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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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Verifying the MKP3 substrate bias in response to CK2 inhibition. For the experiment, A20 cells were stimulated for 10 min with anti-IgG in the presence or absence of casein kinase inhibitor DRB (20 μm). Cells were then fixed and stained for ERK1/2 and MKP3 (top panel), MEK1/2 and MKP3 (middle panel), or MKP1 and MKP3 (bottom panel) and observed under laser scanning confocal microscope (“Experimental Procedures”). Merged images for co-localization between green (ERK1/2, MEK1/2, or MKP1) and red (MKP3) are shown in the figure. The quantitative differences between DRB-treated and -untreated cells for co-localization with MKP3-MEK1/2, MKP3-ERK1/2, and MKP3-MKP1 upon anti-IgG treatment are shown on the right-hand side. Values are the average (±S.E.) of more than at least 40 cells (***, p < 0.0005; **, p < 0.005).
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Figure 4: Verifying the MKP3 substrate bias in response to CK2 inhibition. For the experiment, A20 cells were stimulated for 10 min with anti-IgG in the presence or absence of casein kinase inhibitor DRB (20 μm). Cells were then fixed and stained for ERK1/2 and MKP3 (top panel), MEK1/2 and MKP3 (middle panel), or MKP1 and MKP3 (bottom panel) and observed under laser scanning confocal microscope (“Experimental Procedures”). Merged images for co-localization between green (ERK1/2, MEK1/2, or MKP1) and red (MKP3) are shown in the figure. The quantitative differences between DRB-treated and -untreated cells for co-localization with MKP3-MEK1/2, MKP3-ERK1/2, and MKP3-MKP1 upon anti-IgG treatment are shown on the right-hand side. Values are the average (±S.E.) of more than at least 40 cells (***, p < 0.0005; **, p < 0.005).

Mentions: The results in Fig. 3C, therefore, provide experimental support for our proposal that MKP3 displays differential substrate specificity depending upon its phosphorylation status. To further establish this, however, we also employed an alternate and more direct approach. Here, A20 cells were stimulated either in the presence or absence of the CK2α inhibitor, DRB. Subsequently, these cells were analyzed by confocal microscopy to determine the effects of CK2α inhibition on the co-localization of MKP3 with ERK, MEK, and MKP1. Fig. 4 reveals that in stimulated cells inhibition of CK2α led to a substantial increase in the extent of co-localization between MKP3 and ERK. In contrast, co-localization of MKP3 with both MEK and MKP1 was significantly reduced (Fig. 4). Essentially similar results were obtained in experiments where, instead of inhibition, CK2α was also specifically silenced through targeted siRNA (not shown). At one level these results provide direct evidence for the novel links between MKP3 and MEK and between MKP3 and MKP1 identified by our model. In addition to this, however, the observed alterations in the relative extents of co-localization of MKP3 with ERK, MEK, and MKP1, induced by the inhibition of CK2α-dependent phosphorylation of MKP3, also provide experimental confirmation for our proposal that the substrate specificity of MKP3 is influenced by its phosphorylation status. Collectively, therefore, the consistency between predicted and experimentally obtained phosphorylation profiles of the various signaling intermediates described above as well as the experimental confirmation of the new MKP3-dependent regulatory links proposed provide experimental support for the model depicted in Fig. 1A.


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)

Verifying the MKP3 substrate bias in response to CK2 inhibition. For the experiment, A20 cells were stimulated for 10 min with anti-IgG in the presence or absence of casein kinase inhibitor DRB (20 μm). Cells were then fixed and stained for ERK1/2 and MKP3 (top panel), MEK1/2 and MKP3 (middle panel), or MKP1 and MKP3 (bottom panel) and observed under laser scanning confocal microscope (“Experimental Procedures”). Merged images for co-localization between green (ERK1/2, MEK1/2, or MKP1) and red (MKP3) are shown in the figure. The quantitative differences between DRB-treated and -untreated cells for co-localization with MKP3-MEK1/2, MKP3-ERK1/2, and MKP3-MKP1 upon anti-IgG treatment are shown on the right-hand side. Values are the average (±S.E.) of more than at least 40 cells (***, p < 0.0005; **, p < 0.005).
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Verifying the MKP3 substrate bias in response to CK2 inhibition. For the experiment, A20 cells were stimulated for 10 min with anti-IgG in the presence or absence of casein kinase inhibitor DRB (20 μm). Cells were then fixed and stained for ERK1/2 and MKP3 (top panel), MEK1/2 and MKP3 (middle panel), or MKP1 and MKP3 (bottom panel) and observed under laser scanning confocal microscope (“Experimental Procedures”). Merged images for co-localization between green (ERK1/2, MEK1/2, or MKP1) and red (MKP3) are shown in the figure. The quantitative differences between DRB-treated and -untreated cells for co-localization with MKP3-MEK1/2, MKP3-ERK1/2, and MKP3-MKP1 upon anti-IgG treatment are shown on the right-hand side. Values are the average (±S.E.) of more than at least 40 cells (***, p < 0.0005; **, p < 0.005).
Mentions: The results in Fig. 3C, therefore, provide experimental support for our proposal that MKP3 displays differential substrate specificity depending upon its phosphorylation status. To further establish this, however, we also employed an alternate and more direct approach. Here, A20 cells were stimulated either in the presence or absence of the CK2α inhibitor, DRB. Subsequently, these cells were analyzed by confocal microscopy to determine the effects of CK2α inhibition on the co-localization of MKP3 with ERK, MEK, and MKP1. Fig. 4 reveals that in stimulated cells inhibition of CK2α led to a substantial increase in the extent of co-localization between MKP3 and ERK. In contrast, co-localization of MKP3 with both MEK and MKP1 was significantly reduced (Fig. 4). Essentially similar results were obtained in experiments where, instead of inhibition, CK2α was also specifically silenced through targeted siRNA (not shown). At one level these results provide direct evidence for the novel links between MKP3 and MEK and between MKP3 and MKP1 identified by our model. In addition to this, however, the observed alterations in the relative extents of co-localization of MKP3 with ERK, MEK, and MKP1, induced by the inhibition of CK2α-dependent phosphorylation of MKP3, also provide experimental confirmation for our proposal that the substrate specificity of MKP3 is influenced by its phosphorylation status. Collectively, therefore, the consistency between predicted and experimentally obtained phosphorylation profiles of the various signaling intermediates described above as well as the experimental confirmation of the new MKP3-dependent regulatory links proposed provide experimental support for the model depicted in Fig. 1A.

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