Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

Apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L) has been shown to exert important functions during various immunological processes. The involvement of the death adaptor proteins FADD/MORT1, TRADD, and RIP and the apoptosis-initiating caspases-8 and -10 in death signaling by the two death-inducing TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are controversial. Analysis of the native TRAIL death-inducing signaling complex (DISC) revealed ligand-dependent recruitment of FADD/MORT1 and caspase-8. Differential precipitation of ligand-stimulated TRAIL receptors demonstrated that FADD/MORT1 and caspase-8 were recruited to TRAIL-R1 and TRAIL-R2 independently of each other. FADD/MORT1- and caspase-8-deficient Jurkat cells expressing only TRAIL-R2 were resistant to TRAIL-induced apoptosis. Thus, FADD/MORT1 and caspase-8 are essential for apoptosis induction via TRAIL-R2.

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This study provides the first genetic evidence that caspase-8 occupies an essential and apical position in the Fas signaling pathway and suggests that caspase-8 may participate broadly in multiple apoptotic pathways.

We show here that caspase-8 is required for the death of primary rat neurons induced by an expanded polyglutamine repeat (Q79). Expression of Q79 recruited and activated caspase-8. Inhibition of caspase-8 blocked polyglutamine-induced cell death. Coexpression of Q79 with the caspase inhibitor CrmA, a dominant-negative mutant of FADD (FADD DN), Bcl-2, or Bcl-xL, but not an N-terminally tagged Bcl-xL, prevented the recruitment of caspase-8 and inhibited polyglutamine-induced cell death. Furthermore, Western blot analysis revealed the presence of activated caspase-8 in the insoluble fraction of affected brain regions from Huntington's disease (HD) patients but not in those from neurologically unremarkable controls, suggesting the relocation and activation of caspase-8 during the pathogenesis of HD. These results suggest an essential role of caspase-8 in HD-related neural degenerative diseases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70 S6k . Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3(glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1␤-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.Fas (APO-1/CD95) encodes a transmembrane type I receptor belonging to the tumor necrosis factor (TNF) receptor superfamily which includes TNF receptors 1 and 2 (TNFR1 and TNFR2), nerve growth factor (NGF) receptor, CD27, CD30, CD40, and OX40 (47, 67; reviewed in references 65 and 80). The cognate Fas ligand is a type II transmembrane protein belonging to the TNF family (83). The Fas receptor system has been extensively studied as a model of apoptosis, since crosslinking of the Fas receptor with Fas ligand or specific agonist antibodies results in rapid programmed cell death (47,89,97).Fas-induced apoptosis plays an important role in T-and B-cell homeostasis in the immune system and participates in T-cell activation-induced cell death (5,22,48), the elimination of autoreactive B cells (71), and the maintenance of sites of immune privilege (1). lpr mice defective in Fas (93), gld mice defective in Fas ligand (84), and humans with mutations in Fas (34, 73) all develop an autoimmune, lymphoproliferative disorder, indicating that Fas-induced death plays an important role in the peripheral deletion of lymphocytes.The essential signalling events linking Fas receptor crosslinking to apoptosis have been the subject of intense investigation. The intracellular domain of Fas contains an approximately 70-amino-acid "death domain" required for the induction...

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets key cell cycle regulatory proteins for degradation. Blockade of APC activity causes mitotic arrest. Recent evidence suggests that the APC may have roles outside the cell cycle. Several studies indicate that ubiquitin plays an important role in regulating synaptic strength. We previously showed that ubiquitin is directly conjugated to GLR-1, a C. elegans non-NMDA (N-methyl-D-aspartate) class glutamate receptor (GluR), resulting in its removal from synapses. By contrast, endocytosis of rodent AMPA GluRs is apparently regulated by ubiquitination of associated scaffolding proteins. Relatively little is known about the E3 ligases that mediate these effects. We examined the effects of perturbing APC function on postmitotic neurons in the nematode C. elegans. Temperature-sensitive mutations in APC subunits increased the abundance of GLR-1 in the ventral nerve cord. Mutations that block clathrin-mediated endocytosis blocked the effects of the APC mutations, suggesting that the APC regulates some aspect of GLR-1 recycling. Overexpression of ubiquitin decreased the density of GLR-1-containing synapses, and APC mutations blunted this effect. APC mutants had locomotion defects consistent with increased synaptic strength. This study defines a novel function for the APC in postmitotic neurons.

The polarized trafficking of axonal and dendritic proteins is essential for the structure and function of neurons. Cyclin-dependent kinase-5 (CDK-5) and its activator CDKA-1/p35 regulate diverse aspects of nervous system development and function. Here, we show that CDK-5 and CDKA-1/p35 are required for the polarized distribution of neuropeptide-containing dense-core vesicles (DCVs) in C. elegans cholinergic motor neurons. In cdk-5 or cdka-1/p35 mutants, the predominantly axonal localization of DCVs containing INS-22 neuropeptides was disrupted and DCVs accumulated in dendrites. Time-lapse microscopy in DB class motor neurons revealed decreased trafficking of DCVs in axons and increased trafficking and accumulation of DCVs in cdk-5 mutant dendrites. The polarized distribution of several axonal and dendritic markers, including synaptic vesicles, was unaltered in cdk-5 mutant DB neurons. We found that microtubule polarity is plus-end out in axons and predominantly minus-end out in dendrites of DB neurons. Surprisingly, cdk-5 mutants had increased amounts of plus-end-out microtubules in dendrites, suggesting that CDK-5 regulates microtubule orientation. However, these changes in microtubule polarity are not responsible for the increased trafficking of DCVs into dendrites. Genetic analysis of cdk-5 and the plus-end-directed axonal DCV motor unc-104/KIF1A suggest that increased trafficking of UNC-104 into dendrites cannot explain the dendritic DCV accumulation. Instead, we found that mutations in the minus-end-directed motor cytoplasmic dynein, completely block the increased DCVs observed in cdk-5 mutant dendrites without affecting microtubule polarity. We propose a model where CDK-5 regulates DCV polarity by both promoting DCV trafficking in axons and preventing dynein-dependent DCV trafficking into dendrites.