Pin1 in Neuronal Apoptosis

While the role of the prolyl isomerase Pin1 in dividing cells has long been recognized, Pin1’s function in postmitotic neurons is poorly understood. We have identified a novel mechanism by which Pin1 mediates activation of the mitochondrial cell death machinery specifically in neurons. This perspective presents a sophisticated signaling pathway that triggers neuronal apoptosis upon JNK-mediated phosphorylation of the BH3-only protein BIMEL at serine 65. Pin1 is enriched at the mitochondria in neurons together with BIMEL and components of a neuron-specific JNK signaling complex and functions as a molecular switch that couples the phosphorylation of BIMEL by JNK to apoptosis specifically in neurons. We discuss how these findings relate to our understanding of the development of the nervous system and the pathogenesis of neurologic disorders.     

Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery

A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.     

Pin1 promotes cell death in NGF-dependent neurons through a mechanism requiring c-Jun activity

Developing neurons deprived of trophic support undergo apoptosis mediated by activation of c-Jun N-terminal kinases (JNK) and c-Jun, induction of the Bcl-2 homology 3-only protein Bim_EL, Bax-dependent loss of mitochondrial cytochrome c , and caspase activation. However, the mechanisms that regulate each of these events are only partially understood. Here we show that the prolyl isomerase Pin1 functions as a positive regulator of neuronal death through a c-Jun-dependent mechanism. Ectopic Pin1 promoted caspase-dependent death of NGF-maintained neurons that was associated with an accumulation of Ser⁶³-phosphorylated c-Jun in neuronal nuclei and was partially dependent on Bax. Downregulating Pin1 prior to NGF withdrawal suppressed the accumulation of phosphorylated c-Jun, inhibited the release of cytochrome c , and significantly delayed cell death. Pin1 knockdown inhibited NGF deprivation-induced death to a similar extent in Bim (+/+) and Bim (−/−) neurons. The protective effect of Pin1 knockdown was significantly greater than that caused by loss of Bim and nearly identical to that caused by a dominant negative form of c-Jun. Finally, cell death induced by ectopic Pin1 was largely blocked by expression of dominant negative c-Jun. These results suggest a novel mechanism by which Pin1 promotes cell death involving activation of c-Jun.     

The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: identification of the nuclear pore complex as a potential target of the JNK pathway

c-Jun is induced in many neuronal death paradigms. A critical step in c-Jun regulation involves phosphorylation of Ser63/Ser73 located in the NH2-terminal transactivation domain. To determine the importance of this phosphorylation for neuronal apoptosis, we analyzed the sympathetic neurons of mice carrying a mutant c-Jun gene that lacks Ser63/Ser73 phosphorylation sites (jun aa). Trophic factor-deprivation or DNA damage-induced death was significantly delayed in jun aa/aa neurons. Neuronal c-Jun induction was only partially inhibited, demonstrating that phosphorylation of Ser63/73 is not required for c-Jun activation. The inductions of proapoptotic BH3-only proteins, Bim and PUMA/Bbc3, were delayed during neuronal apoptosis in mutant neurons. These results demonstrate that NH2-terminal c-Jun phosphorylation is important, but not necessary, for the induction of proapoptotic genes and neuronal apoptosis. Thus, additional JNK substrates may be critical for neuronal death. As potential mediators, we identified additional nuclear MLK/JNK substrates, including Nup214 subunit of the nuclear pore complex.     

Phosphorylation of Bcl-associated death protein (Bad) by erythropoietin-activated c-Jun N-terminal protein kinase 1 contributes to survival of erythropoietin-dependent cells

The glycoprotein erythropoietin (Epo) is a hematopoietic cytokine necessary for the survival of erythrocytes from immature erythroid cells. The mitogen-activated c-Jun N-terminal kinase 1 (JNK1) plays an important role in the proliferation and survival of erythroid cells in response to Epo. However, the precise mechanism of JNK1 activation promoting erythroid cell survival is incompletely understood. Here, we reported that JNK1 is required for Epo-mediated cell survival through phosphorylation and inactivation of the pro-apoptotic, Bcl-2 homology domain 3 (BH3)-only Bcl-associated death protein (Bad). Upon Epo withdrawal, HCD57 cells, a murine Epo-dependent cell line, displayed increased apoptotic cell death that was associated with decreased JNK1 activity. Epo withdrawal-induced apoptosis was promoted by inhibition of JNK1 activity but suppressed by expression of a constitutively active JNK1. Furthermore, Epo-activated JNK1 phosphorylated Bad at threonine 201, thereby inhibiting the association of Bad with the anti-apoptotic molecule B-cell lymphoma-extra large (Bcl-X(L)). Replacement of threonine 201 by alanine in Bad promoted Epo withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK1 contributes to the survival of erythroid cells.     

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (ΔΨm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X_L, Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X_L sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.     

Inhibition of JNK by overexpression of the JNL binding domain of JIP-1 prevents apoptosis in sympathetic neurons

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

Extracellular signal-regulated kinases 1/2 are serum-stimulated "Bim(EL) kinases" that bind to the BH3-only protein Bim(EL) causing its phosphorylation and turnover

Bim, a "BH3-only" protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of Bim(EL), targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim(EL) phosphorylation remained unclear. We now show that Bim(EL) is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of Bim(EL) occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim(EL), but not Bim(S) or Bim(L), in vitro, and mutation of Ser(65) to alanine blocks the phosphorylation of Bim(EL) by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GST-Bim(EL), but not GST-Bim(L) or GST-Bim(S), in vitro. ERK1/2 also binds to full-length Bim(EL) in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim(EL) protein. Our findings provide new insights into the post-translational regulation of Bim(EL) and the role of the ERK1/2 pathway in cell survival signaling.     

Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.     

BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons

The Bcl-2 family of proteins are key regulators of programmed cell death. A distinct subfamily of BH3-only molecules has been identified, but their exact mechanism of action remains unclear. Here we show that the BH3-only Bcl-2 family members, Dp5/Hrk and Bim, are induced upstream of the Bax checkpoint in neuronal apoptosis in a manner that shows significant dependence on JNK signaling. We also show that Dp5 and other BH3-only proteins kill cerebellar granule neurons in a Bax-dependent manner. These studies demonstrate that BH3-only members do not act independently in their proapoptotic activities but rather require the action of multidomain proapoptotic Bcl-2 family members to produce cell death.     

CD47 and the 19 kDa interacting protein-3 (BNIP3) in T cell apoptosis

CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Deltapsim). Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPalpha1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis.     

Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGD-dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis.     

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).