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.

Fig. 1.

ERK phosphorylation in vitro. (A) Schematic representation of the input-output response of ERK MAP kinase. The distributive phosphorylation model leads to switch-like response. On the other hand, no model is currently validated for the graded response of ERK, which is observed in mammalian cells and yeast. (B) His-tagged MEK1-SDSE and GST-ERK2-KR were incubated with ATP for the indicated periods, followed by Phos-tag Western blotting analysis. All phospho-isoforms of ERK were detected by anti-GST rabbit antibody (upper) and IRDye680-conjugated anti-rabbit IgG antibody as a secondary antibody. The positions of the phospho-isoforms of ERK are indicated at right (SI Appendix: Fig. S4). pTpY-ERK was simultaneously detected by anti-pTpY-ERK mouse antibody (middle) and IRDye800-conjugated anti-mouse IgG antibody as a secondary antibody. The lower image is the merged image (Red: ERK, Green: pTpY-ERK). (C) The phospho-isoforms of ERK were quantified and plotted with SD (n = 3).

Fig. 2.

Numerical and experimental analyses of the distributive phosphorylation model. (A) Schematic representation of the distributive model (see SI Appendix: Fig. S8A in detail). (B and C) Results of numerical simulations are represented based on experimentally determined parameters (SI Appendix: Tables S1 and S2). (B) Concentrations of np-ERK (blue), pY-ERK (green), pTpY-ERK (red), and pT-ERK (orange) are plotted against time. The kcat/Km value of cRaf kinase for MEK phosphorylation (Raf_kphos) is 1.0 × 10⁶ [/M/ sec ]. (C) Concentrations of the phospho-isoforms of ERK are plotted against the Raf_kphos value at time = 10 (min). (D and E) After serum starvation, HeLa cells were stimulated with 1.0 × 10^-5 g/L EGF at the indicated time period. (D) Cell lysates were separated by SDS-polyacrylamide gel containing Phos-tag and probed with an anti-ERK antibody. (E) The amounts of np-ERK2 (blue), pY-ERK2 (green), and pTpY-ERK2 (red) are plotted against time after EGF stimulation with SD (n = 3). (F and G) After serum starvation, HeLa cells were stimulated with the indicated concentration of EGF for 7.5 min. (F) Cell lysates were analyzed by Phos-tag Western blot analysis as in (D). (G) The amounts of np-ERK2 (blue), pY-ERK2 (green), and pTpY-ERK2 (red) are plotted as a function of the EGF concentration with SD (n = 5).

Fig. 3.

Computational simulation of the processive phosphorylation model. (A) Schematic representation of the processive model (see SI Appendix: Fig. S8B in detail). (B) Results of numerical simulations are represented based on experimentally determined parameters (SI Appendix: Tables S1 and S2). Time series of np-ERK (blue), pY-ERK (green), pTpY-ERK (red), and pT-ERK (orange) concentrations are plotted at Raf_kphos value = 1.0 × 10⁶ [/M/ sec ]. (C) Concentrations of the phospho-isoforms of ERK are plotted as a function of Raf_kphos value at time = 10 (min).

Fig. 4.

Validation of the processive model by pY-ERK dynamics. (A and B) Numerical simulations were performed based on the distributive model (A) and the processive model (B) in the absence (left column) or presence (right column) of the inhibition of all phosphatase reactions. Time series of np-ERK (blue), pY-ERK (green), pTpY-ERK (red), and pT-ERK (orange) concentrations are plotted at Raf_kphos value = 1.0 × 10⁶ [/M/ sec ]. Maximal levels of pY-ERK without inhibition are indicated by gray dashed lines. (C and D) After serum starvation, HeLa cells were treated with mock or 1.0 × 10^-7 M Calyculin A and 1.0 × 10^-4 M bpV for 2 min, followed by 1.0 × 10^-5 g/L EGF for the indicated time period. (C) Cell lysates were separated by electrophoresis in SDS-polyacrylamide gels containing Phos-tag and probed with an anti-ERK antibody. (D) Time series of np-ERK2 (blue), pY-ERK2 (green), and pTpY-ERK2 (red) concentrations without (left) or with (right) phosphatase inhibitors are plotted after EGF stimulation with SD (n = 4).

Fig. 5.

Validation of the processive model by input-output response. (A and B) Serum-starved HeLa cells were stimulated with the different concentration of EGF (blue, 0.01; cyan, 0.03; green, 0.1; yellow, 0.3; orange, 1.0; pink, 3.0; and red, 10( × 10^-6 [g/L])) or 2.0 × 10^-5 M U0126 (black) for 5 min, and then stained with anti-pTpY-ERK1/2 antibody. (A) Fluorescent signals in each cell are represented in a histogram, demonstrating unimodal distributions (n > 90 cells). (B) The average fluorescent intensities are plotted as a function of EGF concentration with SD. Hill coefficient value, nH, was obtained by curve fitting with standard Hill equation (black line). (C) The average of fraction of pTpY-ERK are plotted as a function of EGF concentration with SD (n = 5). This data is same as shown in Fig. 2G (red line). (D) The average of fraction of pTpY-ERK was plotted as a function of ratio value of phosphorylated MEK1/2 to total MEK1/2 with SD (n = 5). (E and F) The pTpY-ERK concentration at time = 10 (min) after stimulation in distributive (E) and processive (F) model are plotted as a function of phosphorylated MEK concentration.

Fig. 6.

Effect of molecular crowding on processive phosphorylation. (A and B) ERK was phosphorylated by MEK in the absence (A) or presence (B) of 15% (w/v) polyethylene glycol (PEG) in vitro and were subjected to Phos-tag Western blotting analysis. (C and D) The concentrations of phospho-isoforms of ERK in (A and B) are plotted as dots against time. Concentration of constitutively active mutant of MEK (1.0 × 10^-7 M) used in these experiments is indicated by gray dashed lines. The same experiments were repeated four times with reproducible results, and representative results are shown. Lines represent the concentration of phospho-isoforms of ERK obtained by fitting the processive model in (E) with the experimental data. The fitted parameters of processivity (p) and crowding factor (c) are represented. (E) Schematic representation of processive phosphorylation. p and c indicate processivity (0 < p < 1) and crowding factor, respectively. (F) Schematic view of quasi-processive model under the condition of molecular crowding.