Stimulus-coupled spatial restriction of extracellular signal-regulated kinase 1/2 activity contributes to the specificity of signal-response pathways

Abstract

Current understanding of cell regulatory systems suggests a diverse array of extracellular stimuli commonly recruit a limited cadre of core signal transduction modules to drive discrete stimulus-specific responses. One such module is the Raf-MEK-extracellular signal-regulated kinase (ERK) kinase cascade. Little information exists about how this pathway can be appropriately coupled to discrete cell biological processes. Contributing factors may include regulation of the duration, amplitude, and/or subcellular compartmentalization of active ERK1/2. To define properties of ERK1/2 that may help mediate stimulus-selective signal propagation, we have examined the dynamic behavior of native ERK1/2 activation at the single-cell level. In primary human cell cultures, ERK1/2 activation is not an all-or-none response. Instead, the amount of active ERK1/2 in individual cells accumulated in proportion to the concentration of external stimulus. The variable degree of ERK1/2 activation correlated well with the degree of ERK1/2 effector activation. Therefore, the relative amplitude of ERK1/2 activation within a cell can be modulated and may contribute to the generation of stimulus-specific biological responses. Importantly, we also found that the capacity of active ERK1/2 to accumulate in the nucleus and drive immediate-early gene expression is dependent upon the nature of the inductive signal, but independent of the amplitude of ERK1/2 activation. Therefore, nuclear accumulation of active ERK1/2 is a discrete regulated step that can direct the function of the kinase in response to specific stimuli.

FIG. 1.

Analysis of ERK1/2 activation at single-cell resolution. (A) Following stimulation for 5 min with EGF at the indicated concentrations, HeLa cells were immunostained or immunoblotted with the dual phospho-ERK1/2 antibody as described in Materials and Methods. Phospho-ERK1/2 immunofluorescence signal intensities from individual cells were calculated as described in Materials and Methods. Histograms derived from greater than 50 cells per condition are shown for a representative experiment. Five additional independent experiments showed similar results. Representative images of phospho-ERK1/2-immunostained cells are shown on the right together with Western analysis of whole-cell lysates. PP-ERK indicates dually phosphorylated active ERK1/2; T-ERK indicates total ERK1/2. (B) The per-cell fluorescence intensity from phospho-ERK1/2-immunostained primary HFFs, stimulated for 5 min with the indicated concentrations of EGF or PMA, was analyzed as for panel A. The histograms shown are a representative experiment from four independent experiments showing similar results. (C) The per-cell fluorescence intensity from phospho-ERK1/2-immunostained primary HFFs, stimulated for 45 min with the indicated concentrations of EGF or PMA, was analyzed as in panel A. (D) Representative images of dual phospho-ERK1/2 staining in stimulated HFFs at indicated ligand concentrations and times. Similar localization patterns were observed at 2 h poststimulation (data not shown).

FIG. 1.

Analysis of ERK1/2 activation at single-cell resolution. (A) Following stimulation for 5 min with EGF at the indicated concentrations, HeLa cells were immunostained or immunoblotted with the dual phospho-ERK1/2 antibody as described in Materials and Methods. Phospho-ERK1/2 immunofluorescence signal intensities from individual cells were calculated as described in Materials and Methods. Histograms derived from greater than 50 cells per condition are shown for a representative experiment. Five additional independent experiments showed similar results. Representative images of phospho-ERK1/2-immunostained cells are shown on the right together with Western analysis of whole-cell lysates. PP-ERK indicates dually phosphorylated active ERK1/2; T-ERK indicates total ERK1/2. (B) The per-cell fluorescence intensity from phospho-ERK1/2-immunostained primary HFFs, stimulated for 5 min with the indicated concentrations of EGF or PMA, was analyzed as for panel A. The histograms shown are a representative experiment from four independent experiments showing similar results. (C) The per-cell fluorescence intensity from phospho-ERK1/2-immunostained primary HFFs, stimulated for 45 min with the indicated concentrations of EGF or PMA, was analyzed as in panel A. (D) Representative images of dual phospho-ERK1/2 staining in stimulated HFFs at indicated ligand concentrations and times. Similar localization patterns were observed at 2 h poststimulation (data not shown).

FIG. 1.

