Figure 1.
JNK MAP kinase pathway was restored by replacing JIP1-JNK docking interaction with PDZ interaction domains.
A. The wiring strategy of the JNK MAP kinase pathway is illustrated. The diagram shows a conventional JIP1-dependent JNK MAP kinase pathway (left). A JIP1* scaffold containing mutations to disrupt docking interaction with JNK leads to release of JNK from the scaffold complex and, therefore, a decrease in the signaling response (middle). The functional scaffold complex is reassembled by re-recruiting missing JNK to the scaffold complex using fusions of syn-JIP1* and JNK1-nNOS, resulting in the restoration of JNK signaling (right). To recruit JNK to JIP1*, syn and nNOS PDZ domains were fused to JIP1* and JNK, respectively. B. JIP1, JIP1* or syn-JIP1* scaffold was co-expressed with Flag-JNK1-nNOS in 293T cells. Myc-tagged scaffold proteins were immunoprecipitated using an anti-myc antibody conjugated to agarose. The bound Flag-JNK1-nNOS was detected by immunoblotting. C. To verify that the alternatively assembled scaffold complex restored the signaling response, dual-phosphorylation of JNK1-nNOS in the whole cell lysates was detected by western blot analysis using an anti-phospho JNK antibody. Expression of HA-MLK3 was used to stimulate the JIP1-mediated JNK pathway in 293T cells [13]. Each experiment described here was performed in triplicate.
Figure 2.
JNK MAP kinase pathway was restored by re-recruiting JNK using JBDs.
A. The diagram illustrates the reassembly of JIP1 scaffold complex by re-recruiting the missing JNK using JBDs. Peptides encoding JBDs were fused to the N-terminus of JIP1* to recruit JNK to the scaffold complex. The box shows the alignment of the amino acid sequences of the following JBDs used in this experiment: mouse JIP1 (156–164), human MKK4 (38–48), and mouse GR (571–582). Basic (+) and hydrophobic (Φ) residues in the consensus sequence were indicated. Conserved hydrophobic residues among the sequences were boxed in gray. Arginine and proline residues mutated in JIP1* were indicated in red. B. Flag-JNK1-nNOS was co-expressed with JIP1 variants (JIP1, JIP1*, syn-JIP1*, JBDJIP1-JIP1*, JBDMKK4-JIP1* or JBDGR-JIP1*) in 293T cells. Interactions between JNK1-nNOS and JIP1 variants were analyzed as described in Figure 1B. C. Dual phosphorylation of Flag-JNK1-nNOS in the whole cell lysates was analyzed as described above to monitor the JNK signaling restored by reassembly of JIP1 complex via JBDs. Expression of HA-MLK3 was used to stimulate JIP1-mediated JNK pathway in 293T cells [13]. D and E. JNK signaling mediated by JIP1 scaffold reassembly was monitored by an in-gel kinase assay to measure the catalytic activity of activated JNK in the whole cell lysates. cJun protein was used as a substrate for JNK. Expressed proteins and phosphorylated JNK were examined by immunoblotting. Samples were separated in a gel containing GST-cJun, and catalytic activity of JNK was quantified by measuring the radioactivity incorporated in GST-cJun using a liquid scintillation counter. Each experiment was performed in triplicate and repeated at least three times.
Figure 3.
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.