Approaches for Monitoring Signal Transduction Changes in Normal and Cancer Cells

  • Paul Dent
  • Philip B. Hylemon
  • Steven Grant
  • Paul B. Fisher
Part of the Methods in Molecularbiology™ book series (MIMB, volume 383)


This chapter will describe methods to assess the activities of protein kinases. Initial studies in the 1950s and 1960s in the field of glucose metabolism examined the activities of several highly specific protein and carbohydrate kinases in cell lysates or isolated cell fractions. As more protein kinases were discovered in the 1980s and 1990s, coupled with the development of immunoprecipitating antibodies, in vitro kinase assays of isolated kinase proteins using γ-32P ATP became a standard procedure. In the 1990s, antibodies were developed that recognize specific sites of regulatory phosphorylation on a variety of protein kinases (phospho-specific antibodies), which have been used to assess kinase activity indirectly through immunoblotting. In this chapter, Methodologies to perform immune complex protein kinase assays and immunoblotting using phospho-specific antibodies against regulatory sites of phosphorylation in protein kinases will be described. The strengths and weaknesses of each approach in determining protein kinase activity will also be discussed.

Key Words

Kinase phosphorylation phosphor-specific immunoprecipitation ERK1/2 MAP kinase JNK1/2 p38 AKT Raf-1 B-Raf cdk 


  1. 1.
    Auer, K. L., Park, J. S., Seth, P., et al. (1998) Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice. Biochem. J. 336, 551–560.PubMedGoogle Scholar
  2. 2.
    Auer, K. L., Contessa, J., Brenz-Verca, S., et al. (1998) The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes. Mol. Biol. Cell 9, 561–573.PubMedGoogle Scholar
  3. 3.
    Carter, S., Auer, K. L., Reardon, D. B., et al. (1998) Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells. Oncogene 16, 2787–2796.CrossRefPubMedGoogle Scholar
  4. 4.
    Contessa, J. N., Hampton, J., Lammering, G., et al. (2002) Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells. Oncogene 21, 4032–4041.CrossRefPubMedGoogle Scholar
  5. 5.
    Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789.CrossRefPubMedGoogle Scholar
  6. 6.
    Cross, D. A., Watt, P. W., Shaw, M., et al. (1997) Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. FEBS Lett. 406, 211–215.CrossRefPubMedGoogle Scholar
  7. 7.
    Dent, P., Haser, W., Haystead, T. A., Vincent, L. A., Roberts, T. M., and Sturgill, T. W. (1992) Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science 257, 1404–1407.CrossRefPubMedGoogle Scholar
  8. 8.
    Dent, P., Chow, Y. H., Wu, J., Morrison, D. K., Jove, R., and Sturgill, T. W. (1994) Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases. Biochem. J. 303, 105–112.PubMedGoogle Scholar
  9. 9.
    Dent, P., Jelinek, T., Morrison, D. K., Weber, M. J., and Sturgill, T. W. (1995) Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases. Science 268, 1902–1906.CrossRefPubMedGoogle Scholar
  10. 10.
    Dent, P., Reardon, D. B., Morrison, D. K., and Sturgill, T. W. (1995) Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro. Mol. Cell. Biol. 15, 4125–4135.PubMedGoogle Scholar
  11. 11.
    Dent, P., Reardon, D. B., Park, J. S., et al. (1999) Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. Mol. Biol. Cell 10, 2493–2506.PubMedGoogle Scholar
  12. 12.
    Gupta, S., Natarajan, R., Payne, S. G., et al. (2004) Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor activation in primary hepatocytes. Role of acidic sphingomyelinase-mediated ceramide generation in FAS receptor activation. J. Biol. Chem. 279, 5821–5828.CrossRefPubMedGoogle Scholar
  13. 13.
    Gupta, S., Stravitz, R. T., Dent, P., and Hylemon, P. B. (2001) Down-regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun N-terminal kinase pathway. J. Biol. Chem. 276, 15,816–15,822.CrossRefPubMedGoogle Scholar
  14. 14.
    Han, S. I., Studer, E., Gupta, S., et al. (2004) Bile acids enhance the activity of the insulin receptor and glycogen synthase in primary rodent hepatocytes. Hepatology 39, 456–463.CrossRefPubMedGoogle Scholar
  15. 15.
    McCubrey, J. A., Steelman, L. S., Hoyle, P. E., et al. (1998) Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. Leukemia 12, 1903–1929.CrossRefPubMedGoogle Scholar
  16. 16.
    Park, J. S., Carter, S., Reardon, D. B., Schmidt-Ullrich, R., Dent, P., and Fisher, P. B. (1999) Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells. Mol. Biol. Cell 10, 4231–4246.PubMedGoogle Scholar
  17. 17.
    Qiao, L., Studer, E., Leach, K., et al. (2001) Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase signaling module enhances DCA-induced apoptosis Mol. Biol. Cell 12, 2629–2645.PubMedGoogle Scholar
  18. 18.
    Qiao, L., Leach, K., McKinstry, R., et al. (2001) Hepatitis B virus X protein increases expression of p21(Cip-1/WAF1/MDA6) and p27(Kip-1) in primary mouse hepatocytes, leading to reduced cell cycle progression. Hepatology 34, 906–917.CrossRefPubMedGoogle Scholar
  19. 19.
    Qiao, L., Han, S. I., Fang, Y., et al. (2003) Bile acid regulation of C/EBP beta, CREB, and c-Jun function, via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates the apoptotic response of hepatocytes. Mol. Cell Biol. 23, 3052–3066.CrossRefPubMedGoogle Scholar
  20. 20.
    Tombes, R. M., Auer, K. L., Mikkelsen, R., et al. (1998) The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. Biochem. J. 330, 1451–1460.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Paul Dent
    • 1
  • Philip B. Hylemon
    • 2
  • Steven Grant
    • 3
  • Paul B. Fisher
    • 4
    • 5
    • 6
    • 7
  1. 1.Department of BiochemistryVirginia Commonwealth UniversityRichmond
  2. 2.Department of Microbiology and ImmunologyVirginia Commonwealth UniversityRichmond
  3. 3.Department of MedicineVirginia Commonwealth UniversityRichmond
  4. 4.Herbert Irving Comprehensive Cancer CenterCollege of Physicians and Surgeons, Columbia University Medical CenterNew York
  5. 5.Department of PathologyCollege of Physicians and Surgeons, Columbia University Medical CenterNew York
  6. 6.Department of NeurosurgeryCollege of Physicians and Surgeons, Columbia University Medical CenterNew York
  7. 7.Department of UrologyCollege of Physicians and Surgeons, Columbia University Medical CenterNew York

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