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Integration of a phosphatase cascade with the mitogen-activated protein kinase pathway provides for a novel signal processing function.

Chaudhri VK, Kumar D, Misra M, Dua R, Rao KV - J. Biol. Chem. (2009)

Bottom Line: Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway.Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses.Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.

View Article: PubMed Central - PubMed

Affiliation: Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

ABSTRACT
We mathematically modeled the receptor-dependent mitogen-activated protein kinase (MAPK) signaling by incorporating the regulation through cellular phosphatases. Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway. A novel regulatory motif was, thus, generated, providing for the combinatorial control of each MAPK intermediate. This ensured a non-linear mode of signal transmission with the output being shaped by the balance between the strength of input signal and the activity gradient along the phosphatase axis. Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses. Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.

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Related in: MedlinePlus

The ERK phosphorylation response is proportional to the stimulus strength. Panel A shows the concordance obtained between experiment (red diamonds, values are the mean ± S.D., n = 3) and simulation (black line) examining the time course of ERK phosphorylation obtained upon stimulation of A20 cells with a saturating (25 μg/ml) concentration of anti-IgG. Panel B shows the results of an in silico analysis estimating the magnitude of ERK phosphorylation (ppERK) obtained after stimulation of cells with varying anti-IgG concentrations. Panel C gives the corresponding results of an experiment where A20 cells were stimulated for 10 min, with the indicated doses of anti-IgG. ERK phosphorylation was then determined in lysates by Western blot analysis (see supplemental Fig. S4). Values are the mean (±S.D.) of three independent experiments and are shown here as normalized raw signal intensity (pSignal intensity). A semi-log plot of these results yielded a Hill coefficient of 0.6 and 0.56 for simulated and the experimental results, respectively. Stimulated cells were also subjected to staining for intracellular phospho-ERK using antibodies specific for double-phosphorylated ERK followed by fluorescein isothiocyanate-labeled secondary antibodies. Stained cells were then analyzed by flow cytometry, and the results are shown in panel D. Depicted here are the profiles obtained for cells stimulated with either 0.1 (black line), 0.5 (green line), 5 (pink line), or 25 μg/ml (blue line) of anti-IgG. The profile for unstimulated cells overlapped with that for cells stimulated with 0.1 μg/ml of ligand. For the negative control, cells were stained with rabbit IgG instead of the anti-phospho-ERK antibody.
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Figure 2: The ERK phosphorylation response is proportional to the stimulus strength. Panel A shows the concordance obtained between experiment (red diamonds, values are the mean ± S.D., n = 3) and simulation (black line) examining the time course of ERK phosphorylation obtained upon stimulation of A20 cells with a saturating (25 μg/ml) concentration of anti-IgG. Panel B shows the results of an in silico analysis estimating the magnitude of ERK phosphorylation (ppERK) obtained after stimulation of cells with varying anti-IgG concentrations. Panel C gives the corresponding results of an experiment where A20 cells were stimulated for 10 min, with the indicated doses of anti-IgG. ERK phosphorylation was then determined in lysates by Western blot analysis (see supplemental Fig. S4). Values are the mean (±S.D.) of three independent experiments and are shown here as normalized raw signal intensity (pSignal intensity). A semi-log plot of these results yielded a Hill coefficient of 0.6 and 0.56 for simulated and the experimental results, respectively. Stimulated cells were also subjected to staining for intracellular phospho-ERK using antibodies specific for double-phosphorylated ERK followed by fluorescein isothiocyanate-labeled secondary antibodies. Stained cells were then analyzed by flow cytometry, and the results are shown in panel D. Depicted here are the profiles obtained for cells stimulated with either 0.1 (black line), 0.5 (green line), 5 (pink line), or 25 μg/ml (blue line) of anti-IgG. The profile for unstimulated cells overlapped with that for cells stimulated with 0.1 μg/ml of ligand. For the negative control, cells were stained with rabbit IgG instead of the anti-phospho-ERK antibody.

Mentions: An in silico prediction of the time-dependent profile of ERK phosphorylation was consistent with the experimentally obtained results (Fig. 2A). Furthermore, a similar in silico analysis of ERK phosphorylation in response to varying ligand concentrations yielded an incremental dose-response response profile (Fig. 2B). This prediction of a graded ERK output could also be experimentally confirmed by stimulating A20 cells with increasing concentrations of anti-IgG (supplemental Fig. S4). Importantly, ERK response to ligand dose was found to be graded regardless of whether it was monitored at the level of the cell population by Western blot analysis (Fig. 2C) or at the level of single cells through intracellular staining for phospho-ERK and detection by flow cytometry (Fig. 2D).


