Through specific interactions with members of the tumor necrosis receptor (TNFR) family, adapter molecules such as the serine/threonine (Ser/Thr) kinase RIP mediate divergent signaling pathways including NF-B activation and cell death. In this study, we have identified and characterized a novel 61-kDa protein kinase related to RIP that is a component of both the TNFR-1 and the CD40 signaling complexes. Receptor interacting protein-2 (RIP2) contains an N-terminal domain with homology to Ser/Thr kinases and a C-terminal caspase activation and recruitment domain (CARD), a homophilic interaction motif that mediates the recruitment of caspase death proteases. Overexpression of RIP2 signaled both NF-B activation and cell death. Mutational analysis revealed the pro-apoptotic function of RIP2 to be restricted to its C-terminal CARD domain, whereas the intact molecule was necessary for NF-B activation. RIP2 interacted with other members of the TNFR-1 signaling complex, including inhibitor of apoptosis protein cIAP1 and with members of the TNFRassociated factor (TRAF) family, specifically TRAF1, TRAF5, and TRAF6, but not with TRAF2, TRAF3, or TRAF4. These TRAF interactions mediate the recruitment of RIP2 to receptor signaling complexes.

Inhibiting the production of amyloid-beta by antagonising gamma-secretase activity is currently being pursued as a therapeutic strategy for Alzheimer's disease (AD). However, early pre-clinical studies have demonstrated that disruption of presenilin-dependent gamma-secretase alters many presenilin-dependent processes, leading to early lethality in several AD model organisms. Subsequently, transgenic animal studies have highlighted several gross developmental side effects arising from presenilin deficiency. Partial knockdown or tissue-specific knockout of presenilins has identified the skin, vascular and immune systems as very sensitive to loss of presenilin functions. A more appreciative understanding of presenilin biology is therefore demanded if gamma-secretase is to be pursued as a therapeutic target. Herein we review the current understanding of gamma-secretase complexes; their regulation, abundance of interacting partners and diversity of substrates. We also discuss regulation of the gamma-secretase complexes, with an emphasis on the functional role of presenilins in cell biology.

The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic processing to generate a functional N-and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser 397 , which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3␤ (GSK-3␤) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser 397 position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3␤. Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF⅐CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser 397 by GSK-3␤ regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF⅐CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.

In this study we have shown that redistribution of the lipid composition of the external and internal leaflets of the PM during apoptosis results in two main alterations in the cell surface, externalisation of PS, and a looser packing of the lipid hydrophobic head groups in the external leaflet. Significantly, neither of these alterations can be directly implicated in the mechanism of apoptotic cell shrinkage, however they do have functions in other phases of the apoptotic process. Progressional studies involving morphological and flow cytometric evaluation, and DNA gel electrophoresis revealed that apoptotic cell shrinkage is associated with a decrease in [Na + ] i and [K + ] i which occurs after visualisation of chromatin condensation and internucleosomal DNA fragmentation, and prior to apoptotic body formation. When apoptotic cultures were supplemented with inhibitors of the Na + , K + -ATPase pump or the Ca 2+ -dependent K + channel, essentially all of the respective Na + or K + efflux during apoptosis can be inhibited, suggesting that essentially all of the Na + and K + efflux can be ascribed to active pumping via the Na + , K + -ATPase pump and the Ca 2+ -dependent K + channel.

The elimination of cells by programmed cell death is a fundamental event in development where multicellular organisms regulate cell numbers or eliminate cells that are functionally redundant or potentially detrimental to the organism. The evolutionary conservation of the biochemical and genetic regulation of programmed cell death across species has allowed the genetic pathways of programmed cell death determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster to act as models to delineate the genetics and regulation of cell death in mammalian cells. These studies have identified cell autonomous and non-autonomous mechanisms that regulate of cell death and reveal that developmental cell death can either be a pre-determined cell fate or the consequence of insufficient cell interactions that normally promote cell survival.

It is apparent that the underlying events in apoptosis are evolutionary conserved (1). First, there is similarity between the genetic, biochemical, and morphological events associated with apoptosis that are highly conserved between species (2, 3). Second, several of the key signaling and effector molecules have highly conserved counterparts that are functionally interchangeable between heterologous species (4 -6). Finally, an increasing number of viral proteins that inhibit apoptosis exhibit anti-apoptotic activity in both vertebrates and invertebrates (7-12).Three Drosophila melanogaster genes, reaper (rpr), grim, and hid, have been identified as key regulators of apoptosis during Drosophila embryogenesis (13-15). Deletion of a chromosomal segment encoding rpr, grim, and hid results in the loss of apoptotic cell death during fly development (16,17). Loss of rpr alone inhibits nearly all apoptosis during Drosophila embryogenesis and attenuates cell death in response to several external inducers. Overexpression of rpr as a transgene in Drosophila eyes or by transfection in a Drosophila cell line results in apoptosis (10, 13). Apoptosis induced by Reaper, a surprisingly small protein of 65 amino acid residues, involves activation of caspases because death is inhibited by specific caspase inhibitors (13,18,19). Grim is predicted to encode a 138-amino acid polypeptide and, like rpr, expression of grim RNA coincides with the onset of apoptosis during embryonic development, and its overexpression induces extensive apoptosis in transgenic flies and cell lines (15). Reaper and Grim appear to function independently of each other, as cell death induced by Grim does not require expression of reaper (15). Both proteins do, however, share a highly homologous N-terminal motif.The inhibitor of apoptosis proteins (IAP) 1 are a family of highly conserved anti-apoptotic proteins first identified in baculovirus by their ability to substitute functionally for the cell death inhibitor p35 (8,20,21). Baculovirus IAPs, namely Op-IAP and Cp-IAP, also block apoptosis in insect SF-21 cells induced by rpr expression (19) and actinomycin D (8). Additionally, Cp-IAP partially inhibits Reaper-induced cell killing in the Drosophila developing eye (10), and Op-IAP can significantly inhibit HeLa cell killing induced by mammalian caspase-1 (22) and the mammalian receptor-associated death adaptor molecule FADD (23). Cellular homologs of IAPs have also been described in humans (24) and Drosophila (10) that, like the baculovirus IAPs, block apoptosis in response to different stimuli.The first discovered human IAP, the neuronal apoptosis inhibitory protein (NAIP), was identified based on its role in the neurodegenerative disorder spinal muscular atrophy (SMA) (25). Recently, four other human homologs, c-IAP1, c-IAP2, X-IAP, and survivin, have been identified, and all demonstrate anti-apoptotic activity (23, 24, 26 -28). An important structural feature of all IAPs is an N-terminal motif termed the baculovirus IAP repeat (BIR). Human c-IAPs were orig...

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The majority of cases with early onset familial Alzheimer's disease have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 protein is a component of a high molecular weight membrane-bound complex that also contains ␤-catenin. The physiological relevance of the association between PS1 and ␤-catenin remains controversial. In this study, we report the identification and functional characterization of a highly conserved glycogen synthase kinase-3␤ consensus phosphorylation site within the hydrophilic loop domain of PS1. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, indicates that PS1 residues Ser 353 and Ser 357 are glycogen synthase kinase-3␤ targets. Substitution of one or both of these residues greatly reduces the ability of PS1 to associate with ␤-catenin. By disrupting this interaction, we demonstrate that the association between PS1 and ␤-catenin has no effect on A␤ peptide production, ␤-catenin stability, or cellular susceptibility to apoptosis. Significantly, in the absence of PS1/␤-catenin association, we found no alteration in ␤-catenin signaling using induction of this pathway by exogenous expression of Wnt-1 or ␤-catenin and a Tcf/ Lef transcriptional assay. These results argue against a pathologically relevant role for the association between PS1 and ␤-catenin in familial Alzheimer's disease.