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. 1997 Oct 15;327(Pt 2):481–486. doi: 10.1042/bj3270481

Carboxymethylation of nuclear protein serine/threonine phosphatase X.

S Kloeker 1, J C Bryant 1, S Strack 1, R J Colbran 1, B E Wadzinski 1
PMCID: PMC1218819  PMID: 9359419

Abstract

Specific rabbit polyclonal antibodies against peptides corresponding to the highly homologous protein serine/threonine phosphatase 2A and X catalytic subunits (PP2A/C and PPX/C respectively) were used to investigate the cellular and subcellular distribution of PP2A/C and PPX/C, as well as their methylation state. Immunoblots of rat tissue extracts revealed a widespread distribution of these enzymes but particularly high levels of PP2A/C and PPX/C in brain and testes respectively. In addition, immunoblots of subcellular fractions and immunocytochemical analyses of rat brain sections demonstrated that PPX/C is predominantly localized to the nucleus, whereas PP2A/C is largely cytoplasmic. Treatment of nuclear extracts with alkali resulted in increased PPX/C immunoreactivity to a polyclonal antibody directed against the C-terminus; no change in PPX immunoreactivity was observed using an antibody against an internal peptide. Alkali treatment of brain and liver cytosolic and nuclear extracts did not change the molecular mass or the isoelectric point of PPX/C. Furthermore, tritiated PPX/C was immunoprecipitated from COS cell extracts incubated with the methyl donor S-adenosyl-l-[methyl-3H]methionine. Thus the increase in immunoreactivity probably results from removal of a carboxymethyl group from PPX/C, as has been shown previously for PP2A/C [Favre, Zolnierowicz, Turowski and Hemmings (1994) J. Biol. Chem. 269, 16311-16317]. Together, our results indicate that the PPX catalytic subunit is a predominantly nuclear phosphatase and is methylated at its C-terminus.

