Multifunctional reaper: sixty-five amino acids of fury.
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Amino Acids↗
Biomedical subjects
S Kornbluth
Publications and source records attributed to S Kornbluth.
Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper.
Protein folding mediated by the Hsp70 family of molecular chaperones requires both ATP and the co-chaperone Hdj-1. BAG-1 was recently identified as a bcl-2-interacting, anti-apoptotic protein that binds to the ATPase domain of Hsp70 and prevents the release of the substrate. While this suggested that cells had the potential to modulate Hsp70-mediated protein folding, physiological regulators of BAG-1 have yet to be identified. We report here that the apoptotic regulator Scythe, originally isolated through binding to the potent apoptotic inducer Reaper, shares limited sequence identity with BAG-1 and inhibits Hsp70- mediated protein refolding. Scythe-mediated inhibition of Hsp70 is reversed by Reaper, providing evidence for the regulated reversible inhibition of chaperone activity. As Scythe functions downstream of Reaper in apoptotic induction, these findings suggest that Scythe/Reaper may signal apoptosis, in part through regulating the folding and activity of apoptotic signaling molecules.
Adenosine Triphosphatases↗
Mitochondria in apoptosis and human disease.
Apoptosis is a process of cell suicide whereby individual cells are destroyed while preserving the integrity and architecture of surrounding tissue. This targeted cell destruction is critical both in physiological contexts as well as pathological states. It seems increasingly evident that mitochondria play an important role in the regulation of programmed cell death via release of proapoptotic agents and/or disruption of cellular energy metabolism. The mechanisms of mitochondrial involvement are beginning to be elucidated, and may involve the participation of bcl-2 family members, reactive oxygen species, and caspases. As part of a central mechanism of amplification of the apoptotic signal, mitochondria may be an appropriate target for therapeutic agents designed to modulate apoptosis. This review focuses on recent advances in understanding mitochondrial mechanisms in apoptosis and the involvement of these pathways in human disease.
Animals↗
Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts.
Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230-241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.
Amino Acid Sequence↗
Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites.
Entry into mitosis is regulated by the Cdc2 kinase complexed to B-type cyclins. We and others recently reported that cyclin B1/Cdc2 complexes, which appear to be constitutively cytoplasmic during interphase, actually shuttle continually into and out of the nucleus, with the rate of nuclear export exceeding the import rate (). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid nuclear accumulation of Cdc2/cyclin B1. Although it has recently been reported that phosphorylation of 4 serines within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at G(2)/M, the role that individual phosphorylation sites play in this process has not been examined (, ). We report here that phosphorylation of a single serine residue (Ser(113) of Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence and, when phosphorylated, prevents binding of the nuclear export factor, CRM1. In contrast, analysis of phosphorylation site mutants suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin B1/Cdc2 characteristic of G(2)/M. Additionally, binding of cyclin B1 to importin-beta, the factor known to be responsible for the slow interphase nuclear entry of cyclin B1, appears to be unaffected by the phosphorylation state of cyclin B. These data suggest that a distinct import factor must be recruited to enhance nuclear entry of Cdc2/cyclin B1 at the G(2)/M transition.
Active Transport, Cell Nucleus↗
Requirement of the prolyl isomerase Pin1 for the replication checkpoint.
The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.
Animals↗
A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch.
We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein. Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants. Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts. While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop. These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events.
Adaptor Proteins, Signal Transducing↗
Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.
Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.
Animals↗
Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import.
Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends upon preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Here we report that Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and that progesterone treatment abrogates this binding. We demonstrate that Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation.
14-3-3 Proteins↗
Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.
Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.
Animals↗
All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners.
Progression through the cell cycle is governed by the periodic activation and inactivation of cyclin-dependent kinase complexes (CDK-cyclins). Although the enzymatic activity of these complexes is regulated tightly, it has recently been demonstrated that an additional facet of cell-cycle control involves the modulation of CDK-cyclin subcellular localization. Recent discoveries include the identification of nuclear transport factors responsible for ferrying some of the CDK-cyclins in and out of the nucleus, the demonstration that phosphorylation can regulate these transport processes and the establishment of potential links between cell-cycle checkpoints and the control of CDK-cyclin subcellular localization.
Animals↗
Mitochondria at the crossroad of apoptotic cell death.
