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Reassembly of JIP1 scaffold complex in JNK MAP kinase pathway using heterologous protein interactions.

Moon J, Park SH - PLoS ONE (2014)

Bottom Line: When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued.Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death.These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seoul National University, Seoul, Korea.

ABSTRACT
Formation of signaling protein complexes is crucial for proper signal transduction. Scaffold proteins in MAP kinase pathways are thought to facilitate complex assembly, thereby promoting efficient and specific signaling. To elucidate the assembly mechanism of scaffold complexes in mammals, we attempted to rationally rewire JIP1-dependent JNK MAP kinase pathway via alternative assembly of JIP1 complex. When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued. Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death. These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.

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JNK signaling restored by alternative assembly of JIP complex was able to induce cell death and alter subcellular localization of JNK.Cell death responses were measured by counting cells stained with SYTOX Green. The relative ratios of cell death are plotted for JNK1-nNOS and JNK2-nNOS. The data in the bar graphs are the mean ±SD of triplicate experiments. A. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with JIP1 variants in 293T cells. Dead cells were counted after 24 hours of transfection. Expression of HA-MLK3 was used to stimulate JIP1-mediated JNK pathway in 293T cells [13]. B. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with variants of the JIP1 scaffold in JIP1−/− MEF cells. Cell death responses of MEF cells were examined after 20 hours of stimulation by glucose starvation. C. DIC and green fluorescence images were obtained for the MEF cells described above using a fluorescence microscope. A scale bar corresponding to 100 µm is indicated. D. Recruitment of JNK1 to JIP1 variants (JIP1, JIP1*, syn-JIP1* or JBDJIP1-JIP1*) via heterologous interaction modules was monitored in 293T cells. Cells were stimulated by expression of HA-MLK3 and were fixed with paraformaldehyde after 24 hours of transfection. JIP1 variants tagged with GFP were visualized by monitoring green fluorescence. Flag-tagged JNK1-nNOS was visualized using an anti-Flag antibody and TRITC-linked secondary antibody. Localization of JIP1 variants and JNK1-nNOS was examined from fluorescence images obtained using a confocal microscope. A scale bar corresponding to 10 µm is indicated. The images were obtained from more than 100 cells and all the cells examined exhibited similar patterns, of which representative images were shown here and in the Figure S6 in File S1.
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pone-0096797-g003: JNK signaling restored by alternative assembly of JIP complex was able to induce cell death and alter subcellular localization of JNK.Cell death responses were measured by counting cells stained with SYTOX Green. The relative ratios of cell death are plotted for JNK1-nNOS and JNK2-nNOS. The data in the bar graphs are the mean ±SD of triplicate experiments. A. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with JIP1 variants in 293T cells. Dead cells were counted after 24 hours of transfection. Expression of HA-MLK3 was used to stimulate JIP1-mediated JNK pathway in 293T cells [13]. B. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with variants of the JIP1 scaffold in JIP1−/− MEF cells. Cell death responses of MEF cells were examined after 20 hours of stimulation by glucose starvation. C. DIC and green fluorescence images were obtained for the MEF cells described above using a fluorescence microscope. A scale bar corresponding to 100 µm is indicated. D. Recruitment of JNK1 to JIP1 variants (JIP1, JIP1*, syn-JIP1* or JBDJIP1-JIP1*) via heterologous interaction modules was monitored in 293T cells. Cells were stimulated by expression of HA-MLK3 and were fixed with paraformaldehyde after 24 hours of transfection. JIP1 variants tagged with GFP were visualized by monitoring green fluorescence. Flag-tagged JNK1-nNOS was visualized using an anti-Flag antibody and TRITC-linked secondary antibody. Localization of JIP1 variants and JNK1-nNOS was examined from fluorescence images obtained using a confocal microscope. A scale bar corresponding to 10 µm is indicated. The images were obtained from more than 100 cells and all the cells examined exhibited similar patterns, of which representative images were shown here and in the Figure S6 in File S1.

Mentions: Chronic activation of JNK signaling is known to induce apoptosis [10], [11], [27], and therefore, we attempted to test whether cell death response can be triggered by reassembly of JIP1 scaffold complex. When 293T cells expressing the appropriate scaffold and JNK fusions were stimulated, the presence of JIP1* resulted in a dramatic decrease in cell death response compared to JIP1 [28]. However, both syn-JIP1* and JBDJIP1-JIP1* were able to fully restore the cell death response with JNK1-nNOS or JNK2-NOS (Figure 3A). To rule out any effects from endogenous JIP1, we also examined cell death in JIP1−/− MEF cells stimulated by glucose starvation [30]. As JIP1 is known to be involved in JNK activation by glucose starvation, the cell death response could be triggered when a functional JIP complex was assembled in the MEF cells. Similar to the results from the experiments in 293T cells, syn-JIP1* and JBDJIP1-JIP1* resulted in the full restoration of cell death responses in JIP1−/− MEF cells (Figure 3B, C). Although syn-JIP1* was less efficient than JBDJIP1-JIP1* in JNK phosphorylation (Figure 2D), it generated similar levels of cell death response. These data demonstrated that JNK signaling by restored by reassembly of JIP1 scaffold complex is sufficient for causing physiological changes in cells.


Reassembly of JIP1 scaffold complex in JNK MAP kinase pathway using heterologous protein interactions.