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Selected References

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  1. Brautigan D. L. Protein phosphatases. Recent Prog Horm Res. 1994;49:197–214. doi: 10.1016/b978-0-12-571149-4.50014-3. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Chelsky D., Sobotka C., O'Neill C. L. Lamin B methylation and assembly into the nuclear envelope. J Biol Chem. 1989 May 5;264(13):7637–7643. [PubMed] [Google Scholar]
  4. Chen J., Martin B. L., Brautigan D. L. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science. 1992 Aug 28;257(5074):1261–1264. doi: 10.1126/science.1325671. [DOI] [PubMed] [Google Scholar]
  5. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Depaoli-Roach A. A., Park I. K., Cerovsky V., Csortos C., Durbin S. D., Kuntz M. J., Sitikov A., Tang P. M., Verin A., Zolnierowicz S. Serine/threonine protein phosphatases in the control of cell function. Adv Enzyme Regul. 1994;34:199–224. doi: 10.1016/0065-2571(94)90017-5. [DOI] [PubMed] [Google Scholar]
  8. Dignam J. D. Preparation of extracts from higher eukaryotes. Methods Enzymol. 1990;182:194–203. doi: 10.1016/0076-6879(90)82017-v. [DOI] [PubMed] [Google Scholar]
  9. Favre B., Zolnierowicz S., Turowski P., Hemmings B. A. The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo. J Biol Chem. 1994 Jun 10;269(23):16311–16317. [PubMed] [Google Scholar]
  10. Fukada Y., Matsuda T., Kokame K., Takao T., Shimonishi Y., Akino T., Yoshizawa T. Effects of carboxyl methylation of photoreceptor G protein gamma-subunit in visual transduction. J Biol Chem. 1994 Feb 18;269(7):5163–5170. [PubMed] [Google Scholar]
  11. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [PubMed] [Google Scholar]
  12. Guo H., Damuni Z. Autophosphorylation-activated protein kinase phosphorylates and inactivates protein phosphatase 2A. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2500–2504. doi: 10.1073/pnas.90.6.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lee J., Chen Y., Tolstykh T., Stock J. A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6043–6047. doi: 10.1073/pnas.93.12.6043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee J., Stock J. Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase. J Biol Chem. 1993 Sep 15;268(26):19192–19195. [PubMed] [Google Scholar]
  15. Mayer-Jaekel R. E., Hemmings B. A. Protein phosphatase 2A--a 'ménage à trois'. Trends Cell Biol. 1994 Aug;4(8):287–291. doi: 10.1016/0962-8924(94)90219-4. [DOI] [PubMed] [Google Scholar]
  16. Mumby M. C., Green D. D., Russell K. L. Structural characterization of cardiac protein phosphatase with a monoclonal antibody. Evidence that the Mr = 38,000 phosphatase is the catalytic subunit of the native enzyme(s). J Biol Chem. 1985 Nov 5;260(25):13763–13770. [PubMed] [Google Scholar]
  17. Mumby M. C., Walter G. Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol Rev. 1993 Oct;73(4):673–699. doi: 10.1152/physrev.1993.73.4.673. [DOI] [PubMed] [Google Scholar]
  18. Ohguro H., Fukada Y., Takao T., Shimonishi Y., Yoshizawa T., Akino T. Carboxyl methylation and farnesylation of transducin gamma-subunit synergistically enhance its coupling with metarhodopsin II. EMBO J. 1991 Dec;10(12):3669–3674. doi: 10.1002/j.1460-2075.1991.tb04934.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pallas D. C., Shahrik L. K., Martin B. L., Jaspers S., Miller T. B., Brautigan D. L., Roberts T. M. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell. 1990 Jan 12;60(1):167–176. doi: 10.1016/0092-8674(90)90726-u. [DOI] [PubMed] [Google Scholar]
  20. Pallas D., Solomon F. Cytoplasmic microtubule-associated proteins: phosphorylation at novel sites is correlated with their incorporation into assembled microtubules. Cell. 1982 Sep;30(2):407–414. doi: 10.1016/0092-8674(82)90238-0. [DOI] [PubMed] [Google Scholar]
  21. Posada J., Cooper J. A. Molecular signal integration. Interplay between serine, threonine, and tyrosine phosphorylation. Mol Biol Cell. 1992 Jun;3(6):583–592. doi: 10.1091/mbc.3.6.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Saitoh Y., Yamamoto H., Ushio Y., Miyamoto E. Characterization of polyclonal antibodies to brain protein phosphatase 2A and immunohistochemical localization of the enzyme in rat brain. Brain Res. 1989 Jun 12;489(2):291–301. doi: 10.1016/0006-8993(89)90862-7. [DOI] [PubMed] [Google Scholar]
  23. Shenolikar S. Protein serine/threonine phosphatases--new avenues for cell regulation. Annu Rev Cell Biol. 1994;10:55–86. doi: 10.1146/annurev.cb.10.110194.000415. [DOI] [PubMed] [Google Scholar]
  24. Strack S., Wadzinski B. E., Ebner F. F. Localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the hindbrain and spinal cord of the rat. J Comp Neurol. 1996 Nov 4;375(1):66–76. doi: 10.1002/(SICI)1096-9861(19961104)375:1<66::AID-CNE4>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Turowski P., Fernandez A., Favre B., Lamb N. J., Hemmings B. A. Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression. J Cell Biol. 1995 Apr;129(2):397–410. doi: 10.1083/jcb.129.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wadzinski B. E., Eisfelder B. J., Peruski L. F., Jr, Mumby M. C., Johnson G. L. NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells. J Biol Chem. 1992 Aug 25;267(24):16883–16888. [PubMed] [Google Scholar]
  28. Wera S., Hemmings B. A. Serine/threonine protein phosphatases. Biochem J. 1995 Oct 1;311(Pt 1):17–29. doi: 10.1042/bj3110017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Xie H., Clarke S. Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain. J Biol Chem. 1994 Jan 21;269(3):1981–1984. [PubMed] [Google Scholar]
  30. da Cruz e Silva O. B., da Cruz e Silva E. F., Cohen P. T. Identification of a novel protein phosphatase catalytic subunit by cDNA cloning. FEBS Lett. 1988 Dec 19;242(1):106–110. doi: 10.1016/0014-5793(88)80995-5. [DOI] [PubMed] [Google Scholar]

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