Pharmacologic inhibitors of MKK1 and MKK2.

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