Integration of a phosphatase cascade with the mitogen-activated protein kinase pathway provides for a novel signal processing function.

Chaudhri VK, Kumar D, Misra M, Dua R, Rao KV - J. Biol. Chem. (2009)

The ERK phosphorylation response is proportional to the stimulus strength. Panel A shows the concordance obtained between experiment (red diamonds, values are the mean ± S.D., n = 3) and simulation (black line) examining the time course of ERK phosphorylation obtained upon stimulation of A20 cells with a saturating (25 μg/ml) concentration of anti-IgG. Panel B shows the results of an in silico analysis estimating the magnitude of ERK phosphorylation (ppERK) obtained after stimulation of cells with varying anti-IgG concentrations. Panel C gives the corresponding results of an experiment where A20 cells were stimulated for 10 min, with the indicated doses of anti-IgG. ERK phosphorylation was then determined in lysates by Western blot analysis (see supplemental Fig. S4). Values are the mean (±S.D.) of three independent experiments and are shown here as normalized raw signal intensity (pSignal intensity). A semi-log plot of these results yielded a Hill coefficient of 0.6 and 0.56 for simulated and the experimental results, respectively. Stimulated cells were also subjected to staining for intracellular phospho-ERK using antibodies specific for double-phosphorylated ERK followed by fluorescein isothiocyanate-labeled secondary antibodies. Stained cells were then analyzed by flow cytometry, and the results are shown in panel D. Depicted here are the profiles obtained for cells stimulated with either 0.1 (black line), 0.5 (green line), 5 (pink line), or 25 μg/ml (blue line) of anti-IgG. The profile for unstimulated cells overlapped with that for cells stimulated with 0.1 μg/ml of ligand. For the negative control, cells were stained with rabbit IgG instead of the anti-phospho-ERK antibody.
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Figure 2: The ERK phosphorylation response is proportional to the stimulus strength. Panel A shows the concordance obtained between experiment (red diamonds, values are the mean ± S.D., n = 3) and simulation (black line) examining the time course of ERK phosphorylation obtained upon stimulation of A20 cells with a saturating (25 μg/ml) concentration of anti-IgG. Panel B shows the results of an in silico analysis estimating the magnitude of ERK phosphorylation (ppERK) obtained after stimulation of cells with varying anti-IgG concentrations. Panel C gives the corresponding results of an experiment where A20 cells were stimulated for 10 min, with the indicated doses of anti-IgG. ERK phosphorylation was then determined in lysates by Western blot analysis (see supplemental Fig. S4). Values are the mean (±S.D.) of three independent experiments and are shown here as normalized raw signal intensity (pSignal intensity). A semi-log plot of these results yielded a Hill coefficient of 0.6 and 0.56 for simulated and the experimental results, respectively. Stimulated cells were also subjected to staining for intracellular phospho-ERK using antibodies specific for double-phosphorylated ERK followed by fluorescein isothiocyanate-labeled secondary antibodies. Stained cells were then analyzed by flow cytometry, and the results are shown in panel D. Depicted here are the profiles obtained for cells stimulated with either 0.1 (black line), 0.5 (green line), 5 (pink line), or 25 μg/ml (blue line) of anti-IgG. The profile for unstimulated cells overlapped with that for cells stimulated with 0.1 μg/ml of ligand. For the negative control, cells were stained with rabbit IgG instead of the anti-phospho-ERK antibody.
Mentions: An in silico prediction of the time-dependent profile of ERK phosphorylation was consistent with the experimentally obtained results (Fig. 2A). Furthermore, a similar in silico analysis of ERK phosphorylation in response to varying ligand concentrations yielded an incremental dose-response response profile (Fig. 2B). This prediction of a graded ERK output could also be experimentally confirmed by stimulating A20 cells with increasing concentrations of anti-IgG (supplemental Fig. S4). Importantly, ERK response to ligand dose was found to be graded regardless of whether it was monitored at the level of the cell population by Western blot analysis (Fig. 2C) or at the level of single cells through intracellular staining for phospho-ERK and detection by flow cytometry (Fig. 2D).

Bottom Line: Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway.Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses.Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.

View Article: PubMed Central - PubMed

Affiliation: Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

ABSTRACT
We mathematically modeled the receptor-dependent mitogen-activated protein kinase (MAPK) signaling by incorporating the regulation through cellular phosphatases. Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway. A novel regulatory motif was, thus, generated, providing for the combinatorial control of each MAPK intermediate. This ensured a non-linear mode of signal transmission with the output being shaped by the balance between the strength of input signal and the activity gradient along the phosphatase axis. Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses. Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.

Show MeSH
Related in: MedlinePlus