Moon J, Park SH - PLoS ONE (2014)

JNK signaling restored by alternative assembly of JIP complex was able to induce cell death and alter subcellular localization of JNK.Cell death responses were measured by counting cells stained with SYTOX Green. The relative ratios of cell death are plotted for JNK1-nNOS and JNK2-nNOS. The data in the bar graphs are the mean ±SD of triplicate experiments. A. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with JIP1 variants in 293T cells. Dead cells were counted after 24 hours of transfection. Expression of HA-MLK3 was used to stimulate JIP1-mediated JNK pathway in 293T cells [13]. B. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with variants of the JIP1 scaffold in JIP1−/− MEF cells. Cell death responses of MEF cells were examined after 20 hours of stimulation by glucose starvation. C. DIC and green fluorescence images were obtained for the MEF cells described above using a fluorescence microscope. A scale bar corresponding to 100 µm is indicated. D. Recruitment of JNK1 to JIP1 variants (JIP1, JIP1*, syn-JIP1* or JBDJIP1-JIP1*) via heterologous interaction modules was monitored in 293T cells. Cells were stimulated by expression of HA-MLK3 and were fixed with paraformaldehyde after 24 hours of transfection. JIP1 variants tagged with GFP were visualized by monitoring green fluorescence. Flag-tagged JNK1-nNOS was visualized using an anti-Flag antibody and TRITC-linked secondary antibody. Localization of JIP1 variants and JNK1-nNOS was examined from fluorescence images obtained using a confocal microscope. A scale bar corresponding to 10 µm is indicated. The images were obtained from more than 100 cells and all the cells examined exhibited similar patterns, of which representative images were shown here and in the Figure S6 in File S1.
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Related In: Results  -  Collection

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pone-0096797-g003: JNK signaling restored by alternative assembly of JIP complex was able to induce cell death and alter subcellular localization of JNK.Cell death responses were measured by counting cells stained with SYTOX Green. The relative ratios of cell death are plotted for JNK1-nNOS and JNK2-nNOS. The data in the bar graphs are the mean ±SD of triplicate experiments. A. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with JIP1 variants in 293T cells. Dead cells were counted after 24 hours of transfection. Expression of HA-MLK3 was used to stimulate JIP1-mediated JNK pathway in 293T cells [13]. B. Flag-JNK1-nNOS or Flag-JNK2-nNOS was co-expressed with variants of the JIP1 scaffold in JIP1−/− MEF cells. Cell death responses of MEF cells were examined after 20 hours of stimulation by glucose starvation. C. DIC and green fluorescence images were obtained for the MEF cells described above using a fluorescence microscope. A scale bar corresponding to 100 µm is indicated. D. Recruitment of JNK1 to JIP1 variants (JIP1, JIP1*, syn-JIP1* or JBDJIP1-JIP1*) via heterologous interaction modules was monitored in 293T cells. Cells were stimulated by expression of HA-MLK3 and were fixed with paraformaldehyde after 24 hours of transfection. JIP1 variants tagged with GFP were visualized by monitoring green fluorescence. Flag-tagged JNK1-nNOS was visualized using an anti-Flag antibody and TRITC-linked secondary antibody. Localization of JIP1 variants and JNK1-nNOS was examined from fluorescence images obtained using a confocal microscope. A scale bar corresponding to 10 µm is indicated. The images were obtained from more than 100 cells and all the cells examined exhibited similar patterns, of which representative images were shown here and in the Figure S6 in File S1.
Mentions: Chronic activation of JNK signaling is known to induce apoptosis [10], [11], [27], and therefore, we attempted to test whether cell death response can be triggered by reassembly of JIP1 scaffold complex. When 293T cells expressing the appropriate scaffold and JNK fusions were stimulated, the presence of JIP1* resulted in a dramatic decrease in cell death response compared to JIP1 [28]. However, both syn-JIP1* and JBDJIP1-JIP1* were able to fully restore the cell death response with JNK1-nNOS or JNK2-NOS (Figure 3A). To rule out any effects from endogenous JIP1, we also examined cell death in JIP1−/− MEF cells stimulated by glucose starvation [30]. As JIP1 is known to be involved in JNK activation by glucose starvation, the cell death response could be triggered when a functional JIP complex was assembled in the MEF cells. Similar to the results from the experiments in 293T cells, syn-JIP1* and JBDJIP1-JIP1* resulted in the full restoration of cell death responses in JIP1−/− MEF cells (Figure 3B, C). Although syn-JIP1* was less efficient than JBDJIP1-JIP1* in JNK phosphorylation (Figure 2D), it generated similar levels of cell death response. These data demonstrated that JNK signaling by restored by reassembly of JIP1 scaffold complex is sufficient for causing physiological changes in cells.

Bottom Line: When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued.Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death.These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seoul National University, Seoul, Korea.

ABSTRACT
Formation of signaling protein complexes is crucial for proper signal transduction. Scaffold proteins in MAP kinase pathways are thought to facilitate complex assembly, thereby promoting efficient and specific signaling. To elucidate the assembly mechanism of scaffold complexes in mammals, we attempted to rationally rewire JIP1-dependent JNK MAP kinase pathway via alternative assembly of JIP1 complex. When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued. Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death. These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.

Show MeSH