Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (

A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.The c-Jun N-terminal/stress-activated kinases (JNKs) 3 appear to play major roles in mediating apoptotic death of both neuronal and nonneuronal cells (1). A cascading pathway has been identified that leads to JNK activation in response to apoptotic stimuli. In this cascade, the GTP-bound form of Rac-1 and/or Cdc42 initiate a sequence of kinase phosphorylations/activations consisting of the mixed lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7 and JNKs (2-5). JNKs activated in this manner then promote death by phosphorylation/activation of the transcription factor c-Jun (1, 6) and/or the BH3-only domain protein BIM (7,8).POSH (plenty of SH3 domains) was identified as a binding partner of GTP-Rac1 (9), and its overexpression causes the death of both nonneuronal (9) and neuronal cells (10) by activating the JNK pathway. Consistent with a required role in apoptotic death paradigms, both antisense RNA and small interfering RNA directed against POSH transcripts protect neuronal PC12 cells and sympathetic neurons from death caused by NGF withdrawal (10). Moreover, antisense directed to POSH interferes with the elevation of intracellular phospho-Jun levels in NGF-deprived cells, thus implicating endogenous POSH in activation of JNKs and consequent c-Jun phosphorylation. The in vivo importance of POSH in cell death and in the apoptotic JNK pathway was recently demonstrated by a study in which knockdown of POSH by infusion of antisense oligonucleotides lead to neuroprotection from cerebral ischemia as well as attenuated activation of the JNK signaling pathway (11).Although many individual components of the apoptotic JNK pathway have been uncovered, questions remain about the molecular means by which they are brought into contact in cells and by which they are directed toward induction of death. Here, we show that the major components of the pathway are assembled in a multiprotein complex anchored by two interacting molecular scaffolds, POSH and JIP (JNKinteracting protein), and that formation of this complex is required for JNK activation and induction of apoptosis. EXPE...

Background: Scaffold proteins, such as the pro-apoptotic scaffold POSH (Plenty of SH3s), organize MAP kinase pathways into functional modules. Results: Sh3rf2 promotes the degradation of POSH and prevents apoptosis in multiple cell types. Conclusion: Sh3rf2 antagonizes POSH-JNK signaling under basal conditions and provides a "brake" on apoptosis. Significance: Sh3rf2 may provide a target in neoplasia and apoptosis involving POSH such as trophic factor deprivation.

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The c-Jun N-terminal kinase (JNK) pathway plays an important role in neuronal apoptosis both during normal CNS development and following stroke in adult animals. As with other MAP kinase pathways, scaffold proteins regulate JNK signaling. The scaffold protein POSH (Plenty of SH3s) enhances JNK activation and apoptosis. We identified a POSH homologue, POSH2, which was cloned from rat brain and is present in cortical neurons in vitro. POSH2 mRNA is expressed in a variety of tissues including brain, and this distribution partially overlaps with that of POSH. POSH2 overexpression promotes JNK activation in HEK293 cells and promotes apoptosis in neuronal PC12 cells, which is blocked by a dominant-negative c-Jun. Finally POSH2 contains a functional RING domain and enhances the stability of coexpressed mixed-lineage kinases. These results indicate that POSH2 may regulate JNK activation and consequent apoptosis under conditions of increased expression.