Skip to main content
SpringerLink
Log in

Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into Mammalian Cell Proteins

A Useful Tool for Studying Protein Prenylation

  • Protocol
Protein Lipidation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 116))

  • 453 Accesses

Abstract

Prenylated proteins comprise a diverse family of proteins that are post-translationally modified by either a farnesyl group or one or more geranylgeranyl groups (1–3). Recent studies suggest that members of this family are involved in a number of cellular processes, including cell signaling (4–6), differentiation (7–9), proliferation (10–12), cytoskeletal dynamics (13–15), and endocytic and exocytic transport (4,16,17). The authors’ studies have focused on the role of prenylated proteins in the cell cycle (18). Exposure of cultured cells to competitive inhibitors (statins) of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase not only blocks the biosynthesis of mevalonic acid (MVA), the biosynthetic precursor of both farnesyl and geranylgeranyl groups, but pleiotropically inhibits DNA replication and cell-cycle progression (10,18–20). Both phenomena can be prevented by the addition of exogenous MVA (10,18,19). The authors have observed that all-trans-geranylgeraniol (GGOH) and, in a few cases, all-trans-farnesol (FOH) can prevent the statin-induced inhibition of DNA synthesis (21). In an effort to understand the biochemical basis of these effects, the authors have developed methods for the labeling and two-dimensional gel analysis of prenylated proteins that should be widely applicable.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Zhang, F. L. and Casey, P. J. (1996) Protein Prenylation: Molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65, 241–269.

    Article  PubMed  CAS  Google Scholar 

  2. Glomset, J. A., Gelb, M. H., and Farnsworth, C. C. (1990) Prenyl proteins in eukariotic cells: a new type of membrane anchor. Trends Biochem. Sci. 15, 139–142.

    Article  PubMed  CAS  Google Scholar 

  3. Maltese, W. A. (1990) Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J. 4, 3319–3328.

    PubMed  CAS  Google Scholar 

  4. Glomset, J. A. and Farnsworth, C. C. (1994) Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes. Annu. Rev. Cell Biol. 10, 181–205.

    Article  PubMed  CAS  Google Scholar 

  5. Casey, P. J., Moomaw, J. F. Zhang, F. L., Higgins, J. B., and Thissen, J. A. (1994) Prenylation and G protein signaling, in Recent Progress in Hormone Research, Academic, New York.

    Google Scholar 

  6. Inglese, J., Koch, W. J., Touhara, K., and Lefkowitz, R. J. (1995) Gβγ interactions with pH domains and Ras-MPK signaling pathways. Trends Biochem. Sci. 20, 151–156.

    Article  PubMed  CAS  Google Scholar 

  7. Marshall, M. S. (1995) Ras target proteins in eukaryotic cells. FASEB J. 9, 1311–1318.

    PubMed  CAS  Google Scholar 

  8. Kato, K., Cox, A. D., Hisaka, M. M., Graham, S. M., Buss, J. E., and Der, C. J. (1992) Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc. Natl. Acad. Sci. USA 89, 6403–6407.

    Article  PubMed  CAS  Google Scholar 

  9. Boguski, M. S. and McCormick, F. (1993) Proteins regulating Ras and its relatives. Nature 365, 643–654.

    Article  Google Scholar 

  10. Jakobisiak, M., Bruno, S., Skiersky, J. S., and Darzynkiewicz, Z. (1991) Cell cycle-specific effects of lovastatin. Proc. Natl. Acad. Sci. USA 88, 3628–3632.

    Article  PubMed  CAS  Google Scholar 

  11. Taylor, S. J. and Shalloway, D. (1996) Cell cycle-dependent activation of Ras. Curr. Biol. 6, 1621–1627.

    Article  PubMed  CAS  Google Scholar 

  12. Olson, M. F., Ashworth, A., and Hall, A. (1995) An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269, 1270–1272.

    Article  PubMed  CAS  Google Scholar 

  13. Fenton, R. G., Kung, H., Longo, D. L., and Smith, M. R. (1992) Regulation of intracellular actin polymerization by prenylated cellular proteins. J. Cell Biol. 117, 347–356.

    Article  PubMed  CAS  Google Scholar 

  14. Pittler, S. J., Fliester, S. J., Fisher, P. L., Keller, R. K., and Rapp, L. M. In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure. J. Cell Biol. 130, 431–439.

    Google Scholar 

  15. Tapon, N. and Hall, A. (1997) Rho, Rac, and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Curr. Opin. Cell Biol. 9, 86–92.

    Article  PubMed  CAS  Google Scholar 

  16. Zerial, M. and Stenmark, H. (1993) Rab GTPases in vesicular transport. Curr. Opin. Cell Biol. 5, 613–620.

    Article  PubMed  CAS  Google Scholar 

  17. Novick, P. and Brennwald, P. (1993) Friends and family: the role of the Rab GTPases in vesicular traffic. Cell 75, 597–601.

    Article  PubMed  CAS  Google Scholar 

  18. Raiteri, M., Arnaboldi, L., McGeady, P., Gelb, M. H., Verri, D., Tagliabue, C., Quarato, P., et al. (1997) Pharmacological control of the mevalonate pathway: effect on smooth muscle cell proliferation. J. Pharmacol. Exp. Ther. 281, 1144–1153.

