Processive phosphorylation of ERK MAP kinase in mammalian cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 31; pp. 12675 - 12680
• Main Authors: Aoki, Kazuhiro, Yamada, Masashi, Kunida, Katsuyuki, Yasuda, Shuhei, Matsuda, Michiyuki
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 02.08.2011; National Acad Sciences
• Subjects: Algorithms; Amino Acid Sequence; Antibodies; Biological Sciences; Blotting, Western; Cell Nucleus - metabolism; Cells; Coefficients; Computer Simulation; Cytoplasm - metabolism; Extracellular Signal-Regulated MAP Kinases - genetics; Extracellular Signal-Regulated MAP Kinases - metabolism; Fluorescence Resonance Energy Transfer; HEK293 Cells; HeLa Cells; Humans; Input output; Kinetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mammals; MAP Kinase Kinase 1 - genetics; MAP Kinase Kinase 1 - metabolism; MAP Kinase Signaling System; mitogen-activated protein kinase; Mitogen-Activated Protein Kinase 1 - genetics; Mitogen-Activated Protein Kinase 1 - metabolism; Modeling; Models, Biological; Phosphorylation; post-translational modification; Protein Kinases - genetics; Protein Kinases - metabolism; Proteins; RNA Interference; Scaffolds; Simulations; Western blotting
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1104030108

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.