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Pharmacologic Inhibitors of MKK1 and MKK2

Methods in Enzymology, ISSN: 0076-6879, Vol: 332, Page: 417-431
2001
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Article Description

This chapter describes the methods to measure drug inhibition of MAP kinase kinases (MKK) activity and activation using purified recombinant proteins in vitro. It also discusses the drug effects on extracellular signal regulated kinases (ERK) activation in intact mammalian cells. Activation of MKK or ERK in response to cell stimulation involves phosphorylation at residues located within the activation lip of each kinase. One approach is to compare the effects using more than one drug and to compare the dose dependence of response inhibition with that of kinase inhibition, where discrepancies in IC 50 may rule out regulatory relationships. A second approach is to test whether overexpression of constitutively active mutant MKK1 modulates cellular targets in a manner that opposes the effects of MKK1/2 inhibitors. Opposing behavior under conditions in which the pathway is turned on versus off provides confirmation of regulation by MKK-ERK. A third approach—as yet unexplored with MKK1/2—is to identify mutations that render it impervious to inhibitors and then examine the effects of overexpressing such mutants on responses that are normally blocked by inhibitor. Mutant overexpression should provide a dominant mechanism, bypassing inhibitor action of endogenous kinase.

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