title: Processive phosphorylation of ERK MAP kinase in mammalian cells.
abstract: The mitogen-activated protein (MAP) kinase pathway is comprised of a three-tiered kinase cascade. The distributive kinetic mechanism of two-site MAP kinase phosphorylation inherently generates a nonlinear switch-like response. However, a linear graded response of MAP kinase has also been observed in mammalian cells, and its molecular mechanism remains unclear. To dissect these input-output behaviors, we quantitatively measured the kinetic parameters involved in the MEK (MAPK/ERK kinase)-ERK MAP kinase signaling module in HeLa cells. Using a numerical analysis based on experimentally determined parameters, we predicted in silico and validated in vivo that ERK is processively phosphorylated in HeLa cells. Finally, we identified molecular crowding as a critical factor that converts distributive phosphorylation into processive phosphorylation. We proposed the term quasi-processive phosphorylation to describe this mode of ERK phosphorylation that is operated under the physiological condition of molecular crowding. The generality of this phenomenon may provide a new paradigm for a diverse set of biochemical reactions including multiple posttranslational modifications.
authors.affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. k-aoki@lif.kyoto-u.ac.jp
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamada
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Kunida
authors.forename: Shuhei
authors.initials: S
authors.lastname: Yasuda
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D003198:Computer Simulation; D003593:Cytoplasm / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference
chemical_list: D008164:Luminescent Proteins; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D048049:Extracellular Signal-Regulated MAP Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1