Knock-down of POSH expression is neuroprotective through down-regulating activation of the MLK3-MKK4-JNK pathway following cerebral ischaemia in the rat hippocampal CA1 subfield

We investigated the expression and subcellular localization of the multidomain protein POSH (plenty of SH3s) by immunohistochemistry and western blot analysis, as well as its role in the selective activation of mixed-lineage kinases (MLKs) 3, MAP kinase kinase (MKK) 4, c-Jun N-terminal kinases (JNKs) and the c-Jun signalling cascade in the rat hippocampal CA1 region following cerebral ischaemia. Our results indicated that the cytosol immunoreactivity of POSH was strong in the CA1-CA3 pyramidal cell but weak in the DG granule cell of the rat hippocampus both in sham control and after reperfusion. Co-immunoprecipitation experiments showed that the interactions of MLK3, MKK4 and phospho-JNKs with POSH were persistently enhanced during the early (30 min) and the later reperfusion period (from 1 to 3 days) compared with sham controls. Consistently, MLK3-MKK4-JNK activation was rapidly increased with peaks both at 30 min and 3 days of reperfusion. Intracerebroventricular infusion of POSH antisense oligodeoxynucleotides (AS-ODNs) not only significantly reduced the protein level of POSH, markedly decreased its interactions with MLK3, MKK4 and phospho-JNKs, but also attenuated the activation of the JNK signalling pathway. In addition, infusion of POSH AS-ODNs significantly increased the neuronal density in the CA1 region at 5 days of reperfusion. Our results suggest that POSH might serve as a scaffold mediating JNK signalling activation in the hippocampal CA1 region following cerebral ischaemia, and POSH AS-ODNs exerts its protective effects on ischaemic injury through a mechanism of inhibition of the MLK3-MKK4-JNK signalling pathway, involving c-Jun and caspase 3 activation.     

Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop

A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death.     

POSH acts as a scaffold for a multiprotein complex that mediates JNK activation in apoptosis

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.     

Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs

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.     

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.     

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.     

Akt2 negatively regulates assembly of the POSH-MLK-JNK signaling complex

We demonstrate that POSH, a scaffold for the JNK signaling pathway, binds to Akt2. A POSH mutant that is unable to bind Akt2 (POSH W489A) exhibits enhanced-binding to MLK3, and this increase in binding is accompanied by increased activation of the JNK signaling pathway. In addition, we show that the association of MLK3 with POSH is increased upon inhibition of the endogenous phosphatidylinositol 3-kinase/Akt signaling pathway. Thus, the assembly of an active JNK signaling complex by POSH is negatively regulated by Akt2. Further, the level of Akt-phosphorylated MLK3 is reduced in cells expressing the Akt2 binding domain of POSH, which acts as a dominant interfering protein. Taken together, our results support a model in which Akt2 binds to a POSH-MLK-MKK-JNK complex and phosphorylates MLK3; phosphorylation of MLK3 by Akt2 results in the disassembly of the JNK complex bound to POSH and down-regulation of the JNK signaling pathway.     

The MLK Family Mediates c-Jun N-Terminal Kinase Activation in Neuronal Apoptosis

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 (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.     

Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.     

Tyrosine phosphorylation of HPK1 by activated Src promotes ischemic brain injury in rat hippocampal CA1 region

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that HPK1 is involved in c-Jun NH2-terminal kinase (JNK) signaling pathway by sequential activation of MLK3-MKK7-JNK3 after cerebral ischemia. Here, we used 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) and MK801 to investigate the events upstream of HPK1 in ischemic brain injury. Immunoprecipitation and immunoblot results showed that PP2 and MK801 significantly decreased the activation of Src, HPK1, MLK3, JNK3 and c-Jun, respectively, during ischemia/reperfusion. Histology and TUNEL staining showed PP2 or MK801 protects against neuron death after brain ischemia. We speculate that this unique signaling pathway through the tyrosine phosphorylation of HPK1 promotes ischemic brain injury by activated Src via N-methyl-d-aspartate receptor and, ultimately, the activation of the MLK3-MKK7-JNK3 pathway after cerebral ischemia.     

NRAGE,a p75 neurotrophin receptor-interacting protein,induces caspase activation and cell death through a JNK-dependent mitochondrial pathway

The p75 neurotrophin receptor (p75NTR) mediates signaling events leading to activation of the JNK pathway and cell death in a variety of cell types. We recently identified NRAGE, a protein that directly interacts with the p75NTR cytosolic region and facilitates p75NTR-mediated cell death. For the present study, we developed an inducible recombinant NRAGE adenovirus to dissect the mechanism of NRAGE-mediated apoptosis. Induced NRAGE expression resulted in robust activation of the JNK pathway that was not inhibited by the pharmacological mixed lineage kinase (MLK) inhibitor CEP1347. NRAGE induced cytosolic accumulation of cytochrome c, activation of Caspases-3, -9 and -7, and caspase-dependent cell death. Blocking JNK and c-Jun action by overexpression of the JNK-binding domain of JIP1 or dominant-negative c-Jun ablated NRAGE-mediated caspase activation and NRAGE-induced cell death. These findings identify NRAGE as a p75NTR interactor capable of inducing caspase activation and cell death through a JNK-dependent mitochondrial apoptotic pathway.     

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.     

POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila

Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. Here, we show that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs resulted in roughened or melanized eyes, respectively. These phenotypes were enhanced by co-overexpression of POSH. We found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway.