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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 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.
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pone-0096797-g001: 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.

Mentions: A major role of scaffold proteins in cell signaling is thought to be tethering of signaling components into an active complex via non-covalent interactions [3], [4], [24]. To investigate the assembly rules of JIP1 scaffold complex, we attempted to wire the JIP1-dependent JNK MAP kinase signaling pathway by replacing JIP1-JNK docking interaction using heterologous interaction modules (Figure 1A). A mutant of JIP1 (JIP1*), containing point mutations R156G and P157G in the JBD, was constructed to disrupt JIP1-JNK docking interaction [25]. The binding affinity of JIP1* for JNK was significantly decreased compared to wild-type JIP1 (Figure 1B). To recruit missing JNK to the scaffold complex, JIP1* and JNK were fused to PDZ domains from syntrophin (syn) and neuronal nitric oxide synthase (nNOS), respectively. When the fusions of syn-JIP1* and JNK1-nNOS were co-expressed in 293T cells, the interaction between JIP1* and JNK was successfully restored via PDZ interactions (Figure 1B). To determine whether the recruitment of JNK to JIP1* via a PDZ interaction module is sufficient for restoring signaling output, the dual-phosphorylation of JNK was monitored in cells expressing JNK1-nNOS along with JIP1, JIP1* or syn-JIP1*. Upon stimulation by MLK3 over-expression, which is known to trigger JIP1-dependent JNK signaling in 293T cells [13], [26]–[28], JIP1* led to a noticeable decrease in JNK phosphorylation, which was presumably due to the defective docking interaction. Surprisingly, syn-JIP1* expressed along with JNK1-nNOS was able to restore phosphorylation of JNK comparable to that by wild-type JIP1 (Figure 1C). Similar results were observed in COS7 cells, where the JIP1-JNK docking interaction was replaced by the identical set of PDZ domains (Figure S1 in File S1). Together, these data indicate that the JIP1-JNK docking interaction is highly modular and that the pathway connectivity of JNK signaling can be restored via alternative assembly of the JIP1 complex using heterologous protein interactions.


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

Moon J, Park SH - PLoS ONE (2014)

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.
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pone-0096797-g001: 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.
Mentions: A major role of scaffold proteins in cell signaling is thought to be tethering of signaling components into an active complex via non-covalent interactions [3], [4], [24]. To investigate the assembly rules of JIP1 scaffold complex, we attempted to wire the JIP1-dependent JNK MAP kinase signaling pathway by replacing JIP1-JNK docking interaction using heterologous interaction modules (Figure 1A). A mutant of JIP1 (JIP1*), containing point mutations R156G and P157G in the JBD, was constructed to disrupt JIP1-JNK docking interaction [25]. The binding affinity of JIP1* for JNK was significantly decreased compared to wild-type JIP1 (Figure 1B). To recruit missing JNK to the scaffold complex, JIP1* and JNK were fused to PDZ domains from syntrophin (syn) and neuronal nitric oxide synthase (nNOS), respectively. When the fusions of syn-JIP1* and JNK1-nNOS were co-expressed in 293T cells, the interaction between JIP1* and JNK was successfully restored via PDZ interactions (Figure 1B). To determine whether the recruitment of JNK to JIP1* via a PDZ interaction module is sufficient for restoring signaling output, the dual-phosphorylation of JNK was monitored in cells expressing JNK1-nNOS along with JIP1, JIP1* or syn-JIP1*. Upon stimulation by MLK3 over-expression, which is known to trigger JIP1-dependent JNK signaling in 293T cells [13], [26]–[28], JIP1* led to a noticeable decrease in JNK phosphorylation, which was presumably due to the defective docking interaction. Surprisingly, syn-JIP1* expressed along with JNK1-nNOS was able to restore phosphorylation of JNK comparable to that by wild-type JIP1 (Figure 1C). Similar results were observed in COS7 cells, where the JIP1-JNK docking interaction was replaced by the identical set of PDZ domains (Figure S1 in File S1). Together, these data indicate that the JIP1-JNK docking interaction is highly modular and that the pathway connectivity of JNK signaling can be restored via alternative assembly of the JIP1 complex using heterologous protein interactions.

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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