Journal:
Article Title: Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase
doi: 10.1038/sj.emboj.7601111
Figure Lengend Snippet: PKC and PI 3-kinase cooperatively mediate Ebp1 and nuclear Akt interaction. (A) In vitro binding assay. Glutathione beads-conjugated GST-Ebp1 wt, S360A and S360D were incubated with lysate of 293 cells, transfected with HA-Akt (K179A) and stimulated with or without EGF. In the absence of EGF, S360D selectively binds to Akt; in contrast, neither wt nor S360A binds. After EGF treatment, both wt and S360D interact with Akt; however, S360A does not associate with Akt. (B) Ebp1 colocalizes with Akt in the nucleus. PC12 cells were treated with NGF for 45 min, then stained with anti-Ebp1 and anti-Akt antibodies, respectively. Both Ebp1 and Akt colocalize in the nucleus (white arrows, upper panel). HEK293 cells were transfected with GFP-Ebp1 constructs. All constructs reside in the cytoplasm. However, both GFP-Ebp1 wild-type and S360A localize in the nucleolus, whereas S360D uniformly distribute in the nucleus (white arrows, lower panel). (C) Nuclear but not plasma membrane Akt selectively binds to Ebp1 wild type and S360D mutant in PC12 cells. Ebp1 cell lines were infected with adenovirus expressing myristoylated-Akt or nuclear Akt, respectively. Nuclear Akt-NLS binds to S360D mutant stronger than Ebp1 wild type; however, it does not interact with S360A mutant. By contrast, plasma membrane HA-tagged Akt-myr displays negligible interaction with Ebp1 (upper panel). The expression of infected Akt constructs in PC12 cells was verified (lower panels). (D) Nuclear but not plasma membrane Akt prevents DNA fragmentation. Ebp1 cell lines were infected with Akt-myr or nuclear Akt adenovirus, respectively. The nuclei were isolated and analyzed in the active apoptosome. DNA fragmentation occurs in the nuclei from Akt/myr-infected cells regardless of Ebp1 wild type or mutants; in contrast, nuclear Akt-infected wild-type Ebp1 and S360D strongly antagonize DNA fragmentation, while S360A fails, and this effect seems independent of Akt kinase activity. The experiments were repeated three times, and similar DNA fragmentation patterns were observed. (E) Inhibition of PI 3-kinase or PKC abolishes Akt and Ebp1 interaction. Myc-NLS-Akt-CA stably transfected PC12 cells were pretreated with wortmannin (20 nM), GF109203X (10 μM) or PMA (10 μM) for 30 min, respectively, then stimulated with NGF. Compared to control, NGF stimulation yields Akt/Ebp1 interaction, which is disrupted by PI 3-kinase or PKC inhibitor. Interestingly, PMA also elicits the association (upper panel). The precipitated Akt phosphorylation status correlates with its binding effects (lower panel). (F) PI 3-kinase and PKC signaling cooperatively regulate NGF's antiapoptotic effect. Myc-NLS-Akt-CA cells were pretreated with 20 nM wortmannin, 10 μM GF109203X or wortmannin+GF109203X, then incubated with or without NGF. Apoptosis was initiated by 250 nM staurosporine for 24 h. DNA fragmentation reveals when both inhibitors were employed, but much lesser extent DNA fragmentation occurs while wortmannin or GF109203X alone was used. The faint DNA cleavage in control condition was completely inhibited by NGF. (G) Ebp1 and its N-terminal 1–136 truncate strongly bind to active CAD. Various purified GST-Ebp1 fragments were incubated with His-DFF45/40, pretreated with or without active caspase-3. The proteins associated with glutathione beads were analyzed with anti-CAD antibody. (H) NGF provokes Ebp1 to associate with CAD. PC12 cells were pretreated with NGF, followed by staurosporine incubation for 16 or 24 h. CAD was immunoprecipitated with agarose-conjugated beads. Ebp1 binds to CAD upon NGF treatment. Staurosporine does not alter Ebp1 to bind active CAD.
