|
|
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© The Rockefeller University Press,
0021-9525/2000//1391 $5.00
The Journal of Cell Biology, Volume 151, Number 7,
, 2000 1391-1400
Original Article |
Wee1-Regulated Apoptosis Mediated by the Crk Adaptor Protein in Xenopus Egg Extracts
kornb001{at}mc.duke.edu
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.
Key Words: apoptosis • caspase • Crk • Wee1 • Xenopus
© 2000 Government
 |
Introduction |
|---|
 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
|---|
Although apoptotic signaling pathways differ between cell types, in most cells the key executioners of the apoptotic process are members of a protease family known as the caspases (Alnemri 1997). These proteases, which share homology with the Caenorhabditis elegans death gene product, CED-3, are synthesized in a zymogenic form and activated either by proximity-induced autoprocessing or cleavage in trans by other caspases. Once activated, caspases undermine cellular integrity by cleaving key cellular substrates such as nuclear lamins and gelsolin (for review see Thornberry and Lazebnik 1998).
In many apoptotic cells, caspase activation is preceded by release of cytochrome c from the intermembrane space of the mitochondria to the cytoplasm (for review see Green and Reed 1998). Once released into the cytosol, cytochrome c serves as an activating cofactor in a multimeric structure known as the "apoptosome," comprised of a caspase (caspase-9), an ATP-binding protein (Apaf-1), and ATP (or dATP) (Liu et al. 1996; Li et al. 1997; Zou et al. 1999). The apoptosome, once activated, induces proteolytic activation of procaspase-9, which subsequently cleaves and activates a workhorse of execution, caspase-3. Hence, the mitochondrial release of cytochrome c is a paramount site of regulation of programmed cell death, in particular, by the Bcl-2 family of proteins (for review see Gross et al. 1999). Proapoptotic members of the family, such as Bax, promote the release of cytochrome c, while antiapoptotic members, such as Bcl-xL, inhibit translocation of cytochrome c from the mitochondrial intermembrane space to the cytosol.
Apoptosis has been very well conserved in metazoans. Not only are homologous apoptotic signaling molecules conserved among flies, frogs, worms, and mammals, but apoptotic regulators in one system can often functionally substitute for those in another (e.g., Hengartner and Horvitz 1994; Newmeyer et al. 1994; Evans et al. 1997a; Kuwana et al. 1998; Dorstyn et al. 1999). In keeping with this evolutionary conservation of the apoptotic program, the dramatic biochemical and morphological events of apoptosis can be recapitulated in a cell-free system derived from eggs of the frog, Xenopus laevis (Newmeyer et al. 1994; Evans et al. 1997b; Kluck et al. 1997b). When these extracts are incubated at room temperature, hallmark apoptotic activities, such as mitochondrial cytochrome c release, caspase activation, cleavage of apoptotic substrates, and DNase activation can be observed. Furthermore, nuclei added to these extracts undergo morphological changes characteristic of apoptosis, including chromatin condensation, membrane vesiculation, and ultimately, complete nuclear fragmentation. Although the physiological basis for this apoptotic program has not been precisely defined, it has been speculated that these extracts serve as an in vitro model for oocyte atresia, wherein matured oocytes that are not laid as eggs are reabsorbed by apoptotic cell death (Hughes and Gorospe 1991; Smith et al. 1991; Tilly et al. 1992; Newmeyer et al. 1994).
Characterization of Xenopus egg extracts by several laboratories has established that the egg extract displays appropriate biochemical responses to common inhibitors of apoptosis such as peptide inhibitors of caspases (ZVAD, YVAD, DEVD, among others) and Bcl-2 (Newmeyer et al. 1994; Kluck et al. 1997a,Kluck et al. 1997b). Additionally, previous work has demonstrated that this system is responsive to proapoptotic signaling molecules such as Drosophila Reaper and human caspase-8 (Evans et al. 1997a; Kuwana et al. 1998). Moreover, it has been firmly established that mitochondrial cytochrome c release is critical for apoptosis in this system, as in other systems (Kluck et al. 1997a,Kluck et al. 1997b).
In analyzing the requirements for in vitro apoptosis in this system, we and others have previously demonstrated a role for phosphotyrosine signaling pathways in the early events of apoptosis (Liu et al. 1994; Migita et al. 1994; Evans et al. 1997b; Farschon et al. 1997). Building on this, we found that the adaptor protein, Crk, is required for the in vitro apoptosis in this system: immunodepletion of Crk or addition of anti-Crk antisera, rendered the extracts unable to undergo apoptosis (Evans et al. 1997b). Consistent with these results, we found that the isolated Src homology (SH) 2 domain of Crk, but not analogous domains from other adaptor proteins, could inhibit apoptosis, presumably by acting as a dominant negative inhibitor of endogenous Crk–phosphotyrosine signaling interactions. Additionally, depleting extracts of Crk SH2-binding proteins (using the glutathione S-transferase [GST]–Crk SH2 domain bound to glutathione-Sepharose) effectively inhibited apoptosis (Evans et al. 1997b).
