Direct Interaction of the Molecular Scaffolds POSH and JIP Is Required for Apoptotic Activation of JNKs

Abstract

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...

Sh3rf2/POSHER Protein Promotes Cell Survival by Ring-mediated Proteasomal Degradation of the c-Jun N-terminal Kinase Scaffold POSH (Plenty of SH3s) Protein

“…6A, top panel). Consistent with the idea that Sh3rf2 degrades POSH under basal conditions, the time of Sh3rf2 loss is earlier than that previously reported for POSH stabilization following NGF deprivation (19).…”

“…PCR products, not including the start codon, were ligated in-frame into the pcDNA3-GST vector. POSH, MLK3, MLK2, DLK, and JIP constructs have been previously described (17,19) as have Sh3rf2 and the RING mutant of Sh3rf2 (22).…”

“…As part of its scaffold function, POSH interacts directly with members of the MLK (mixed lineage kinase) MKKK family and promotes their activation (16,17). It also interacts indirectly with MKK4/7 and the JNKs through its interaction with the JIPs (19). In this capacity, the POSH-JIP interaction leads to formation of a multiprotein complex (POSH-JIP apoptotic complex or PJAC) that brings together key components of the JNK activation cascade.…”

Scaffold proteins of MAP-kinase modules

“…Thus, POSH functions as a scaffold protein in linking Rac1 and downstream JNK module in an apoptotic pathway. Recently, it has been observed that POSH associates with JIP1 and that this POSH-JIP1 complex is involved in inducing apoptosis (Kukekov et al, 2006). Although both POSH and JIP1 can independently interact with different MAP3Ks, MAP2Ks and JNKs, it has been reasoned that for the efficient activation of JNK module, the cooperation between POSH and JIP1 may be required (Kukekov et al, 2006).…”

Regulation of the Pro-apoptotic Scaffolding Protein POSH by Akt

“…POSH directly binds Rac, MLK, and another scaffold protein, JIP (JNK-interacting protein), which in turn binds MKK4/7 and JNK, to facilitate this pathway. This multiprotein signaling assembly has been termed PJAC, for POSH-JIP apoptotic complex (3). The role of POSH as a scaffold for this signaling complex appears to be critical; in an apoptotic model involving withdrawal of nerve growth factor from cultures of neuronally differentiated PC12 cells or rat primary sympathetic neurons, apoptosis was dramatically reduced by pretreatment with POSH short interfering RNA or antisense oligonucleotides (4).…”