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. 2003 Jul 1;373(Pt 1):91–99. doi: 10.1042/BJ20021598

Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs.

Amanda S Varner 1, Charles E Ducker 1, Zuping Xia 1, Yan Zhuang 1, Mackenzie L De Vos 1, Charles D Smith 1
PMCID: PMC1223475  PMID: 12670300

Abstract

The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity towards the myristoylated substrate peptide but not the farnesylated peptide, while ras -transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the K (m) values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the K (m) for MyrGCK(NBD) was 5-fold lower than the K (m) for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC(50) values of approx. 4 and 1.3 microM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N -ethylmaleimide and thermal denaturation. Overall, these data demonstrate that the enzymic palmitoylation of farnesyl- and myristoyl-containing peptide substrates can be differentiated, suggesting that multiple motif-specific PATs exist.

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Selected References

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

  1. Abraham K. M., Levin S. D., Marth J. D., Forbush K. A., Perlmutter R. M. Thymic tumorigenesis induced by overexpression of p56lck. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3977–3981. doi: 10.1073/pnas.88.9.3977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adnane J., Jackson R. J., Nicosia S. V., Cantor A. B., Pledger W. J., Sebti S. M. Loss of p21WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/knockout mammary cancer model. Oncogene. 2000 Nov 9;19(47):5338–5347. doi: 10.1038/sj.onc.1203956. [DOI] [PubMed] [Google Scholar]
  3. Apolloni A., Prior I. A., Lindsay M., Parton R. G., Hancock J. F. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol. 2000 Apr;20(7):2475–2487. doi: 10.1128/mcb.20.7.2475-2487.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barbacid M. ras oncogenes: their role in neoplasia. Eur J Clin Invest. 1990 Jun;20(3):225–235. doi: 10.1111/j.1365-2362.1990.tb01848.x. [DOI] [PubMed] [Google Scholar]
  5. Bartels D. J., Mitchell D. A., Dong X., Deschenes R. J. Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Oct;19(10):6775–6787. doi: 10.1128/mcb.19.10.6775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bañ M. C., Jackson C. S., Magee A. I. Pseudo-enzymatic S-acylation of a myristoylated yes protein tyrosine kinase peptide in vitro may reflect non-enzymatic S-acylation in vivo. Biochem J. 1998 Mar 1;330(Pt 2):723–731. doi: 10.1042/bj3300723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bhatnagar R. S., Gordon J. I. Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle. Trends Cell Biol. 1997 Jan;7(1):14–20. doi: 10.1016/S0962-8924(97)10044-7. [DOI] [PubMed] [Google Scholar]
  8. Bizzozero O. A., Bixler H. A., Pastuszyn A. Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters. Biochim Biophys Acta. 2001 Feb 9;1545(1-2):278–288. doi: 10.1016/s0167-4838(00)00291-0. [DOI] [PubMed] [Google Scholar]
  9. Blanpain C., Wittamer V., Vanderwinden J. M., Boom A., Renneboog B., Lee B., Le Poul E., El Asmar L., Govaerts C., Vassart G. Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways. J Biol Chem. 2001 Apr 25;276(26):23795–23804. doi: 10.1074/jbc.M100583200. [DOI] [PubMed] [Google Scholar]
  10. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  11. Boutin J. A. Myristoylation. Cell Signal. 1997 Jan;9(1):15–35. doi: 10.1016/s0898-6568(96)00100-3. [DOI] [PubMed] [Google Scholar]
  12. Bouvier M., Loisel T. P., Hebert T. Dynamic regulation of G-protein coupled receptor palmitoylation: potential role in receptor function. Biochem Soc Trans. 1995 Aug;23(3):577–581. doi: 10.1042/bst0230577. [DOI] [PubMed] [Google Scholar]
  13. Böhlen P., Stein S., Dairman W., Udenfriend S. Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophys. 1973 Mar;155(1):213–220. doi: 10.1016/s0003-9861(73)80023-2. [DOI] [PubMed] [Google Scholar]
  14. Camp L. A., Hofmann S. L. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J Biol Chem. 1993 Oct 25;268(30):22566–22574. [PubMed] [Google Scholar]
  15. Camp L. A., Verkruyse L. A., Afendis S. J., Slaughter C. A., Hofmann S. L. Molecular cloning and expression of palmitoyl-protein thioesterase. J Biol Chem. 1994 Sep 16;269(37):23212–23219. [PubMed] [Google Scholar]
  16. Casey P. J. Protein lipidation in cell signaling. Science. 1995 Apr 14;268(5208):221–225. doi: 10.1126/science.7716512. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Coleman R. A., Rao P., Fogelsong R. J., Bardes E. S. 2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes. Biochim Biophys Acta. 1992 Apr 23;1125(2):203–209. doi: 10.1016/0005-2760(92)90046-x. [DOI] [PubMed] [Google Scholar]
  19. Creaser Steffen P., Peterson Blake R. Sensitive and rapid analysis of protein palmitoylation with a synthetic cell-permeable mimic of SRC oncoproteins. J Am Chem Soc. 2002 Mar 20;124(11):2444–2445. doi: 10.1021/ja017671x. [DOI] [PubMed] [Google Scholar]
  20. Das A. K., Dasgupta B., Bhattacharya R., Basu J. Purification and biochemical characterization of a protein-palmitoyl acyltransferase from human erythrocytes. J Biol Chem. 1997 Apr 25;272(17):11021–11025. doi: 10.1074/jbc.272.17.11021. [DOI] [PubMed] [Google Scholar]
  21. Degtyarev M. Y., Spiegel A. M., Jones T. L. Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin. J Biol Chem. 1993 Nov 15;268(32):23769–23772. [PubMed] [Google Scholar]
  22. Dudler T., Gelb M. H. Palmitoylation of Ha-Ras facilitates membrane binding, activation of downstream effectors, and meiotic maturation in Xenopus oocytes. J Biol Chem. 1996 May 10;271(19):11541–11547. doi: 10.1074/jbc.271.19.11541. [DOI] [PubMed] [Google Scholar]
  23. Dunphy J. T., Greentree W. K., Manahan C. L., Linder M. E. G-protein palmitoyltransferase activity is enriched in plasma membranes. J Biol Chem. 1996 Mar 22;271(12):7154–7159. doi: 10.1074/jbc.271.12.7154. [DOI] [PubMed] [Google Scholar]
  24. Dunphy J. T., Linder M. E. Signalling functions of protein palmitoylation. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):245–261. doi: 10.1016/s0005-2760(98)00130-1. [DOI] [PubMed] [Google Scholar]
  25. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  26. Heuckeroth R. O., Towler D. A., Adams S. P., Glaser L., Gordon J. I. 11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase. J Biol Chem. 1988 Feb 15;263(5):2127–2133. [PubMed] [Google Scholar]
  27. Hightower K. E., Casey P. J., Fierke C. A. Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase. Biochemistry. 2001 Jan 30;40(4):1002–1010. doi: 10.1021/bi002237d. [DOI] [PubMed] [Google Scholar]
  28. Hightower K. E., Huang C. C., Casey P. J., Fierke C. A. H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate. Biochemistry. 1998 Nov 3;37(44):15555–15562. doi: 10.1021/bi981525v. [DOI] [PubMed] [Google Scholar]
  29. Lawrence D. S., Zilfou J. T., Smith C. D. Structure-activity studies of cerulenin analogues as protein palmitoylation inhibitors. J Med Chem. 1999 Dec 2;42(24):4932–4941. doi: 10.1021/jm980591s. [DOI] [PubMed] [Google Scholar]
  30. Liu L., Dudler T., Gelb M. H. Purification of a protein palmitoyltransferase that acts on H-Ras protein and on a C-terminal N-Ras peptide. J Biol Chem. 1996 Sep 20;271(38):23269–23276. doi: 10.1074/jbc.271.38.23269. [DOI] [PubMed] [Google Scholar]
  31. McCabe J. B., Berthiaume L. G. Functional roles for fatty acylated amino-terminal domains in subcellular localization. Mol Biol Cell. 1999 Nov;10(11):3771–3786. doi: 10.1091/mbc.10.11.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Michaelson D., Silletti J., Murphy G., D'Eustachio P., Rush M., Philips M. R. Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol. 2001 Jan 8;152(1):111–126. doi: 10.1083/jcb.152.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Michaelson David, Ahearn Ian, Bergo Martin, Young Stephen, Philips Mark. Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi. Mol Biol Cell. 2002 Sep;13(9):3294–3302. doi: 10.1091/mbc.E02-02-0095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mouillac B., Caron M., Bonin H., Dennis M., Bouvier M. Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells. J Biol Chem. 1992 Oct 25;267(30):21733–21737. [PubMed] [Google Scholar]
  35. Mumby S. M., Kleuss C., Gilman A. G. Receptor regulation of G-protein palmitoylation. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2800–2804. doi: 10.1073/pnas.91.7.2800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mumby S. M. Reversible palmitoylation of signaling proteins. Curr Opin Cell Biol. 1997 Apr;9(2):148–154. doi: 10.1016/s0955-0674(97)80056-7. [DOI] [PubMed] [Google Scholar]
  37. Park H. W., Beese L. S. Protein farnesyltransferase. Curr Opin Struct Biol. 1997 Dec;7(6):873–880. doi: 10.1016/s0959-440x(97)80160-1. [DOI] [PubMed] [Google Scholar]
