CONVERSION OF SB 203580-INSENSITIVE MBP KINASE FAMILY MEMBERS TO DRUG-SENSITIVE FORMS BY A SINGLE AMINO-ACID SUBSTITUTION

Background: Specific inhibitors of protein kinases have great therapeu tic potential, but the molecular basis underlying their specificity is only poorly understood, We have investigated the drug SE 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-a ctivated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38 beta 2 but no t other mitogen-activated protein kinase family members. Like inhibito rs of other protein kinases, SE 203580 binds in the ATP-binding pocket of SAPK2a/p38. Results: The SAP kinases SAPK1 gamma/JNK1, SAPK3 and S APK4 are not inhibited by SE 203580, because they have methionine in t he position equivalent to Thr106 in the ATP-binding region of SAPK2a/p 30 and SAPK2b/p38 beta 2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGF beta receptors, we have establi shed that for a protein kinase to be inhibited by SE 203580, the sidec hain of this residue must be no larger than that of threonine, Sensiti vity to inhibition by SE 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I T GF beta receptor, which has serine at the position equivalent to Thr10 6 of SAPK2a/p38 and SAPK2b/p38 beta 2, is inhibited by SE 203580. Conc lusions: These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the prese nce of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhib itor.