Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 May 1;347(Pt 3):845–855.

A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres.

C J Hastie 1, G K Carnegie 1, N Morrice 1, P T Cohen 1
PMCID: PMC1221024  PMID: 10769191

Abstract

Protein phosphatase 4 (PPP4) is a protein serine/threonine phosphatase that has been implicated in microtubule organization at centrosomes. Complexes of PPP4 with high apparent molecular masses (450 and 600 kDa) were purified from mammalian skeletal muscle and testis to near homogeneity. Amino acid sequences derived from a protein component present in both complexes were utilized to identify a human cDNA. The encoded putative PPP4 regulatory subunit (termed PPP4R2), comprising 453 amino acids, had a molecular mass of 50.4 kDa. The interaction of PPP4R2 with PPP4 catalytic subunit (PPP4c) was confirmed by co-sedimentation of PPP4c with PPP4R2 expressed in bacteria and human cells. PPP4c formed a complex of 450 kDa with baculovirus expressed His(6)-tagged PPP4R2. Immunocytological detection of PPP4R2 at centrosomes suggests that it may target PPP4c to this location. Native 450 kDa and 600 kDa PPP4 complexes are inactive, but can be activated by basic proteins, suggesting that PPP4R2 may also regulate the activity of PPP4c at centrosomal microtubule organising centres.

Full Text

The Full Text of this article is available as a PDF (347.3 KB).

Selected References

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

  1. Alessi D. R., Street A. J., Cohen P., Cohen P. T. Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme. Eur J Biochem. 1993 May 1;213(3):1055–1066. doi: 10.1111/j.1432-1033.1993.tb17853.x. [DOI] [PubMed] [Google Scholar]
  2. Armstrong C. G., Browne G. J., Cohen P., Cohen P. T. PPP1R6, a novel member of the family of glycogen-targetting subunits of protein phosphatase 1. FEBS Lett. 1997 Nov 24;418(1-2):210–214. doi: 10.1016/s0014-5793(97)01385-9. [DOI] [PubMed] [Google Scholar]
  3. Barker H. M., Craig S. P., Spurr N. K., Cohen P. T. Sequence of human protein serine/threonine phosphatase 1 gamma and localization of the gene (PPP1CC) encoding it to chromosome bands 12q24.1-q24.2. Biochim Biophys Acta. 1993 Aug 18;1178(2):228–233. doi: 10.1016/0167-4889(93)90014-g. [DOI] [PubMed] [Google Scholar]
  4. Brewis N. D., Cohen P. T. Protein phosphatase X has been highly conserved during mammalian evolution. Biochim Biophys Acta. 1992 Dec 29;1171(2):231–233. doi: 10.1016/0167-4781(92)90129-n. [DOI] [PubMed] [Google Scholar]
  5. Brewis N. D., Street A. J., Prescott A. R., Cohen P. T. PPX, a novel protein serine/threonine phosphatase localized to centrosomes. EMBO J. 1993 Mar;12(3):987–996. doi: 10.1002/j.1460-2075.1993.tb05739.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buendia B., Draetta G., Karsenti E. Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase. J Cell Biol. 1992 Mar;116(6):1431–1442. doi: 10.1083/jcb.116.6.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Centonze V. E., Borisy G. G. Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope. J Cell Sci. 1990 Mar;95(Pt 3):405–411. doi: 10.1242/jcs.95.3.405. [DOI] [PubMed] [Google Scholar]
  8. Chen J., Peterson R. T., Schreiber S. L. Alpha 4 associates with protein phosphatases 2A, 4, and 6. Biochem Biophys Res Commun. 1998 Jun 29;247(3):827–832. doi: 10.1006/bbrc.1998.8792. [DOI] [PubMed] [Google Scholar]
  9. Cohen P. T. Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem Sci. 1997 Jul;22(7):245–251. doi: 10.1016/s0968-0004(97)01060-8. [DOI] [PubMed] [Google Scholar]
  10. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  11. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  12. Dictenberg J. B., Zimmerman W., Sparks C. A., Young A., Vidair C., Zheng Y., Carrington W., Fay F. S., Doxsey S. J. Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J Cell Biol. 1998 Apr 6;141(1):163–174. doi: 10.1083/jcb.141.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feng Y., Hodge D. R., Palmieri G., Chase D. L., Longo D. L., Ferris D. K. Association of polo-like kinase with alpha-, beta- and gamma-tubulins in a stable complex. Biochem J. 1999 Apr 15;339(Pt 2):435–442. [PMC free article] [PubMed] [Google Scholar]
  14. Geissler S., Pereira G., Spang A., Knop M., Souès S., Kilmartin J., Schiebel E. The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 1996 Aug 1;15(15):3899–3911. [PMC free article] [PubMed] [Google Scholar]
  15. Hastie C. J., Cohen P. T. Purification of protein phosphatase 4 catalytic subunit: inhibition by the antitumour drug fostriecin and other tumour suppressors and promoters. FEBS Lett. 1998 Jul 24;431(3):357–361. doi: 10.1016/s0014-5793(98)00775-3. [DOI] [PubMed] [Google Scholar]
  16. Helps N. R., Brewis N. D., Lineruth K., Davis T., Kaiser K., Cohen P. T. Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos. J Cell Sci. 1998 May;111(Pt 10):1331–1340. doi: 10.1242/jcs.111.10.1331. [DOI] [PubMed] [Google Scholar]
  17. Hemmings B. A., Adams-Pearson C., Maurer F., Müller P., Goris J., Merlevede W., Hofsteenge J., Stone S. R. alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry. 1990 Apr 3;29(13):3166–3173. doi: 10.1021/bi00465a002. [DOI] [PubMed] [Google Scholar]
  18. Hu M. C., Tang-Oxley Q., Qiu W. R., Wang Y. P., Mihindukulasuriya K. A., Afshar R., Tan T. H. Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappaB activity. J Biol Chem. 1998 Dec 11;273(50):33561–33565. doi: 10.1074/jbc.273.50.33561. [DOI] [PubMed] [Google Scholar]
  19. Hubbard M. J., Cohen P. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci. 1993 May;18(5):172–177. doi: 10.1016/0968-0004(93)90109-z. [DOI] [PubMed] [Google Scholar]
  20. Kellogg D. R., Moritz M., Alberts B. M. The centrosome and cellular organization. Annu Rev Biochem. 1994;63:639–674. doi: 10.1146/annurev.bi.63.070194.003231. [DOI] [PubMed] [Google Scholar]
  21. Kidd D., Raff J. W. LK6, a short lived protein kinase in Drosophila that can associate with microtubules and centrosomes. J Cell Sci. 1997 Jan;110(Pt 2):209–219. doi: 10.1242/jcs.110.2.209. [DOI] [PubMed] [Google Scholar]
