COMPETITIVE-INHIBITION OF MAP KINASE ACTIVATION BY A PEPTIDE REPRESENTING THE ALPHA(C) HELIX OF ERK

On the basis of the crystal structure of the MEK substrate ERK, we hav e synthesized a 15 amino acid peptide representing the alpha(c) helix of human ERK1. We find this peptide to be an inhibitor of ERK phosphor ylation by its upstream activator MEK. Circular dichroic spectroscopy indicates that the peptide has little secondary structure in aqueous b uffer, but can readily adopt an alpha-helical structure in aprotic sol vent. Steady-state kinetic analysis indicates that the peptide serves as a competitive inhibitor of ERK binding to MEK, with a dissociation constant, K-i, of 0.84 mu M. Together with ATP-competitive inhibitors of MEK, we have used this peptide to define the kinetic mechanism of M EK catalysis. These studies reveal that MEK operates through a bi-bi r andom-ordered sequential mechanism. The synthetic peptide inhibits als o the phosphorylation of p38 and ERK by the upstream activator MKK3, b ut is at least 3-fold less potent as an inhibitor of SEK activation of JNK1. Interestingly, the peptide also showed some ability to inhibit ERK-mediated phosphorylation of myelin basic protein, but was inactive as an inhibitor of the unrelated kinases Raf, Abl, and PKA. These res ults imply that the alpha(c) helix is an important locus of interactio n for the formation of a MEK-ERK complex. The alpha(c) helix cannot, h owever, be the sole determinant of activator selectivity among the MAP kinases. Molecules designed to target the alpha(c) helix binding pock et of MAP kinase activators may provide a novel means of inhibiting th ese signal transducers.