THE MPM-2 ANTIBODY INHIBITS MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITYBY BINDING TO AN EPITOPE CONTAINING PHOSPHOTHREONINE-183

Mitogen-activated protein (MAP) kinases are a family of serine/threoni ne kinases implicated in the control of cell proliferation and differe ntiation. We have found that activated p42(mapk) is a target for the p hosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42(mapk) Binding of the MPM-2 antibody to active p42(mapk) in vitro results in a decrease in p42(mapk) enzymatic activity. The MPM-2 phosphoepitope can be generate d in vitro on bacterially expressed p42(mapk) by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42(mapk) proteins mutated in their regulatory sites shows that phosph orylated Thr-183 is essential for the binding of the MPM-2 antibody. M PM-2 binding to Thr-183 is affected by the amino acid present in the o ther regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalan ine results in strong binding of the MPM-2 antibody, whereas substitut ion with glutamic acid substantially diminishes MPM-2 antibody binding . The MPM-2 phosphoepitope antibody recognizes an amino acid domain in corporating the regulatory phosphothreonine on activated p42(mapk) in eggs during meiosis and in mammalian cultured cells during the G(0) to G(1) transition.