  22. Kloeker S., Bryant J. C., Strack S., Colbran R. J., Wadzinski B. E. Carboxymethylation of nuclear protein serine/threonine phosphatase X. Biochem J. 1997 Oct 15;327(Pt 2):481–486. doi: 10.1042/bj3270481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kloeker S., Wadzinski B. E. Purification and identification of a novel subunit of protein serine/threonine phosphatase 4. J Biol Chem. 1999 Feb 26;274(9):5339–5347. doi: 10.1074/jbc.274.9.5339. [DOI] [PubMed] [Google Scholar]
  24. Kumagai A., Dunphy W. G. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science. 1996 Sep 6;273(5280):1377–1380. doi: 10.1126/science.273.5280.1377. [DOI] [PubMed] [Google Scholar]
  25. Lane H. A., Nigg E. A. Cell-cycle control: POLO-like kinases join the outer circle. Trends Cell Biol. 1997 Feb;7(2):63–68. doi: 10.1016/S0962-8924(96)10051-9. [DOI] [PubMed] [Google Scholar]
  26. Mayer-Jaekel R. E., Baumgartner S., Bilbe G., Ohkura H., Glover D. M., Hemmings B. A. Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila. Mol Biol Cell. 1992 Mar;3(3):287–298. doi: 10.1091/mbc.3.3.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mayer-Jaekel R. E., Ohkura H., Gomes R., Sunkel C. E., Baumgartner S., Hemmings B. A., Glover D. M. The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell. 1993 Feb 26;72(4):621–633. doi: 10.1016/0092-8674(93)90080-a. [DOI] [PubMed] [Google Scholar]
  28. Moorhead G., MacKintosh C., Morrice N., Cohen P. Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography. FEBS Lett. 1995 Apr 3;362(2):101–105. doi: 10.1016/0014-5793(95)00197-h. [DOI] [PubMed] [Google Scholar]
  29. Moorhead G., MacKintosh R. W., Morrice N., Gallagher T., MacKintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography. FEBS Lett. 1994 Dec 12;356(1):46–50. doi: 10.1016/0014-5793(94)01232-6. [DOI] [PubMed] [Google Scholar]
  30. Moritz M., Braunfeld M. B., Sedat J. W., Alberts B., Agard D. A. Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature. 1995 Dec 7;378(6557):638–640. doi: 10.1038/378638a0. [DOI] [PubMed] [Google Scholar]
  31. Moritz M., Zheng Y., Alberts B. M., Oegema K. Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J Cell Biol. 1998 Aug 10;142(3):775–786. doi: 10.1083/jcb.142.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nishiwaki S., Fujiki H., Suganuma M., Nishiwaki-Matsushima R., Sugimura T. Rapid purification of protein phosphatase 2A from mouse brain by microcystin-affinity chromatography. FEBS Lett. 1991 Feb 11;279(1):115–118. doi: 10.1016/0014-5793(91)80264-4. [DOI] [PubMed] [Google Scholar]
  33. Orgad S., Brewis N. D., Alphey L., Axton J. M., Dudai Y., Cohen P. T. The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1. FEBS Lett. 1990 Nov 26;275(1-2):44–48. doi: 10.1016/0014-5793(90)81435-q. [DOI] [PubMed] [Google Scholar]
  34. Ronne H., Carlberg M., Hu G. Z., Nehlin J. O. Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol Cell Biol. 1991 Oct;11(10):4876–4884. doi: 10.1128/mcb.11.10.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  36. Snaith H. A., Armstrong C. G., Guo Y., Kaiser K., Cohen P. T. Deficiency of protein phosphatase 2A uncouples the nuclear and centrosome cycles and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos. J Cell Sci. 1996 Dec;109(Pt 13):3001–3012. doi: 10.1242/jcs.109.13.3001. [DOI] [PubMed] [Google Scholar]
  37. Sobel S. G., Snyder M. A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J Cell Biol. 1995 Dec;131(6 Pt 2):1775–1788. doi: 10.1083/jcb.131.6.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sontag E., Nunbhakdi-Craig V., Bloom G. S., Mumby M. C. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J Cell Biol. 1995 Mar;128(6):1131–1144. doi: 10.1083/jcb.128.6.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tassin A. M., Celati C., Moudjou M., Bornens M. Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin. J Cell Biol. 1998 May 4;141(3):689–701. doi: 10.1083/jcb.141.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tavares A. A., Glover D. M., Sunkel C. E. The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts. EMBO J. 1996 Sep 16;15(18):4873–4883. [PMC free article] [PubMed] [Google Scholar]
  41. Tournebize R., Andersen S. S., Verde F., Dorée M., Karsenti E., Hyman A. A. Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis. EMBO J. 1997 Sep 15;16(18):5537–5549. doi: 10.1093/emboj/16.18.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tung H. Y., Pelech S., Fisher M. J., Pogson C. I., Cohen P. The protein phosphatases involved in cellular regulation. Influence of polyamines on the activities of protein phosphatase-1 and protein phosphatase-2A. Eur J Biochem. 1985 Jun 3;149(2):305–313. doi: 10.1111/j.1432-1033.1985.tb08927.x. [DOI] [PubMed] [Google Scholar]
  43. Webster G. A., Perkins N. D. Transcriptional cross talk between NF-kappaB and p53. Mol Cell Biol. 1999 May;19(5):3485–3495. doi: 10.1128/mcb.19.5.3485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zheng Y., Wong M. L., Alberts B., Mitchison T. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature. 1995 Dec 7;378(6557):578–583. doi: 10.1038/378578a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES