Approaches for Monitoring Signal Transduction Changes in Normal and Cancer Cells

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-³² P 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.