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POSH acts as a scaffold for a multiprotein complex that mediates JNK activation in apoptosis
Zhiheng Xu1, Nickolay V Kukekov, Lloyd A Greene
1Department of Pathology and Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Abstract:
We report that the multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1 as described previously, we find that POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade.
Insights
The protein POSH acts as a scaffold, organizing the JNK pathway crucial for neuronal death. It links activated Rac1 to downstream components, influencing programmed cell death in neurons.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- The c-Jun N-terminal kinase (JNK) pathway is implicated in programmed neuronal death.
- Scaffold proteins play critical roles in signal transduction by organizing signaling complexes.
- The protein POSH (plenty of SH3s) is a multidomain protein with known interactions in signaling pathways.
Purpose of the Study:
- To investigate the role of POSH as a scaffold protein in the JNK pathway of neuronal death.
- To elucidate the specific interactions of POSH within the JNK signaling cascade.
- To determine the functional significance of POSH in mediating neuronal apoptosis.
Main Methods:
- Co-immunoprecipitation assays to assess protein-protein interactions in vivo and in vitro.
- Overexpression studies using dominant-negative constructs and inhibitors to probe pathway function.
- Application of antisense oligonucleotides and small interfering RNA (siRNA) to reduce POSH levels.
- Assessment of c-Jun phosphorylation and neuronal apoptosis as readouts of pathway activity.
Main Results:
- POSH directly binds to mixed-lineage kinases (MLKs) and complexes with MAP kinase kinases (MKKs) 4/7 and c-Jun N-terminal kinases (JNKs).
- Overexpression of POSH enhances apoptotic neuronal death, which can be blocked by inhibiting downstream JNK pathway components.
- Silencing POSH expression using antisense or siRNA suppresses c-Jun phosphorylation and neuronal apoptosis induced by nerve growth factor (NGF) deprivation.
- POSH acts as a central scaffold, linking activated Rac1/Cdc42 to the downstream JNK apoptotic cascade.
Conclusions:
- POSH functions as a critical scaffold protein within the JNK signaling pathway, facilitating neuronal apoptosis.
- POSH integrates upstream signals (activated Rac1) with downstream JNK cascade effectors, promoting programmed neuronal death.
- Targeting POSH may offer a therapeutic strategy for conditions involving excessive neuronal death.