Using protein kinase and protein phosphatase inhibitors to dissect protein phosphorylation pathways.
It is often desirable to attempt to measure and to modulate the activity of the protein kinases and phosphatases responsible for a given phenotypic change. The protocols in this unit describe how to assay a variety of protein kinase activities in complex mixtures, such as whole cell lysates, in order to screen compounds as potential inhibitors of these activities. The effects of known or potential inhibitors can be evaluated by monitoring phenotypic changes in intact cells following treatment with the inhibitor. The activities of the major serine/threonine-specific protein phosphatases in most mammalian cells can be distinguished using a selective inhibitor, okadaic acid. Support protocols describe preparation of cell extracts needed for the kinase assays, and of (32)P-labeled phosphorylase used in the inhibition assays.
Karaszkiewicz
James W
JW
Kirkegaard and Perry Laboratories, Gaithersburg, Maryland.
Henrich
Curtis J
CJ
Life Technologies, Inc, Gaithersburg, Maryland.
D000818:Animals; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects* / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D004791:Enzyme Inhibitors; D011505:Protein-Tyrosine Kinases; D010749:Phosphoprotein Phosphatases