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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)

Abstract

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 

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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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