Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 Feb;7(2):457–469. doi: 10.1002/pro.5560070227

A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry.

P T Jedrzejewski 1, A Girod 1, A Tholey 1, N König 1, S Thullner 1, V Kinzel 1, D Bossemeyer 1
PMCID: PMC2143929  PMID: 9521123

Abstract

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Adavani S. R., Schwarz M., Showers M. O., Maurer R. A., Hemmings B. A. Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit. Eur J Biochem. 1987 Sep 1;167(2):221–226. doi: 10.1111/j.1432-1033.1987.tb13326.x. [DOI] [PubMed] [Google Scholar]
  2. Artigues A., Birkett A., Schirch V. Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase. J Biol Chem. 1990 Mar 25;265(9):4853–4858. [PubMed] [Google Scholar]
  3. Aswad D. W. Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues. J Biol Chem. 1984 Sep 10;259(17):10714–10721. [PubMed] [Google Scholar]
  4. Baker R. T., Varshavsky A. Yeast N-terminal amidase. A new enzyme and component of the N-end rule pathway. J Biol Chem. 1995 May 19;270(20):12065–12074. doi: 10.1074/jbc.270.20.12065. [DOI] [PubMed] [Google Scholar]
  5. Bechtel P. J., Beavo J. A., Krebs E. G. Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1977 Apr 25;252(8):2691–2697. [PubMed] [Google Scholar]
  6. Beebe S. J., Oyen O., Sandberg M., Frøysa A., Hansson V., Jahnsen T. Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase. Mol Endocrinol. 1990 Mar;4(3):465–475. doi: 10.1210/mend-4-3-465. [DOI] [PubMed] [Google Scholar]
  7. Boivin D., Bilodeau D., Béliveau R. Immunochemical characterization of L-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues. Biochem J. 1995 Aug 1;309(Pt 3):993–998. doi: 10.1042/bj3090993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bornstein P., Balian G. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 1977;47:132–145. doi: 10.1016/0076-6879(77)47016-2. [DOI] [PubMed] [Google Scholar]
  9. Bossemeyer D., Engh R. A., Kinzel V., Ponstingl H., Huber R. Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24). EMBO J. 1993 Mar;12(3):849–859. doi: 10.1002/j.1460-2075.1993.tb05725.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brennan T. V., Anderson J. W., Jia Z., Waygood E. B., Clarke S. Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase. J Biol Chem. 1994 Oct 7;269(40):24586–24595. [PubMed] [Google Scholar]
  11. Buechler J. A., Vedvick T. A., Taylor S. S. Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with acetic anhydride: substrate-induced conformational changes. Biochemistry. 1989 Apr 4;28(7):3018–3024. doi: 10.1021/bi00433a042. [DOI] [PubMed] [Google Scholar]
  12. Clarke S. Perspectives on the biological function and enzymology of protein carboxyl methylation reactions in eucaryotic and procaryotic cells. Adv Exp Med Biol. 1988;231:213–228. doi: 10.1007/978-1-4684-9042-8_17. [DOI] [PubMed] [Google Scholar]
  13. Clarke S. Protein carboxyl methyltransferases: two distinct classes of enzymes. Annu Rev Biochem. 1985;54:479–506. doi: 10.1146/annurev.bi.54.070185.002403. [DOI] [PubMed] [Google Scholar]
  14. Davis M. T., Lee T. D. Analysis of peptide mixtures by capillary high performance liquid chromatography: a practical guide to small-scale separations. Protein Sci. 1992 Jul;1(7):935–944. doi: 10.1002/pro.5560010712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duronio R. J., Jackson-Machelski E., Heuckeroth R. O., Olins P. O., Devine C. S., Yonemoto W., Slice L. W., Taylor S. S., Gordon J. I. Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1506–1510. doi: 10.1073/pnas.87.4.1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Engh R. A., Girod A., Kinzel V., Huber R., Bossemeyer D. Crystal structures of catalytic subunit of cAMP-dependent protein kinase in complex with isoquinolinesulfonyl protein kinase inhibitors H7, H8, and H89. Structural implications for selectivity. J Biol Chem. 1996 Oct 18;271(42):26157–26164. doi: 10.1074/jbc.271.42.26157. [DOI] [PubMed] [Google Scholar]
  17. Flatau G., Lemichez E., Gauthier M., Chardin P., Paris S., Fiorentini C., Boquet P. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature. 1997 Jun 12;387(6634):729–733. doi: 10.1038/42743. [DOI] [PubMed] [Google Scholar]
  18. Gamm D. M., Baude E. J., Uhler M. D. The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo. J Biol Chem. 1996 Jun 28;271(26):15736–15742. doi: 10.1074/jbc.271.26.15736. [DOI] [PubMed] [Google Scholar]
  19. Geiger T., Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem. 1987 Jan 15;262(2):785–794. [PubMed] [Google Scholar]
  20. Girod A., Kinzel V., Bossemeyer D. In vivo activation of recombinant cAPK catalytic subunit active site mutants by coexpression of the wild-type enzyme, evidence for intermolecular cotranslational phosphorylation. FEBS Lett. 1996 Aug 5;391(1-2):121–125. doi: 10.1016/0014-5793(96)00717-x. [DOI] [PubMed] [Google Scholar]
  21. Hanakam F., Albrecht R., Eckerskorn C., Matzner M., Gerisch G. Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein. EMBO J. 1996 Jun 17;15(12):2935–2943. [PMC free article] [PubMed] [Google Scholar]
  22. Harootunian A. T., Adams S. R., Wen W., Meinkoth J. L., Taylor S. S., Tsien R. Y. Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. Mol Biol Cell. 1993 Oct;4(10):993–1002. doi: 10.1091/mbc.4.10.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Itaya K., Ui M. A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta. 1966 Sep;14(3):361–366. doi: 10.1016/0009-8981(66)90114-8. [DOI] [PubMed] [Google Scholar]
  25. Jedrzejewski P. T., Lehmann W. D. Detection of modified peptides in enzymatic digests by capillary liquid chromatography/electrospray mass spectrometry and a programmable skimmer CID acquisition routine. Anal Chem. 1997 Feb 1;69(3):294–301. doi: 10.1021/ac9606618. [DOI] [PubMed] [Google Scholar]
  26. Jiménez J. S., Kupfer A., Gani V., Shaltiel S. Salt-induced conformational changes in the catalytic subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase. Use for establishing a connection between one sulfhydryl group and the gamma-P subsite in the ATP site of this subunit. Biochemistry. 1982 Mar 30;21(7):1623–1630. doi: 10.1021/bi00536a024. [DOI] [PubMed] [Google Scholar]
  27. Johnson R. S., Ohguro H., Palczewski K., Hurley J. B., Walsh K. A., Neubert T. A. Heterogeneous N-acylation is a tissue- and species-specific posttranslational modification. J Biol Chem. 1994 Aug 19;269(33):21067–21071. [PubMed] [Google Scholar]
  28. Kinzel V., Hotz A., König N., Gagelmann M., Pyerin W., Reed J., Kübler D., Hofmann F., Obst C., Gensheimer H. P. Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species. Arch Biochem Biophys. 1987 Mar;253(2):341–349. doi: 10.1016/0003-9861(87)90187-1. [DOI] [PubMed] [Google Scholar]
  29. Kinzel V., Kübler D. Single step purification of the catalytic subunit(s) of cyclic 3', 5'-adenosine monophosphate-dependent protein kinase(s) from rat muscle. Biochem Biophys Res Commun. 1976 Jul 12;71(1):257–264. doi: 10.1016/0006-291x(76)90276-x. [DOI] [PubMed] [Google Scholar]
  30. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  31. Kupfer A., Gani V., Jiménez J. S., Shaltiel S. Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3073–3077. doi: 10.1073/pnas.76.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee S. L., Gorman K. B., Steinberg R. A. Methods for studying synthesis, turnover, and phosphorylation of catalytic subunit of cAMP-dependent protein kinase in mammalian cells. Mol Cell Endocrinol. 1996 Feb 5;116(2):233–241. doi: 10.1016/0303-7207(95)03719-5. [DOI] [PubMed] [Google Scholar]
  33. Lee S. L., Steinberg R. A. Pathways for degradation of the catalytic subunit of cAMP-dependent protein kinase differ in wild-type and kinase-negative S49 mouse lymphoma cells. J Biol Chem. 1996 Jul 12;271(28):16553–16558. doi: 10.1074/jbc.271.28.16553. [DOI] [PubMed] [Google Scholar]
  34. Lura R., Schirch V. Role of peptide conformation in the rate and mechanism of deamidation of asparaginyl residues. Biochemistry. 1988 Oct 4;27(20):7671–7677. doi: 10.1021/bi00420a015. [DOI] [PubMed] [Google Scholar]
  35. McLaughlin S., Aderem A. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem Sci. 1995 Jul;20(7):272–276. doi: 10.1016/s0968-0004(00)89042-8. [DOI] [PubMed] [Google Scholar]
  36. Meinkoth J. L., Ji Y., Taylor S. S., Feramisco J. R. Dynamics of the distribution of cyclic AMP-dependent protein kinase in living cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9595–9599. doi: 10.1073/pnas.87.24.9595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murray E. D., Jr, Clarke S. Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues. J Biol Chem. 1984 Sep 10;259(17):10722–10732. [PubMed] [Google Scholar]
  38. Nelson N. C., Taylor S. S. Differential labeling and identification of the cysteine-containing tryptic peptides of catalytic subunit from porcine heart cAMP-dependent protein kinase. J Biol Chem. 1981 Apr 25;256(8):3743–3750. [PubMed] [Google Scholar]
  39. Olsen S. R., Uhler M. D. Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1989 Nov 5;264(31):18662–18666. [PubMed] [Google Scholar]
  40. Peters K. A., Demaille J. G., Fischer E. H. Adenosine 3':5'-monophosphate dependent protein kinase from bovine heart. Characterization of the catalytic subunit. Biochemistry. 1977 Dec 27;16(26):5691–5697. doi: 10.1021/bi00645a007. [DOI] [PubMed] [Google Scholar]