    PubMed  CAS  Google Scholar 

  19. Habenicht, A. J. R., Glomset, J. A., and Ross, R. (1980) Relation of cholesterol and mevalonic acid to the cell cycle in smooth muscle and Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J. Biol. Chem. 255, 5134–5140.

    PubMed  CAS  Google Scholar 

  20. Goldstein, J. L. and Brown, M. S. (1990) Regulation of the mevalonate pathway. Nature 343, 425–430.

    Article  PubMed  CAS  Google Scholar 

  21. Corsini, A., Mazzotti, M., Raiteri, M., Soma, M. R., Gabbiani, G., Fumagalli, R., and Paoletti, R. (1993) Relationship between mevalonate pathway and arterial myocyte proliferation: in vitro studies with inhibitor of HMG-CoA reductase. Atherosclerosis 101, 117–125.

    Article  PubMed  CAS  Google Scholar 

  22. Crick, D. C., Waechter, C. J., and Andres, D. A. (1994) Utilization of geranyl-geraniol for protein isoprenylation in C6 glial cells. Biochem. Biophys. Res. Commun. 205, 955–961.

    Article  PubMed  CAS  Google Scholar 

  23. Crick, D. C., Andres, D. A., and Waechter, C. J. (1995) Farnesol is utilized for protein isoprenylation and the biosynthesis of cholesterol in mammalian cells. Biochem. Biophys. Res. Commun. 211, 590–599.

    Article  PubMed  CAS  Google Scholar 

  24. Danesi, R., McLellan, C. A., and Myers, C. E. (1995) Specific labeling of isoprenylated proteins: application to study inhibitors of the posttranslational farnesylation and geranylgeranylation. Biochem. Biophys. Res. Commun. 206, 637–643.

    Article  PubMed  CAS  Google Scholar 

  25. Shin-ichi, O., Watanabe, M., and Nishino, T. (1996) Identification and characterization of geranylgeraniol kinase and geranylgeranyl phosphate kinase from the Archebacterium Sulfolobus acidocaldarius. J. Biochem. 119, 541–547.

    Google Scholar 

  26. Westfall, D., Aboushadi, N., Shackelford, J. E., and Krisans, S. (1997) Metabolism of farnesol: phosphorylation of farnesol by rat liver microsomial and peroxisomal fractions. Biochem. Biophys. Res. Commun. 230, 562–568.

    Article  PubMed  CAS  Google Scholar 

  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.

    PubMed  CAS  Google Scholar 

  28. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  29. Görg, A., Postel, W., and Günther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546.

    Article  PubMed  Google Scholar 

  30. Jungblutt, P., Thiede, B., Simny-Arndt, U. Müller, E.-C., Wittmann-Liebold, B., and Otto, A. (1997) Resolution power of two-dimensional electrophoresis and identification of proteins from gels. Electrophoresis 17, 839–847.

    Article  Google Scholar 

  31. O’Farrell, P. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.

    PubMed  Google Scholar 

  32. Patton, W. F., Pluskal, M. G., Skea, W. M., Buecker, J. L., Lopez, M. F., Zimmermann, R., Lelanger, L. M., and Hatch, P. D. (1990) Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern repoducibility and polypeptide resolution. BioTech. 8, 518–529.

    CAS  Google Scholar 

  33. Lopez, M. F. and Patton, W. F. (1990) Reproducibility of polypeptide spot positions in two-dimensional gels run using carrier ampholytes in the isolelectric focusing dimension. Electrophoresis 18, 338–343.

    Article  Google Scholar 

  34. Görg, A., Günther, B., Obermaier, C., Posch, A., and Weiss, W. (1995) Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems. Electrophoresis 16, 1079–1086.

    Article  PubMed  Google Scholar 

  35. Schmidt, R. A., Schneider, C. J., and Glomset, J. A. (1984) Evidence for post-translational incorporation of a product of mavalonic acid into Swiss 3T3 cell proteins. J. Biol. Chem. 259, 10,175–10,180.

    PubMed  CAS  Google Scholar 

  36. Sanchez, J.-C., Rouge, V., Pisteur, M., Ravier, F., Tonella, L., Moosmayer, M., Wilkins, M. R., and Hochstrasser, D. F. (1997) Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327.

    Article  PubMed  CAS  Google Scholar 

  37. Huber, L. A., Ullrich, O., Takai, Y., Lütcke, A., Dupree, P., Olkkonen, et al. (1994) Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis. Proc. Natl. Acad. Sci. USA 91, 7874–7878.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of Washington, Seattle, WA

    Alberto Corsini, Christopher C. Farnsworth, Paul McGeady & John A. Glomset

  2. Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA

    Michael H. Gelb

Authors
  1. Alberto Corsini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher C. Farnsworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul McGeady
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael H. Gelb
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John A. Glomset
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Washington, Seattle, WA

    Michael H. Gelb

Rights and permissions

Reprints and permissions

Copyright information

© 1998 Humana Press Inc.

About this protocol

Cite this protocol

Corsini, A., Farnsworth, C.C., McGeady, P., Gelb, M.H., Glomset, J.A. (1998). Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into Mammalian Cell Proteins. In: Gelb, M.H. (eds) Protein Lipidation Protocols. Methods in Molecular Biology, vol 116. Humana Press. https://doi.org/10.1385/1-59259-264-3:125

Download citation

  • DOI: https://doi.org/10.1385/1-59259-264-3:125

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-534-8

  • Online ISBN: 978-1-59259-264-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation