A SINGLE AMINO-ACID SUBSTITUTION MAKES ERK2 SUSCEPTIBLE TO PYRIDINYL IMIDAZOLE INHIBITORS OF P38 MAP KINASE

Mitogen-activated protein (MAP) kinases are serine/threonine kinases t hat mediate intracellular signal transduction pathways. Pyridinyl imid azole compounds block pro-inflammatory cytokine production and are spe cific p38 kinase inhibitors. ERK2 is related to p38 in sequence and st ructure, but is not inhibited by pyridinyl imidazole inhibitors. Cryst al structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a sin gle residue difference at threonine 106 between p38 and other MAP kina ses is sufficient to confer selectivity of pyridinyl imidazoles. We ha ve changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues . The single residue change Q105A in ERK2 enhances the binding of SB20 2190 at least 25,000-fold compared to wild-type ERK2. We report enzyma tic analyses of wild-type ERK2 and the mutant proteins, and the crysta l structure of a pyridinyl imidazole, SB203580, bound to an ERK2 penta mutant, I103L, Q105T, D106H, E109G, T110A. These ATP binding site subs titutions induce low nanomolar sensitivity to pyridinyl imidazoles. Fu rthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.