Assaying protein kinase activity.
Protein kinases, encoded by approx 2% of eukaryotic genes, represent one of the major classes of cell-regulatory molecules. Assessment of the catalytic activity of a specific protein kinase can be an important step in elucidating signal-transduction pathways that affect cell behavior. As an example of approaches taken to measure protein kinase activity, this chapter presents methods useful for determination of the activity of the oncogenic protein-tyrosine kinase v-Src. Included are protocols for heterologous expression of the kinase in yeast Saccharomyces cerevisiae, immunoaffinity purification from yeast cell lysates, kinase reactions using incorporation of 32P into peptide substrates, and quantifying protein kinase activity. The Notes section discusses alternative approaches for assaying the activity of Src recovered from vertebrate cells and it gives recommendations for assaying the activity of the other protein kinases with respect to the substrate specifity and the composition of kinase reaction buffer.
Brábek
Jan
J
Department of Cell and Developmental Biology, Vanderbuilt University School of Medicine, Nashville, TN, USA.
Hanks
Steven K
SK
D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008722:Methods; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011494:Protein Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015398:Signal Transduction
D018076:DNA, Complementary; D009842:Oligopeptides; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)