Article Snippet: Generation and characterization of phosph-Ebp1 S360 anti-sera Rabbit polyclonal anti-phospho-Ebp1 S360 antibody was generated by Covance Research Products Inc.
Techniques: In Vitro, Binding Assay, Incubation, Transfection, Staining, Construct, Clinical Proteomics, Membrane, Mutagenesis, Infection, Expressing, Isolation, Activity Assay, Inhibition, Stable Transfection, Control, Phospho-proteomics, Purification, Immunoprecipitation
Santa Cruz Biotechnology is a verified supplier 
![PKC phosphorylates Ebp1 on serine 360. (A) Diagram of various GST-Ebp1 fragments. (B) In vitro PKC assay. In total, 2 μg of purified GST-Ebp1 fusion proteins was incubated with active PKC in the presence of γ-32P-ATP. Both wild-type Ebp1 and T366A mutant were strongly phosphorylated. Interestingly, fragment 292–394 was strongly phosphorylated (upper panel). The identity of purified recombinant proteins were verified by Coomassie blue staining (lower panel). (C) In vitro PKC assay with C-terminal fragments of Ebp1. S360 and 361 might be the phosphorylation sites (upper panel). Coomassie blue staining of GST-Ebp1 fragments (lower panel). (D) In vitro PKC assay with Ebp1 mutants. S360A mutation disrupts PKC phosphorylation (lower panel). Coomassie blue staining of GST-Ebp1 mutants (upper panel). (E) PKC phosphorylates Ebp1 in cells. PC12 cells were treated with NGF for various times, and the cytoplasmic and nuclear fractions were prepared. NGF stimulation does not significantly alter the cytoplasmic Ebp1 phosphorylation. By contrast, NGF markedly provokes nuclear Ebp1 phosphorylation at 30 min. The purity and identity of each fraction was confirmed by immunoblotting with anti-tubulin and anti-PARP antibodies (second and third panels). NGF provokes both Akt and PKC-δ nuclear translocation (fourth and bottom panels). (F) PKC inhibitors block NGF-elicited Ebp1 phosphorylation in the nucleus. PC12 cells were pretreated with various PKC inhibitors for 30 min, followed by NGF for 30 min, and the cytoplasmic and nuclear fractions were prepared. NGF-provoked nuclear Ebp1 phosphorylation was inhibited by 10 μM GF109203X and 60 nM Go6983, and completely abrogated by 6 μM Rotterlin. By contrast, cytoplasmic Ebp1 phosphorylation is not diminished. The total Ebp1 in the nuclear fraction is verified by immunoblotting (bottom panel). (G) In vitro kinase assay with PKC isoform proteins. GST-Ebp1 (1 μg) and [γ-32P]ATP were incubated with various PKC isoforms at 30°C for 1 h. PKC isoforms were immunoprecipitated from NGF-treated or control PC12 cells. Compared to control, NGF-treated PKC-δ but not -α or -ζ selectively phosphorylates Ebp1. (H) Knocking down of PKC-δ abolishes Ebp1 phosphorylation. PC12 cells were transfected with PKC-δ and -α siRNA, respectively. In 48 h, the cells were stimulated with NGF. Both PKC isoforms were substantially removed upon transfection of their siRNA (left top panel). Depletion of PKC-δ but not PKC-α blocks the phosphorylation of nuclear Ebp1 (left bottom panels). Myc-tagged wild-type Ebp1 and S360A stably transfected PC12 cells were transfected with PKC-δ siRNA. In 48 h, the cells were stimulated with NGF, Ebp1 was immunoprecipitated from nuclear fraction with anti-Myc antibody. Potent Ebp1 phosphorylation was revealed in wild-type Myc-Ebp1 stably transfected cells, which was completely diminished in PKC-δ-knocked down cells. By contrast, no phosphorylation was detected in Myc-Ebp1S360A-transfected cells (right panels).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2972/pmc01462972/pmc01462972__7601111f4.jpg)