Analysis of ERK1/2 activation at single-cell resolution. (A) Following stimulation for 5 min with EGF at the indicated concentrations, HeLa cells were immunostained or immunoblotted with the dual phospho-ERK1/2 antibody as described in Materials and Methods. Phospho-ERK1/2 immunofluorescence signal intensities from individual cells were calculated as described in Materials and Methods. Histograms derived from greater than 50 cells per condition are shown for a representative experiment. Five additional independent experiments showed similar results. Representative images of phospho-ERK1/2-immunostained cells are shown on the right together with Western analysis of whole-cell lysates. PP-ERK indicates dually phosphorylated active ERK1/2; T-ERK indicates total ERK1/2. (B) The per-cell fluorescence intensity from phospho-ERK1/2-immunostained primary HFFs, stimulated for 5 min with the indicated concentrations of EGF or PMA, was analyzed as for panel A. The histograms shown are a representative experiment from four independent experiments showing similar results. (C) The per-cell fluorescence intensity from phospho-ERK1/2-immunostained primary HFFs, stimulated for 45 min with the indicated concentrations of EGF or PMA, was analyzed as in panel A. (D) Representative images of dual phospho-ERK1/2 staining in stimulated HFFs at indicated ligand concentrations and times. Similar localization patterns were observed at 2 h poststimulation (data not shown).

FIG. 2.

Differential responses of ERK1/2 effectors to EGF and PMA stimulation. (A) Whole-cell lysates from serum-starved HFFs stimulated for 5 min with 2.0 nM EGF or 10 nM PMA were immunoblotted with the indicated antibodies. U0126 was used at 10 μM for 45 min prior to stimulation. (B) Forty-five minutes following stimulation of HFFs with the indicated concentrations of EGF or PMA, c-Fos expression was evaluated by immunostaining of fixed cells (top panel) or immunoblotting of whole-cell lysates (bottom panel). The percentage of c-Fos-expressing cells from three independent experiments is shown. Error bars represent the standard deviation from the mean. Representative images from EGF- and PMA-stimulated cells are shown beneath the graph. (C) Cyclin D1 and total ERK1/2 immunoblots from whole-cell lysates of serum-starved (SS) HFFs stimulated for the indicated times with 2 nM EGF or 10 nM PMA.

FIG. 2.

Differential responses of ERK1/2 effectors to EGF and PMA stimulation. (A) Whole-cell lysates from serum-starved HFFs stimulated for 5 min with 2.0 nM EGF or 10 nM PMA were immunoblotted with the indicated antibodies. U0126 was used at 10 μM for 45 min prior to stimulation. (B) Forty-five minutes following stimulation of HFFs with the indicated concentrations of EGF or PMA, c-Fos expression was evaluated by immunostaining of fixed cells (top panel) or immunoblotting of whole-cell lysates (bottom panel). The percentage of c-Fos-expressing cells from three independent experiments is shown. Error bars represent the standard deviation from the mean. Representative images from EGF- and PMA-stimulated cells are shown beneath the graph. (C) Cyclin D1 and total ERK1/2 immunoblots from whole-cell lysates of serum-starved (SS) HFFs stimulated for the indicated times with 2 nM EGF or 10 nM PMA.

FIG. 2.

Differential responses of ERK1/2 effectors to EGF and PMA stimulation. (A) Whole-cell lysates from serum-starved HFFs stimulated for 5 min with 2.0 nM EGF or 10 nM PMA were immunoblotted with the indicated antibodies. U0126 was used at 10 μM for 45 min prior to stimulation. (B) Forty-five minutes following stimulation of HFFs with the indicated concentrations of EGF or PMA, c-Fos expression was evaluated by immunostaining of fixed cells (top panel) or immunoblotting of whole-cell lysates (bottom panel). The percentage of c-Fos-expressing cells from three independent experiments is shown. Error bars represent the standard deviation from the mean. Representative images from EGF- and PMA-stimulated cells are shown beneath the graph. (C) Cyclin D1 and total ERK1/2 immunoblots from whole-cell lysates of serum-starved (SS) HFFs stimulated for the indicated times with 2 nM EGF or 10 nM PMA.

FIG. 3.

Effect of LB on active ERK1/2 localization and accumulation of c-Fos. (A) Following pretreatment with 200-ng/ml LB (+LB) or without LB (−LB), serum-starved HFFs were stimulated with 2 nM EGF or 10 nM PMA for 45 min. Fixed cells were stained with the indicated antibodies. (B) Quantitation of c-Fos-expressing cells treated as in panel A. The percentage of c-Fos-expressing cells in the population is shown normalized to that observed upon PMA stimulation in the absence of LB (arbitrarily set to 100). Error bars represent the standard deviation from the mean from three independent experiments. (C) Whole-cell lysates from HFFs treated as in panel A were immunoblotted with the indicated antibodies.