In the past few years, it has become widely appreciated that apoptotic cell death generally involves activation of a family of proteases, the caspases, which undermine the integrity of the cell by cleavage of critical intracellular substrates. Caspases, which are synthesized as inactive zymogens, are themselves caspase substrates and this cleavage leads to their activation. Hence, the potential exists for cascades of caspases leading to cell death. However, it has been recently recognized that another, perhaps more prominent route to caspase activation, involves the mitochondria. Upon receipt of apoptotic stimuli, either externally or internally generated, cells initiate signaling pathways which converge upon the mitochondria to promote release of cytochrome C to the cytoplasm; cytochrome c, thus released, acts as a potent cofactor in caspase activation. Even cell surface "death receptors" such as Fas, which can trigger direct caspase activation (and potentially a caspase cascade), appear to utilize mitochondria as part of an amplification mechanism; it has been recently demonstrated that activated caspases can cleave key substrates to trigger mitochondrial release of cytochrome c, thereby inducing further caspase activation and amplifying the apoptotic signal. Therefore, mitochondria play a central role in apoptotic cell death, serving as a repository for cytochrome c.
Animals↗
Treatment of spider veins using a 10 millisecond pulse-duration frequency-doubled neodymium YAG laser.
BACKGROUND: The pulsed dye laser has been the standard for treating vascular lesions. Although quite effective for treating facial vessels and port-wine stains, spider veins of the lower extremities are more difficult to treat. Recent studies have shown that lasers with longer pulse durations are more effective at treating spider veins. A new long-pulse frequency-doubled Neodymium:YAG laser has been developed with a 10-ms pulse duration and sufficient energy to enable treatment with a 3- or 4-mm diameter treatment beam. OBJECTIVE: To determine the effectiveness of the long pulse Neodymium:YAG laser for treating spider veins of the lower extremities. METHODS: Spider veins less than 0.75 mm in diameter on the legs of 15 female volunteers were treated in 1 or 2 areas. Treatments were administered through a water-cooled chill tip using the frequency-doubled Neodymium:YAG laser with a 10-ms pulse duration. A dose of 16 J/cm2 was administered, completing 3 passes over each visible vein during each session, for a total of 2 sessions administered 6 weeks apart. Photographs of treatment areas were digitally analyzed for degree of vessel clearance. RESULTS: Computer-based image analysis revealed clearing of over 75% of veins following 2 treatments with 16 J/cm2. Side effects were minimal, and the treatments were well tolerated. CONCLUSIONS: The 532 nm, 10 ms pulse duration, frequency-doubled Neodymium:YAG laser is safe and effective for treating spider veins of the lower extremities less than 0.75 mm in diameter, in patients with Fitzpatrick skin Types I-III.
Adult↗
Scythe: a novel reaper-binding apoptotic regulator.
Reaper is a central regulator of apoptosis in Drosophila melanogaster. With no obvious catalytic activity or homology to other known apoptotic regulators, reaper's mechanism of action has been obscure. We recently reported that recombinant Drosophila reaper protein induced rapid mitochondrial cytochrome c release, caspase activation and apoptotic nuclear fragmentation in extracts of Xenopus eggs. We now report the purification of a 150 kDa reaper-interacting protein from Xenopus egg extracts, which we have named Scythe. Scythe is highly conserved among vertebrates and contains a ubiquitin-like domain near its N-terminus. Immunodepletion of Scythe from extracts completely prevented reaper-induced apoptosis without affecting apoptosis triggered by activated caspases. Moreover, a truncated variant of Scythe lacking the N-terminal domain induced apoptosis even in the absence of reaper. These data suggest that Scythe is a novel apoptotic regulator that is an essential component in the pathway of reaper-induced apoptosis.
Amino Acid Sequence↗
The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.
The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Here we demonstrate that depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. We show biochemically that this phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 was able to bind Pin1, an event we have recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1.
Amino Acid Sequence↗
Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.
Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.
Animals↗
Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c.
Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation. However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated and, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, we added active caspase-8 to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promoted the apoptotic program, culminating in fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produced DNA degradation, but left nuclear membranes intact. Thus, mitochondria were required for complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations of caspase-8 were required to activate downstream caspases. However, when mitochondria were present, the effects of low concentrations of caspase-8 were vastly amplified through cytochrome c-dependent caspase activation. Caspase-8 promoted cytochrome c release indirectly, by cleaving at least one cytosolic substrate. Bcl-2 blocked apoptosis only at the lowest caspase-8 concentrations, potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2.
Animals↗
Regulation of apoptosis in Xenopus egg extracts.
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Animals↗