1093/emboj/20.10.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Umeda M., Manabe Y., Uchimiya H. Phosphorylation of the C2 subunit of the proteasome in rice (Oryza sativa L.). FEBS Lett. 1997 Feb 24;403(3):313–317. doi: 10.1016/s0014-5793(97)00073-2. [DOI] [PubMed] [Google Scholar]
  44. Voges D., Zwickl P., Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem. 1999;68:1015–1068. doi: 10.1146/annurev.biochem.68.1.1015. [DOI] [PubMed] [Google Scholar]
  45. Wójcik Cezary, DeMartino George N. Intracellular localization of proteasomes. Int J Biochem Cell Biol. 2003 May;35(5):579–589. doi: 10.1016/s1357-2725(02)00380-1. [DOI] [PubMed] [Google Scholar]
  46. Yang Y., Früh K., Ahn K., Peterson P. A. In vivo assembly of the proteasomal complexes, implications for antigen processing. J Biol Chem. 1995 Nov 17;270(46):27687–27694. doi: 10.1074/jbc.270.46.27687. [DOI] [PubMed] [Google Scholar]

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

RESOURCES