title: Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase.
abstract: Previous work on the responses of mitogen-activated protein (MAP) kinase cascade components in a Xenopus oocyte extract system demonstrated that p42 MAP kinase (MAPK) exhibits a sharp, sigmoidal stimulus/response curve, rather than a more typical hyperbolic curve. One plausible explanation for this behavior requires the assumption that MAP kinase kinase (MAPKK) carries out its dual phosphorylation of p42 MAPK by a distributive mechanism, where MAPKK dissociates from MAPK between the first and second phosphorylations, rather than a processive mechanism, where MAPKK carries out both phosphorylations before dissociating. Here we have investigated the mechanism through which a constitutively active form of human MAPKK-1 (denoted MAPKK-1 R4F or MAPKK-1*) phosphorylates Xenopus p42 MAPK in vitro. We found that the amount of monophosphorylated MAPK formed during the phosphorylation reaction exceeded the amount of MAPKK-1* present, which would not be possible if the phosphorylation occurred exclusively by a processive mechanism. The monophosphorylated MAPK was phosphorylated predominantly on tyrosine, but a small proportion was phosphorylated on threonine, indicating that the first phosphorylation is usually, but not invariably, the tyrosine phosphorylation. We also found that the rate at which pulse-labeled monophosphorylated MAPK became bisphosphorylated depended on the MAPKK-1* concentration, behavior that is predicted by the distributive model but incompatible with the processive model. These findings indicate that MAPKK-1* phosphorylates p42 MAPK by a two-collision, distributive mechanism rather than a single-collision, processive mechanism, and provide a mechanistic basis for understanding how MAP kinase can convert graded inputs into switch-like outputs.
authors.affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332, USA. ferrell@cmgm.stanford.edu
authors.forename: J E
authors.initials: JE
authors.lastname: Ferrell
authors.forename: R R
authors.initials: RR
authors.lastname: Bhatt
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008956:Models, Chemical; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014981:Xenopus
chemical_list: D013912:Threonine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D014357:Trypsin