  38. Poulio J. F., Béliveau R. Palmitoylation of brain capillary proteins. Int J Biochem Cell Biol. 1995 Nov;27(11):1133–1144. doi: 10.1016/1357-2725(95)00095-7. [DOI] [PubMed] [Google Scholar]
  39. Rajala R. V., Datla R. S., Moyana T. N., Kakkar R., Carlsen S. A., Sharma R. K. N-myristoyltransferase. Mol Cell Biochem. 2000 Jan;204(1-2):135–155. doi: 10.1023/a:1007012622030. [DOI] [PubMed] [Google Scholar]
  40. Resh M. D. Regulation of cellular signalling by fatty acid acylation and prenylation of signal transduction proteins. Cell Signal. 1996 Sep;8(6):403–412. doi: 10.1016/s0898-6568(96)00088-5. [DOI] [PubMed] [Google Scholar]
  41. Roskoski R., Jr, Ritchie P. Role of the carboxyterminal residue in peptide binding to protein farnesyltransferase and protein geranylgeranyltransferase. Arch Biochem Biophys. 1998 Aug 15;356(2):167–176. doi: 10.1006/abbi.1998.0768. [DOI] [PubMed] [Google Scholar]
  42. Roth Amy F., Feng Ying, Chen Linyi, Davis Nicholas G. The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. J Cell Biol. 2002 Oct 7;159(1):23–28. doi: 10.1083/jcb.200206120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Singaraja Roshni R., Hadano Shinji, Metzler Martina, Givan Scott, Wellington Cheryl L., Warby Simon, Yanai Anat, Gutekunst Claire-Anne, Leavitt Blair R., Yi Hong. HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis. Hum Mol Genet. 2002 Nov 1;11(23):2815–2828. doi: 10.1093/hmg/11.23.2815. [DOI] [PubMed] [Google Scholar]
  44. Smith C. D., Zilfou J. T., Stratmann K., Patterson G. M., Moore R. E. Welwitindolinone analogues that reverse P-glycoprotein-mediated multiple drug resistance. Mol Pharmacol. 1995 Feb;47(2):241–247. [PubMed] [Google Scholar]
  45. Towler D. A., Adams S. P., Eubanks S. R., Towery D. S., Jackson-Machelski E., Glaser L., Gordon J. I. Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities. J Biol Chem. 1988 Feb 5;263(4):1784–1790. [PubMed] [Google Scholar]
  46. Towler D. A., Adams S. P., Eubanks S. R., Towery D. S., Jackson-Machelski E., Glaser L., Gordon J. I. Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase. Proc Natl Acad Sci U S A. 1987 May;84(9):2708–2712. doi: 10.1073/pnas.84.9.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ueno K., Suzuki Y. p260/270 expressed in embryonic abdominal leg cells of Bombyx mori can transfer palmitate to peptides. J Biol Chem. 1997 May 23;272(21):13519–13526. doi: 10.1074/jbc.272.21.13519. [DOI] [PubMed] [Google Scholar]
  48. Uittenbogaard A., Smart E. J. Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae. J Biol Chem. 2000 Aug 18;275(33):25595–25599. doi: 10.1074/jbc.M003401200. [DOI] [PubMed] [Google Scholar]
  49. Varner Amanda S., De Vos Mackenzie L., Creaser Steffen P., Peterson Blake R., Smith Charles D. A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity. Anal Biochem. 2002 Sep 1;308(1):160–167. doi: 10.1016/s0003-2697(02)00212-9. [DOI] [PubMed] [Google Scholar]
  50. Veit M., Sachs K., Heckelmann M., Maretzki D., Hofmann K. P., Schmidt M. F. Palmitoylation of rhodopsin with S-protein acyltransferase: enzyme catalyzed reaction versus autocatalytic acylation. Biochim Biophys Acta. 1998 Oct 2;1394(1):90–98. doi: 10.1016/s0005-2760(98)00097-6. [DOI] [PubMed] [Google Scholar]
  51. Webb Y., Hermida-Matsumoto L., Resh M. D. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem. 2000 Jan 7;275(1):261–270. doi: 10.1074/jbc.275.1.261. [DOI] [PubMed] [Google Scholar]
  52. Wedegaertner P. B., Bourne H. R. Activation and depalmitoylation of Gs alpha. Cell. 1994 Jul 1;77(7):1063–1070. doi: 10.1016/0092-8674(94)90445-6. [DOI] [PubMed] [Google Scholar]
  53. Wright D. D., Sefton B. M., Kamps M. P. Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. Mol Cell Biol. 1994 Apr;14(4):2429–2437. doi: 10.1128/mcb.14.4.2429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Xia Z., Smith C. D. Efficient synthesis of a fluorescent farnesylated Ras peptide. J Org Chem. 2001 Jul 27;66(15):5241–5244. doi: 10.1021/jo015526w. [DOI] [PubMed] [Google Scholar]
  55. Yu C. L., Jove R., Burakoff S. J. Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase. J Immunol. 1997 Dec 1;159(11):5206–5210. [PubMed] [Google Scholar]
  56. Zhao Lihong, Lobo Sandra, Dong Xiangwen, Ault Addison D., Deschenes Robert J. Erf4p and Erf2p form an endoplasmic reticulum-associated complex involved in the plasma membrane localization of yeast Ras proteins. J Biol Chem. 2002 Oct 11;277(51):49352–49359. doi: 10.1074/jbc.M209760200. [DOI] [PubMed] [Google Scholar]

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