We report here the surprising finding that the relevant Crk SH2 interacting protein, important for apoptotic signaling in the extract, is the previously well-characterized cell cycle regulator, Wee1 (Nurse and Thuriaux 1980; Igarashi et al. 1991; Parker et al. 1992; McGowan and Russell 1993, McGowan and Russell 1995). Specifically, we have purified Wee1 as a Crk SH2 interactor, demonstrated that Wee1 is required for apoptosis in the extract, and shown that addition of exogenous Wee1 can markedly accelerate apoptosis. Although Wee1 is best known as an inhibitor of the cell cycle kinase, Cdc2, this activity does not seem to be responsible for its apoptotic role, as other similarly potent Cdc2 inhibitors do not enhance apoptotic signaling. Perhaps most importantly, we have found that Wee1's role in apoptosis is linked to Crk signaling. Addition of exogenous Wee1 restored apoptosis to an extract depleted of Crk SH2 interactors, and the ability of Wee1 to accelerate in vitro apoptosis was dependent on the presence of Crk. Collectively, our data suggest that Wee1 and Crk cooperate in a novel pathway of apoptosis in Xenopus egg extracts.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation of Xenopus Egg Extracts
For induction of egg laying, mature female frogs were injected with 100 U of pregnant mare serum gonadotropin (Calbiochem) to induce maturation of oocytes. Subsequently, 3–28 d later, a second injection of human chorionic gonadotropin (Upstate Biotechnology) was given to these frogs. Within 12–20 h after human chorionic gonadotropin injection, eggs were harvested for production of extracts. Jelly coats were removed using a 2% cysteine solution (pH 8.0). Eggs were then washed three times using modified Ringer's solution (MMR: 1 M NaCl, 20 mM KCl, 10 mM MgSO4, 25 mM CaCl 2, 5 mM Hepes, pH 7.8, 0.8 mM EDTA), and finally, washed three times in egg lysis buffer (ELB: 250 mM sucrose, 2.5 mM MgCl2, 1.0 mM dithiothreitol, 50 mM KCl, 10 mM Hepes, pH 7.7). Eggs were packed using low-speed centrifugation at 400 g, and subsequently cytochalasin B (5 µg/ml, final concentration; Calbiochem), aprotinin/leupeptin (5 µg/ml, final concentration), and cycloheximide (50 µg/ml, final concentration) were added. Egg lysis was performed using centrifugation at 10,000 g for 15 min. The crude extracts generated from this protocol were supplemented with an energy regenerating system consisting of 2 mM ATP, 5 µg/ml creatine kinase, and 20 mM phosphocreatine (final concentrations). Recombinant proteins added to these extracts were diluted into protein concentrating buffer (XB: 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM KOH-Hepes, pH 7.7, and 50 mM sucrose) at the indicated protein concentrations.
For visual apoptosis assays, nuclei were formed in extracts by addition of demembranated sperm chromatin (
2,000 nuclei/µl). Extract samples were taken at various time points during a room temperature incubation and subjected to formaldehyde fixation and staining with Hoechst 33258. Changes in nuclear morphology associated with apoptosis were monitored using fluorescence microscopy.
To prepare purified cytosolic extracts, crude interphase extract was ultracentrifuged at 200,000 g (70 min, 4°C) in a Beckman Coulter TL-100 centrifuge using a TLS 55 rotor. The cytosolic fraction was removed and recentrifuged for an additional 25 min at 200,000 g.
Purification of Crk SH2-binding Proteins
25 ml of crude egg extract was incubated with 5 ml of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) coupled to GST or GST–Crk SH2 for 30 min at room temperature using the manufacturer's protocol. The beads were pelleted and washed five times with ELB including 1 mM sodium vanadate and combined in a Bio-Rad Laboratories column. The bound proteins were eluted from the resin with 10 ml (5 x 2 ml) boiling SDS-PAGE buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 0.7 M β-mercaptoethanol). The eluate was loaded into dialysis tubing (8-kD molecular mass cut off) and concentrated by placing Aquacide I (Calbiochem) around the dialysis tubing. When the eluate had been concentrated to 200 µl, it was dialyzed against 62.5 mM Tris, pH 6.8, 0.7 M β-mercaptoethanol overnight, supplemented with bromophenol blue, and loaded on a long 7.5% SDS-PAGE gel. The gel was stained with Pro Blue Colloidol Blue stain (Owl Separation Systems), and relevant protein bands were subjected to peptide microsequencing (as described below).
Peptide Microsequencing
The bands of interest were excised from the one-dimensional gel and digested in-gel with trypsin (Boehringer) in 10 mM Tris, pH 8.0, according to the procedure of Shevchenko et al. 1996 except that alkylation of sulfhydryls was accomplished with 4-vinylpyridine. After incubation at 37°C for 16 h, digests were vortexed and placed in a sonication bath for 5 min. The liquid around the gel pieces was removed and saved. The gel pieces were then extracted with a second aliquot of 10 mM Tris that was pooled with the extracts. Finally, the gel pieces were extracted with acetonitrile, which was combined with the other extracts. Volume and organic content in the in-gel digest extract was reduced by concentration in a Speed Vac (60 µl, final volume). Proteolytic peptides from the in-gel digests were analyzed using a fully automated nanoscale capillary liquid chromatography system (Famos autoinjector/Ultimate chromatograph; LC Packings, Inc.) coupled with a hybrid quadrupole/time-of-flight tandem mass spectrometer (Q-Tof; Micromass, Inc.). 50-µl aliquots of the proteolytic peptides in 0.1% formic acid–water were injected by the auto sampler onto a preconcentration/desalting column (300 µm inner diameter [ID] x 1 mm long, 5 µm PepMap C 18; LC Packings, Inc.) at a flow rate of 25 µl/min. After desalting using a mobile phase of 0.1% formic acid–water, the trapped peptides were backflushed onto a nanoscale capillary liquid chromatography (LC) column (75 µm ID x 15 cm long, 3 µm PepMap C 18; LC Packings, Inc.). A linear mobile phase gradient at 200 nl/min of 2–32% acetonitrile in water (0.1% formic acid in both buffers) over 30 min was used to elute the peptides into the ion source (Z-Spray; Micromass, Inc.) of the mass spectrometer. The electrospray was generated by connecting the LC eluant to nanoelectrospray tips (360 µm outer diameter [OD], 20 µm ID, tapered down to 10 µm OD, 5 µm ID; New Objective, Inc.) which were distally coated with platinum. A 2,000 V potential was applied to the spraytips, giving a stable electrospray across the LC gradient. Data-dependent scanning software (MassLynx; Micromass, Inc.) was used to acquire both molecular weight (MS spectra) and amino acid sequence (production MS/MS spectra) information from the peptides in chromatographic real time. Protein identifications from the dataset were accomplished by using Mascot software (Matrix Sciences) to search a nonredundant database, comparing the experimentally obtained MS data (molecular weight) and MS/MS data (amino acid sequence) with in-silico predictions from the nonredundant database.
DEVDase Assays
To monitor caspase activity, 3-µl aliquots of each extract sample at various time points were incubated with 90 µl of DEVDase buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% glycerol) and the colorimetric peptide substrate Ac-DEVD-pNA (200 mM, final concentration) (BIOMOL Research Labs, Inc.). Enzyme reactions were performed at 37°C for 30–60 min. The absorbance of the colorimetric product was measured at 405 nm using a LabSystems MultiSkan MS microtiter plate reader. As the samples for this assay were analyzed spectrophotometrically, the density of pigment granules in the extracts caused some variation in the background level of absorbance at 405 nm.
Crk and Wee1 Coimmunoprecipitation
Anti–c-Myc, HA, or Crk monoclonal antibody (2.5 µg antibody/sample) was coupled to protein A– Sepharose beads (Sigma-Aldrich) for 1 h at 4°C and washed in ELB twice. The antibody-coupled beads were blocked to prevent nonspecific protein binding in ELB plus 10 mg/ml BSA for 20 min at 4°C and washed twice with ELB. The isolated cytosolic extract was supplemented with 1 mM sodium vanadate and an ATP-regeneration mix and incubated at room temperature for 30 min. 20 µl of antibody-coupled beads was added to 175 µl cytosolic extract and incubated for 1 h at 4°C. The beads were then pelleted and washed four times with ELB containing 1 mM sodium vanadate. Proteins were eluted from beads by boiling in 2x sample buffer and run on a 7.5% SDS-PAGE gel. The gel was transferred to membrane and probed with polyclonal antisera directed against the XWee1 protein.
This same immunoprecipitation was also performed using affinity-purified polyclonal antisera directed against XWee1 (Zymed Laboratories) coupled to protein A–Sepharose (as described above) or affinity-purified rabbit IgG (Jackson ImmunoResearch Laboratories). Proteins eluted from these beads were separated on a 10.5% SDS-PAGE gel and Western blot analysis was performed using a polyclonal antibody directed against Crk (Transduction Laboratories).
Immunodepletion and Antibody Addition Assays
Protein A–Sepharose resin (50-µl aliquots) (Sigma-Aldrich) was washed two times with ELB and subsequently incubated with 1% BSA in ELB for 30 min at 4°C. This resin was then washed two additional times with ELB and then incubated with 10 µg of anti-XWee1 IgG (Zymed Laboratories) (control: 10 µg of rabbit IgG; Jackson ImmunoResearch Laboratories) or 50 µg total protein of polyclonal anti-Crk antiserum (control: 50 µg total protein of preimmune serum) in a 1% BSA/ELB solution (total volume 400 µl). Protein A–IgG complexes were formed at 4°C for 1 h and then washed two times in ELB. Egg extract samples (250 µl) were depleted with the protein A–antibody resins (50 µl) two times for 30 min at 4°C. Depleted extracts were supplemented with the energy-regenerating cocktail (described above), incubated at room temperature for 3–5 h, and subjected to a DEVDase activity assay (as described above).
Crk SH2-binding Protein Pulldown Assays
The GST, Crk wild-type, Crk R38K SH2, or Crk SH2 recombinant proteins were linked to glutathione-Sepharose beads. For pulldown assays analyzed by Western blotting, 25 µl of beads was incubated with 200 µl extract for 30 min at room temperature. The beads were then pelleted and washed three to five times with ELB supplemented with 1 mM sodium vanadate to prevent tyrosine dephosphorylation. The bound proteins were eluted in 2x SDS sample buffer and separated on 7.5% SDS-PAGE gel.
For determining effects of Crk SH2 domain on apoptotic activity, this same technique was used. Extracts were depleted two times on the GST protein resins under the conditions described above. The depleted extracts were then supplemented with the energy-regenerating mix described above and incubated at room temperature for 3–5 h. Samples were taken from these extracts at various time intervals and subjected to a DEVDase assay (as described above).
Immunoblotting
Immunoblotting was performed after SDS-PAGE and transfer to polyvinylidene fluoride membranes (Millipore). Blots were incubated with appropriate primary antisera described above and subsequently with (secondary antibodies) horseradish peroxidase–linked goat anti–rabbit antibody or goat anti–mouse antibody (Jackson ImmunoResearch Laboratories). Blots were developed using an enhanced chemiluminescence kit (Renaissance; Dupont).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
As shown in Fig. 1 B, the XWee1 sequence contains several previously mapped tyrosine phosphorylation sites that conform to the consensus for Crk SH2 binding (YXXP/L) (Birge et al. 1993; Songyang et al. 1993). To confirm that Wee1 could bind to the Crk SH2 domain, the population of Crk-binding proteins was again resolved by SDS-PAGE and examined by immunoblotting with anti-Wee1 sera. As anticipated, the anti-XWee1 sera recognized a band of 68 kD in the GST–Crk pulldown, which was absent from control pulldowns with GST alone (Fig. 2 A). Consistent with a role for tyrosine phosphorylation in mediating the SH2–Wee1 interaction, a mutant form of the Crk SH2 domain (R38K; Mayer and Hanafusa 1990) unable to bind tyrosine phosphorylated substrates was not able to bind Wee1 from egg extracts (Fig. 2 B). In addition, the Crk SH2 domain could not bind a variant of Wee1 mutated so as to change tyrosine to phenylalanine at the known sites of Wee1 tyrosine phosphorylation (Fig. 2 C).
|
|
10 ng/µl) to extracts supplemented with nuclei promoted accelerated membrane blebbing, chromatin condensation, and nuclear fragmentation. As has been seen with other stimuli that accelerate apoptosis in the egg extract, the caspase activation and nuclear fragmentation were preceded by mitochondrial cytochrome c release (data not shown). Consistent with a requirement for mitochondrial cytochrome c release, Wee1 could not promote caspase activation in purified cytosolic extracts lacking mitochondria (Fig. 4 C). However, the addition of exogenous cytochrome c activated caspases in identical extracts, demonstrating their apoptotic potential. While the absolute timing of spontaneous apoptosis varies from extract to extract, in all cases recombinant Wee1 hastened the onset of apoptosis.
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Wee1 Regulates Apoptosis in Xenopus Egg Extracts
For each batch of Xenopus egg extract, there is a characteristic time that elapses before that extract enters apoptosis. Using many different extracts, we found that addition of exogenous recombinant Wee1 consistently and reproducibly accelerated the initiation of apoptosis, as measured by mitochondrial cytochrome c release, caspase activation, and nuclear fragmentation. Consistent with a role for Wee1 in modulating the in vitro apoptosis in Xenopus egg extracts, neutralization of Wee1 function through either antibody addition or immunodepletion severely delayed the onset of apoptosis.
Although incubation of egg extracts at room temperature leads to activation of the apoptotic program, the origin of the apoptotic signal in this system is unknown. The ability of these extracts to undergo apoptosis has been speculated to reflect the in vivo process of oocyte atresia, wherein oocytes that do not receive the appropriate trophic support die by apoptosis (Hughes and Gorospe 1991; Smith et al. 1991; Tilly et al. 1992; Newmeyer et al. 1994). An alternative hypothesis is based on the observation that inhibition of zygotic transcription/translation usually initiated at the midblastula transition (MBT) results in massive apoptosis of embryos at the early gastrula transition. (Hensey and Gautier 1997; Sible et al. 1997; Stack and Newport 1997). The available data on this phenomenon are consistent with the presence of a maternal apoptotic inhibitor that is degraded over time and must be replaced by synthesis of new (or more) zygotic inhibitors at the MBT to prevent apoptosis when the maternal inhibitor is depleted at the early gastrula transition. It is possible that a maternal apoptotic program is unmasked when maternally encoded inhibitors of apoptosis are degraded or otherwise inactivated by incubation of egg extract on the bench.
In Xenopus, Wee1 is present during early oogenesis (stages I–IV), is absent from stage VI oocytes, reappears at meiosis II, and persists throughout gastrulation (Murakami and Vande Woude 1998; Nakajo et al. 2000). Therefore, if Wee1 participates in an in vivo process of oocyte atresia, it might do so early in oogenesis. Perhaps more plausibly, the amount of Wee1 translated as oocytes transit to maturation might determine the propensity of the mature oocyte/egg to undergo apoptosis before laying and fertilization. Indeed, preliminary data suggest that extracts prepared from stage VI oocytes, lacking Wee1, are refractory to apoptosis in vitro (Evans, E.K., and S. Kornbluth, unpublished results).
Although Wee1 plays a preeminent role in regulating entry into mitosis, this function of Wee1 reflects its ability to suppress cyclin-dependent kinases (Parker et al. 1992; McGowan and Russell 1993; Coleman and Dunphy 1994; Murakami and Vande Woude 1998; Murakami et al. 1999; Nakajo et al. 2000; Walter et al. 2000). The role of Cdc2 and its close relative, Cdk2, in modulating apoptosis has been somewhat controversial. Although discrepancies may legitimately be ascribed to cell type differences, active Cdc2 or Cdk2 has been variously reported either to accelerate apoptosis, inhibit apoptosis, be required for apoptosis, or be entirely dispensable for apoptosis (Meikrantz et al. 1994; Norbury et al. 1994; Chen et al. 1995; Ongkeko et al. 1995; Meikrantz and Schlegel 1996; Yao et al. 1996; Zhou et al. 1998). As suppression of Cdc2 by either Myt1 or roscovitine had no effect on the rate of apoptotic progression in our extracts, we consider it unlikely that simple suppression of Cdc2/Cdk2 activities underlies the ability of Wee1 to accelerate apoptosis. Although ectopic expression of Myt1 in oocytes was reportedly unable to function like Wee1 in suppressing germinal vesicle breakdown, in that case the authors suspected that Myt1 was only weakly active either due to expression levels or progesterone-induced down regulation (Nakajo et al. 2000). In our experiments, Wee1 and Myt1 preparations were added exogenously, obviating expression problems, after calibration for equivalent levels of Cdc2–cyclin B-phosphorylating activity; roscovitine concentrations used were also shown to be entirely effective in suppressing cyclin B–induced activation of histone H1–directed kinase activity (data not shown). Therefore, these experiments point to an alternative mode of action for Wee1. It is possible that Wee1 does double duty as a cell cycle regulator and an apoptotic regulator, ideally situating it as a pivotal participant in the decision to proceed to fertilization and embryogenesis or to enter apoptosis.
Wee1 Interacts with a Crk-dependent Apoptotic Signaling Pathway
Previous work aimed at elucidating a pathway of apoptotic signaling in Xenopus egg extracts implicated the adaptor protein Crk and proteins interacting with its SH2 and NH2-terminal SH3 domains (Evans et al. 1997b). Although Wee1 is not a particularly abundant protein in Xenopus egg extracts, in affinity chromatography experiments it emerged as a prominent binding partner of the isolated Crk SH2 domain. Moreover, the ability of exogenous Wee1 to accelerate apoptosis depended on the presence of Crk in the extract.
As the name "adaptor" implies, Crk and proteins like it are believed to function in the physical joining, or colocalization, of distinct classes of molecules, in this case tyrosine phosphorylated SH2 binders and polyproline-containing SH3 interactors (Birge et al. 1996). Most commonly, a membrane-bound tyrosine kinase receptor autophosphorylates after ligand engagement, providing a membrane docking site for an adaptor protein, thereby coconcentrating SH3-bound ligands at the membrane. However, in the case of Wee1–Crk, it would seem that a predominantly nuclear protein, Wee1, interacts with Crk to signal apoptosis. Although Crk has been reported to localize predominantly to focal adhesions in intact cells, we and others have also observed a substantial proportion of Crk in the nucleus of cultured cells, both by immunofluorescence and GFP tagging (Matsuda et al. 1993; Smith, J.J., and S. Kornbluth, unpublished results). Moreover, fluorescein-tagged recombinant Crk concentrates in nuclei in the egg extract (Evans, E.K., and S. Kornbluth, unpublished results). Therefore, it is entirely possible that Crk–Wee1 is part of a proapoptotic signal emanating from the nucleus.
In Xenopus egg extracts, intact nuclei only form upon addition of exogenous chromatin or nuclei. However, even in the absence of added nuclei, light membranes present in the extract form sheets of annulate lamellae containing nuclear pore complexes which are competent to transport macromolecules; hence, even an extract entirely lacking nuclei contains structures which may accumulate normally nuclear proteins. Interestingly, the basal rate of caspase activation in extracts containing added nuclei substantially exceeded that in extracts lacking nuclei (Smith, J.J., and S. Kornbluth, unpublished results), and Wee1 further accelerated apoptosis in nuclei-containing extracts (data not shown).
An alternative model for Wee1–Crk function, independent of nuclear compartmentalization, is that Crk functions to bring Wee1 in close contact with a Crk SH3 binder; conversely, Wee1 might function to juxtapose Crk and one of its (non-Crk) binding partners. We have found that depletion of Crk SH3 interactors from egg extracts (using a GST–Crk SH3 column) inhibits Wee1-induced acceleration of apoptosis, further implicating downstream Crk-mediated signaling pathways in Wee1-dependent apoptotic pathways (Smith, J.J., and S. Kornbluth, unpublished results). Interestingly, Coomassie blue staining of GST–Crk SH2 interactors (run in parallel with the antiphosphotyrosine Western blot shown in Fig. 1) revealed approximately seven specific proteins associated with the SH2 precipitates (data not shown). This suggests the possibility that Crk–Wee1 may be part of a larger multiprotein complex. However, apart from Wee1, none of the Coomassie blue–stainable proteins were quantitatively depleted from the extract in association with the GST–SH2 resin (Evans, E.K., and S. Kornbluth, unpublished results). Distinguishing between the various models of Crk–Wee1 function will require identification of Crk SH3 ligands, or potentially other Wee1 ligands, which are required for apoptosis. Interestingly, if any of these mechanisms are relevant to very early embryonic development in Xenopus, they must be engaged in the beginning of the first embryonic cell cycle, since tyrosine phosphorylation of Wee1, upon which Crk binding depends, disappears after the first cell cycle until after the MBT (Murakami et al. 1999). Alternatively, Wee1 tyrosine phosphorylation may be permissive for engagement of the apoptotic machinery after the MBT.
Crk–Wee1 and Mammalian Cell Apoptosis
Inevitably, the question arises as to whether the Wee1–Crk complex participates in an apoptotic process unique to Xenopus oocytes/eggs/embryos or whether this complex might play a role in mammalian somatic cell apoptosis. While we and others have observed that overexpression of Crk can accelerate apoptosis in intact human tissue culture cells, it is not yet clear if this is a Wee1-regulated event (Parrizas et al. 1997; Smith, J.J., and S. Kornbluth, unpublished results). However, it is attractive to speculate that genotoxic damage might trigger either a Wee1-dependent, checkpoint-mediated cell cycle arrest or a Wee1-regulated apoptotic pathway, depending on the degree of DNA damage. In addition, cell cycle–regulated inhibition of Wee1–Crk complexes may help to prevent the onset of apoptosis when cells are detached from their neighbors and the substratum at the time of entry into mitosis. During interphase, cells that do not have proper matrix or cell–cell interactions will initiate an apoptotic program coined "anoikis" (Frisch and Francis 1994; Frisch et al. 1996). However, this program is not initiated at each mitosis, despite the fact that cells round up and lose physical contact with both the extracellular matrix and surrounding cells. Wee1 tyrosine phosphorylation, thought to result from autophosphorylation, is likely absent at mitosis when Wee1 is inactive, thereby preventing binding of Crk and engagement of a Wee1–Crk apoptotic pathway. These and other possibilities await future examination of mammalian Wee1, Crk, and potential Wee1–Crk complexes in apoptotic regulation.
|
Acknowledgments |
|---|
This work was supported by a grant from the National Institutes of Health to S. Kornbluth (RO1 GM56518). S. Kornbluth is a Scholar of the Leukemia and Lymphoma Society. E.K. Evans was supported by a grant from the Department of Defense Breast Cancer Research Program (DAMD17-97-1-7141).
Submitted: 15 September 2000
Revised: 31 October 2000
Accepted: 6 November 2000
J.J. Smith and E.K. Evans contributed equally to this work.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Alnemri E.S.. Mammalian cell death proteasesa family of highly conserved aspartate specific cysteine proteases, J. Cell. Biochem, 64, 1997, 33–42.[Medline]
Birge R.B., Fajardo J.E., Reichman C., Shoelson S.E., Songyang Z., Cantley L.C. & Hanafusa H.. Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts, Mol. Cell. Biol, 13, 1993, 4648–4656.
Birge R.B., Knudsen B.S., Besser D. & Hanafusa H.. SH2 and SH3-containing adaptor proteinsredundant or independent mediators of intracellular signal transduction, Genes Cells, 1, 1996, 595–613.[Abstract]
Chen G., Shi L., Litchfield D.W. & Greenberg A.H.. Rescue from granzyme B–induced apoptosis by Wee1 kinase, J. Exp. Med, 181, 1995, 2295–2300.
Coleman T.R. & Dunphy W.G.. Cdc2 regulatory factors, Curr. Opin. Cell Biol, 6, 1994, 877–882.[Medline]
Dorstyn L., Colussi P.A., Quinn L.M., Richardson H. & Kumar S.. DRONC, an ecdysone-inducible Drosophila caspase, Proc. Natl. Acad. Sci. USA, 96, 1999, 4307–4312.
Duan H., Orth K., Chinnaiyan A.M., Poirier G.G., Froelich C.J., He W.W. & Dixit V.M.. ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B, J. Biol. Chem, 271, 1996, 16720–16724.
Evans E.K., Kuwana T., Strum S.L., Smith J.J., Newmeyer D.D. & Kornbluth S.. Reaper-induced apoptosis in a vertebrate system, EMBO (Eur. Mol. Biol. Organ.) J, 16, 1997, 7372–7381a.[Medline]
Evans E.K., Lu W., Strum S.L., Mayer B.J. & Kornbluth S.. Crk is required for apoptosis in Xenopus egg extracts, EMBO (Eur. Mol. Biol. Organ.) J, 16, 1997, 230–241b.[Medline]
Farschon D.M., Couture C., Mustelin T. & Newmeyer D.D.. Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-1beta converting enzyme family proteases, and a dense membrane fraction, J. Cell Biol, 137, 1997, 1117–1125.
Frisch S.M. & Francis H.. Disruption of epithelial cell–matrix interactions induces apoptosis, J. Cell Biol, 124, 1994, 619–626.
Frisch S.M., Vuori K., Ruoslahti E. & Chan-Hui P.Y.. Control of adhesion-dependent cell survival by focal adhesion kinase, J. Cell Biol, 134, 1996, 793–799.
Gerber-Huber S., Nardelli D., Haefliger J.A., Cooper D.N., Givel F., Germond J.E., Engel J., Green N.M. & Wahli W.. Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene, Nucleic Acids Res, 15, 1987, 4737–4760.
Green D.R.. Apoptotic pathwayspaper wraps stone blunts scissors, Cell, 102, 2000, 1–4.[Medline]
Green D.R. & Reed J.C.. Mitochondria and apoptosis, Science, 281, 1998, 1309–1312.
Gross A., McDonnell J.M. & Korsmeyer S.J.. BCL-2 family members and the mitochondria in apoptosis, Genes Dev, 13, 1999, 1899–1911.
Hengartner M.O. & Horvitz H.R.. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2, Cell, 76, 1994, 665–676.[Medline]
Hensey C. & Gautier J.. A developmental timer that regulates apoptosis at the onset of gastrulation, Mech. Dev, 69, 1997, 183–195.[Medline]
Hughes F.M. Jr. & Gorospe W.C.. Biochemical identification of apoptosis (programmed cell death) in granulosa cellsevidence for a potential mechanism underlying follicular atresia, Endocrinology, 129, 1991, 2415–2422.
Igarashi M., Nagata A., Jinno S., Suto K. & Okayama H.. Wee1(+)-like gene in human cells, Nature, 353, 1991, 80–83.[Medline]
Kluck R.M., Bossy-Wetzel E., Green D.R. & Newmeyer D.D.. The release of cytochrome c from mitochondriaa primary site for Bcl-2 regulation of apoptosis, Science, 275, 1997, 1132–1136a.
Kluck R.M., Martin S.J., Hoffman B.M., Zhou J.S., Green D.R. & Newmeyer D.D.. Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system, EMBO (Eur. Mol. Biol. Organ.) J, 16, 1997, 4639–4649b.[Medline]
Kornbluth S., Sebastian B., Hunter T. & Newport J.. Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14, Mol. Biol. Cell, 5, 1994, 273–282.[Abstract]
Kuwana T., Smith J.J., Muzio M., Dixit V., Newmeyer D.D. & Kornbluth S.. Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c, J. Biol. Chem, 273, 1998, 16589–16594.
Li P., Nijhawan D., Budihardjo I., Srinivasula S.M., Ahmad M., Alnemri E.S. & Wang X.. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade, Cell, 91, 1997, 479–489.[Medline]
Liu X., Kim C.N., Yang J., Jemmerson R. & Wang X.. Induction of apoptotic program in cell-free extractsrequirement for dATP and cytochrome c, Cell, 86, 1996, 147–157.[Medline]
Liu Y., Bhalla K., Hill C. & Priest D.G.. Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line, Biochem. Pharmacol, 48, 1994, 1265–1272.[Medline]
Matsuda M., Nagata S., Tanaka S., Nagashima K. & Kurata T.. Structural requirement of CRK SH2 region for binding to phosphotyrosine-containing proteins. Evidence from reactivity to monoclonal antibodies, J. Biol. Chem, 268, 1993, 4441–4446.
Mayer B.J. & Hanafusa H.. Mutagenic analysis of the v-Crk oncogenerequirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation, J. Virol, 64, 1990, 3581–3589.
McGowan C.H. & Russell P.. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15, EMBO (Eur. Mol. Biol. Organ.) J, 12, 1993, 75–85.[Medline]
McGowan C.H. & Russell P.. Cell cycle regulation of human WEE1, EMBO (Eur. Mol. Biol. Organ.) J, 14, 1995, 2166–2175.[Medline]
Meikrantz W. & Schlegel R.. Suppression of apoptosis by dominant negative mutants of cyclin-dependent protein kinases, J. Biol. Chem, 271, 1996, 10205–10209.
Meikrantz W., Gisselbrecht S., Tam S.W. & Schlegel R.. Activation of cyclin A-dependent protein kinases during apoptosis, Proc. Natl. Acad. Sci. USA, 91, 1994, 3754–3758.
Migita K., Eguchi K., Kawabe Y., Mizokami A., Tsukada T. & Nagataki S.. Prevention of anti-CD3 monoclonal antibody-induced thymic apoptosis by protein tyrosine kinase inhibitors, J. Immunol, 153, 1994, 3457–3465.[Abstract]
Mueller P.R., Coleman T.R., Kumagai A. & Dunphy W.G.. Myt1a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15, Science, 270, 1995, 86–90.
Murakami M.S. & Vande Woude G.F.. Analysis of the early embryonic cell cycles of Xenopus; regulation of cell cycle length by Xe-wee1 and Mos, Development, 125, 1998, 237–248.[Abstract]
Murakami M.S., Copeland T.D. & Vande Woude G.F.. Mos positively regulates Xe-Wee1 to lengthen the first mitotic cell cycle of Xenopus, Genes Dev, 13, 1999, 620–631.
Nakajo N., Yoshitome S., Iwashita J., Iida M., Uto K., Ueno S., Okamoto K. & Sagata N.. Absence of Wee1 ensures the meiotic cell cycle in Xenopus oocytes, Genes Dev, 14, 2000, 328–338.
Newmeyer D.D., Farschon D.M. & Reed J.C.. Cell-free apoptosis in Xenopus egg extractsinhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria, Cell, 79, 1994, 353–364.[Medline]
Norbury C., MacFarlane M., Fearnhead H. & Cohen G.M.. Cdc2 activation is not required for thymocyte apoptosis, Biochem. Biophys. Res. Commun, 202, 1994, 1400–1406.[Medline]
Nurse P. & Thuriaux P.. Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe, Genetics, 96, 1980, 627–637.
Ongkeko W., Ferguson D.J., Harris A.L. & Norbury C.. Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage, J. Cell Sci, 108, 1995, 2897–2904.[Abstract]
Parker L.L., Atherton-Fessler S., Piwnica-Worms H., Lee M.S., Ogg S., Falk J.L., Swenson K.I., Featherstone C. & Russell P.. p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15, Proc. Natl. Acad. Sci. USA, 89, 1992, 2917–2921.
Parrizas M., Blakesley V.A., Beitner-Johnson D. & Le Roith D.. The proto-oncogene Crk-II enhances apoptosis by a Ras-dependent, Raf-1/MAP kinase-independent pathway, Biochem. Biophys. Res. Commun, 234, 1997, 616–620.[Medline]
Shevchenko A., Wilm M., Vorm O. & Mann M.. Mass spectrometric sequencing of protein silver-stained polyacrylamide gels, Anal. Chem, 68, 1996, 850–858.[Medline]
Sible J.C., Anderson J.A., Lewellyn A.L. & Maller J.L.. Zygotic transcription is required to block a maternal program of apoptosis in Xenopus embryos, Dev. Biol, 189, 1997, 335–346.[Medline]
Smith L.D., Xu W.L. & Varnold R.L.. Oogenesis and oocyte isolation, Methods Cell Biol, 36, 1991, 45–60.[Medline]
Songyang Z., Shoelson S.E., Chaudhuri M., Gish G., Pawson T., Haser W.G., King F., Roberts T., Ratnofsky S. & Lechleider R.J.. SH2 domains recognize specific phosphopeptide sequences, Cell, 72, 1993, 767–778.[Medline]
Stack J.H. & Newport J.W.. Developmentally regulated activation of apoptosis early in Xenopus gastrulation results in cyclin A degradation during interphase of the cell cycle, Development, 124, 1997, 3185–3195.[Abstract]
Thornberry N.A. & Lazebnik Y.. Caspasesenemies within, Science, 281, 1998, 1312–1316.
Tilly J.L., Billig H., Kowalski K.I. & Hsueh A.J.. Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism, Mol. Endocrinol, 6, 1992, 1942–1950.
Vaux D.L. & Korsmeyer S.J.. Cell death in development, Cell, 96, 1999, 245–254.[Medline]
Wahli W.. Evolution and expression of vitellogenin genes, Trends Genet, 4, 1988, 227–232.[Medline]
Wallace R.A.. Vitellogenesis and oocyte growth in nonmammalian vertebrates, Dev. Biol., 1, 1985, 127–177.
Walter S.A., Guadagno S.N. & Ferrell J.E. Jr.. Activation of Wee1 by p42 MAPK in vitro and in cycling Xenopus egg extracts, Mol. Biol. Cell, 11, 2000, 887–896.
Yao S.L., McKenna K.A., Sharkis S.J. & Bedi A.. Requirement of p34cdc2 kinase for apoptosis mediated by the Fas/APO-1 receptor and interleukin 1beta-converting enzyme-related proteases, Cancer Res, 56, 1996, 4551–4555.
Zhou B.B., Li H., Yuan J. & Kirschner M.W.. Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells, Proc. Natl. Acad. Sci. USA, 95, 1998, 6785–6790.
Zou H., Henzel W.J., Liu X., Lutschg A. & Wang X.. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3, Cell, 90, 1997, 405–413.[Medline]
Zou H., Li Y., Liu X. & Wang X.. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9, J. Biol. Chem, 274, 1999, 11549–11556.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
Related Article
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