  41. Qi M., Zhuo M., Skålhegg B. S., Brandon E. P., Kandel E. R., McKnight G. S., Idzerda R. L. Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1571–1576. doi: 10.1073/pnas.93.4.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Robinson A. B., Rudd C. J. Deamidation of glutaminyl and asparaginyl residues in peptides and proteins. Curr Top Cell Regul. 1974;8(0):247–295. doi: 10.1016/b978-0-12-152808-9.50013-4. [DOI] [PubMed] [Google Scholar]
  43. Roepstorff P., Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984 Nov;11(11):601–601. doi: 10.1002/bms.1200111109. [DOI] [PubMed] [Google Scholar]
  44. Schmidt G., Sehr P., Wilm M., Selzer J., Mann M., Aktories K. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature. 1997 Jun 12;387(6634):725–729. doi: 10.1038/42735. [DOI] [PubMed] [Google Scholar]
  45. Scott J. D. Cyclic nucleotide-dependent protein kinases. Pharmacol Ther. 1991;50(1):123–145. doi: 10.1016/0163-7258(91)90075-w. [DOI] [PubMed] [Google Scholar]
  46. Shoji S., Parmelee D. C., Wade R. D., Kumar S., Ericsson L. H., Walsh K. A., Neurath H., Long G. L., Demaille J. G., Fischer E. H. Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1981 Feb;78(2):848–851. doi: 10.1073/pnas.78.2.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Showers M. O., Maurer R. A. A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1986 Dec 15;261(35):16288–16291. [PubMed] [Google Scholar]
  48. Stephenson R. C., Clarke S. Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J Biol Chem. 1989 Apr 15;264(11):6164–6170. [PubMed] [Google Scholar]
  49. Stewart A. E., Arfin S. M., Bradshaw R. A. Protein NH2-terminal asparagine deamidase. Isolation and characterization of a new enzyme. J Biol Chem. 1994 Sep 23;269(38):23509–23517. [PubMed] [Google Scholar]
  50. Szendrei G. I., Fabian H., Mantsch H. H., Lovas S., Nyéki O., Schön I., Otvos L., Jr Aspartate-bond isomerization affects the major conformations of synthetic peptides. Eur J Biochem. 1994 Dec 15;226(3):917–924. doi: 10.1111/j.1432-1033.1994.t01-1-00917.x. [DOI] [PubMed] [Google Scholar]
  51. Toner-Webb J., van Patten S. M., Walsh D. A., Taylor S. S. Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1992 Dec 15;267(35):25174–25180. [PubMed] [Google Scholar]
  52. Tyler-Cross R., Schirch V. Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. J Biol Chem. 1991 Nov 25;266(33):22549–22556. [PubMed] [Google Scholar]
  53. Uhler M. D., Carmichael D. F., Lee D. C., Chrivia J. C., Krebs E. G., McKnight G. S. Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1300–1304. doi: 10.1073/pnas.83.5.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Uhler M. D., Chrivia J. C., McKnight G. S. Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1986 Nov 25;261(33):15360–15363. [PubMed] [Google Scholar]
  55. Van Patten S. M., Fletcher W. H., Walsh D. A. The inhibitor protein of the cAMP-dependent protein kinase-catalytic subunit interaction. Parameters of complex formation. J Biol Chem. 1986 Apr 25;261(12):5514–5523. [PubMed] [Google Scholar]
  56. Van Patten S. M., Hotz A., Kinzel V., Walsh D. A. The inhibitor protein of the cyclic AMP-dependent protein kinase-catalytic subunit interaction. Composition of multiple complexes. Biochem J. 1988 Dec 15;256(3):785–789. doi: 10.1042/bj2560785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Walsh D. A., Van Patten S. M. Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J. 1994 Dec;8(15):1227–1236. doi: 10.1096/fasebj.8.15.8001734. [DOI] [PubMed] [Google Scholar]
  58. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  59. Wiemann S., Kinzel V., Pyerin W. Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase. Biochim Biophys Acta. 1992 Nov 15;1171(1):93–96. doi: 10.1016/0167-4781(92)90144-o. [DOI] [PubMed] [Google Scholar]
  60. Wiemann S., Kinzel V., Pyerin W. Isoform C beta 2, an unusual form of the bovine catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1991 Mar 15;266(8):5140–5146. [PubMed] [Google Scholar]
  61. Wiemann S., Voss H., Kinzel V., Pyerin W. Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts. Biochim Biophys Acta. 1991 Jun 13;1089(2):254–256. doi: 10.1016/0167-4781(91)90018-h. [DOI] [PubMed] [Google Scholar]
  62. Wilcox C., Hu J. S., Olson E. N. Acylation of proteins with myristic acid occurs cotranslationally. Science. 1987 Nov 27;238(4831):1275–1278. doi: 10.1126/science.3685978. [DOI] [PubMed] [Google Scholar]
  63. Wright H. T. Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Crit Rev Biochem Mol Biol. 1991;26(1):1–52. doi: 10.3109/10409239109081719. [DOI] [PubMed] [Google Scholar]
  64. Yonemoto W., Garrod S. M., Bell S. M., Taylor S. S. Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1993 Sep 5;268(25):18626–18632. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES