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Figure 1. Purification of Crk SH2 binding proteins. (A) Crk SH2 binding proteins were isolated as described in Materials and Methods, resolved using SDS-PAGE, and processed for Western blot analysis using an antiphosphotyrosine antibody. Lane 1, GST beads plus extract; lane 2, GSTâSH2 beads plus ELB; lane 3, GSTâSH2 beads plus extract. (B) Microsequencing analysis identified Wee1 as the specific 68-kD major Crk SH2 binding protein. Mass spectrometric analysis yielded two peptides that match exactly the sequence of the XWee1 protein. The first peptide, IGAGEFGSVFK, corresponds to amino acids 216â226 in the Wee1 protein sequence, and the second peptide obtained from microsequencing, ANEILQEDY, matches amino acids 395â403 (bold and underlined). Putative Crk SH2 binding consensus sequences (YKTL, amino acids 110â113; YSQL, amino acids 403â406) are indicated by bold text.
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Figure 7. Wee1 restores apoptotic activity to Crk SH2âdepleted extracts. GSTâCrk SH2 domain bound to glutathione-Sepharose was used to deplete egg extracts (two times, 30 min, 4°C). Recombinant Wee1 (20 ng/μl, final concentration) or XB (control) was added to depleted extracts at a 1:10 dilution (vol/vol). The depleted extracts were also supplemented with energy-regenerating cocktail, incubated at room temperature. Samples were taken at the indicated times and subjected to a DEVDase assay.
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Figure 2. Wee1 binds to the Crk SH2 domain. (A) GST-fused Crk SH2 domain linked to glutathione-Sepharose was used to precipitate Crk SH2 binding proteins from egg extracts. GST bound to glutathione-Sepharose resin was used as a control for nonspecific protein binding. Bead-bound material was washed in ELB to remove nonspecific protein binding. Bound proteins were eluted in SDS-PAGE sample buffer and detected by Western blot with polyclonal anti-Wee1 antisera. (B) A mutant form of the Crk SH2 domain (R38K), which does not bind tyrosine phosphoryalted proteins, does not bind Wee1. Resins consisting of GST (lane 2), GSTâSH2 (lane 4), or GSTâ-SH2R38K (lane 6) bound to glutathione-Sepharose were incubated with Xenopus egg extract. The bead-bound material was washed several times with ELB to remove nonspecific protein binding and then resolved by SDS-PAGE. Wee1 binding was detected by immunoblotting with an affinity-purified anti-XWee1 antibody. GST, GSTâSH2, and GSTâSH2R38K resins that were not incubated with extract (lanes 1, 3, and 5, respectively) were also resolved on this gel as negative controls. (C) Tyrosine phosphorylation of Wee1 is required for its interaction with Crk SH2 domain. Lysates were generated from baculovirus-infected Sf9 cells expressing either wild-type (wt) XWee1 or a mutant form of XWee1 in which three tyrosines (Y-3 Wee1; Y90, Y103, and Y110) were mutated to phenylalanine. These lysates were incubated with either GST (lanes 2 and 3) or GSTâCrk SH2 domain (lanes 5 and 6) bound to glutathione-Sepharose. Bead-bound material was washed several times with ELB to remove nonspecific protein binding and subsequently resolved using SDS-PAGE. Western blot analysis, using an affinity-purified polyclonal anti-XWee1 antibody, was used to determine whether XWee1 (lanes 2 and 5) or Y-3 Wee1 (lanes 3 and 6) bound to either of the two resins. As negative controls, GST resin (lane 1) and GSTâCrk SH2 resin (lane 4), which were not incubated with extract, were also resolved on this gel.
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Figure 3. Endogenous Crk and Wee1 physically interact in egg extracts. (A) Antiâc-Myc, HA, or Crk monoclonal antibodies were bound to protein AâSepharose and used to precipitate bound proteins from egg extract as described in Materials and Methods. Bead-bound material was washed in ELB to remove nonspecific protein binding, and then resuspended in SDS-sample buffer and processed for Western blotting using a polyclonal Wee1 antibody for detection. (B) Reciprocal immunoprecipitation was performed using a polyclonal Wee1 antibody or control IgG bound to protein AâSepharose. Samples were processed for Western blotting using an anti-Crk monoclonal antibody for protein detection.
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Figure 4. Addition of exogenous Wee1 accelerates apoptosis in egg extracts. (A) His-tagged XWee1 produced in baculovirus-infected Sf9 cells (20 or 10 ng/μl, final concentration) or XB was added to egg extracts. During a room temperature incubation, extract samples (3 μl) were taken at the indicated times and processed for DEVD-pNA cleavage activity. (B) His-tagged Wee1 (20 ng/μl, final concentration; right) or XB (buffer control; left) was added at 1:10 (vol/vol) to extracts supplemented with nuclei (â¼2,000 nuclei/μl). Extract samples (2 μl) were taken at various time intervals, fixed with formaldehyde, and stained (chromatin) with Hoechst 33258. Fluorescence microscopy was used to visualize the nuclear morphological changes associated with apoptosis. (C) His-Wee1 (20 ng/μl, final concentration), purified cytochrome c (1.5 ng/μl, final concentration), or XB was added to purified cytosolic egg extracts lacking heavy membrane components, including mitochondria, at a 1:10 (vol/vol) dilution. During a room temperature incubation, extract samples were taken at the indicated times and processed for DEVD-pNA cleavage activity.
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Figure 5. Wee1, but not Myt1, accelerates caspase activation in egg extracts. Recombinant GSTâMyt1 and His-Wee1 were diluted into XB such that the amount of Cdc2-phosphorylating activity of each preparation was equal. These kinases, normalized for activity, were then added to egg extracts at a 1:10 dilution (vol/vol). These treated extracts were incubated at room temperature and subjected to a DEVDase assay with extract samples taken at the indicated times.
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Figure 6. Endogenous Wee1 is required for apoptosis in egg extracts. (A) Egg extracts were depleted of endogenous Wee1 using affinity-purified polyclonal anti-Wee1 IgG; or mock depleted using purified rabbit IgG (control) bound to protein AâSepharose (two consecutive depletions; 4°C, 30 min). Depleted extracts were incubated at room temperature and subjected to a DEVDase activity assay with extract samples taken at the indicated times. (B) Depletion of endogenous Wee1 was confirmed by Western blot analysis of (2 μl) samples of depleted extracts using the polyclonal Wee1 antibody for protein detection. The numbers 1 and 2 represent the first or second rounds of depletion, respectively. (C) The affinity-purified polyclonal anti-Wee1 IgG or control IgG was added (50 ng IgG/μl extract) at a 1:10 (vol/vol) dilution. These extracts were supplemented with energy-regenerating cocktail, incubated at room temperature, and subjected to a DEVDase activity assay with extract samples taken at the indicated times. (D) Affinity-purified anti-XWee1 IgG or control rabbit IgG was added to egg extracts (final concentration 20 ng/μl). Recombinant Wee1 (final protein concentration 20 ng/μl) or XB was added to the extracts at 1:10 (vol/vol) dilution. These extracts were subsequently treated with an energy-regenerating cocktail and incubated at room temperature. Extract samples (3 μl) were taken at the indicated times and subjected to a DEVDase assay.
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Figure 8. Wee1 signals on a Crk-mediated apoptotic pathway. Egg extracts were depleted with immune complexes formed from polyclonal anti-Crk antisera or preimmune sera bound to protein AâSepharose (two consecutive depletions; 4°C, 30 min). Recombinant His-tagged Wee1 (10 ng/μl, final concentration) or XB (control) was added to the depleted extracts at a 1:10 dilution (vol/vol). Extracts were incubated at room temperature, and extract samples were taken at the indicated times in order to assay DEVD-pNA cleavage activity.
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