Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase

Abstract

Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1.

Figure 1

Purification of Ebp1 from NGF-treated PC12 cell nuclear extract. (A) DNA fragmentation assay. Various amount of nuclear extract was preincubated with active apoptosomes containing His-DFF45/40, pretreated with 100 ng active caspase-3, for 10 min at 4°C. The control nuclei from PC12 cells were added and incubated for another 40 min. The fragmented DNA was extracted and resolved on 2% agarose (upper panel). The purity of His-tagged DFF45/40 was verified by Coomassie blue staining (lower panel). (B) Purification chart. DNA fragmentation inhibitory activity was eluted from the columns with NaCl with the indicated concentrations. (C) Mono Q column purification of Ebp1. DNA fragmentation assay with various fractions from Mono Q column reveals that fraction #28 contains the inhibitory proteins. (D) Silver staining of purified proteins. The identity of each band from fraction #28 is described. The proteins were also analyzed with anti-phospho-Ebp1 S360 antibody. Ebp1 distribution in the fractions correlates with its inhibitory activity.

Figure 2

Ebp1 inhibits DNA fragmentation activity of CAD. (A) Immunodepletion of Ebp1 abolishes the inhibitory activity of nuclear extract. The immunodepleted supernatant was analyzed with DNA fragmentation assay. Compared to the control IgG, immunodepletion of Ebp1 diminishes the inhibitory activity (upper left panel, lanes 3 and 4). However, adding back 3 μg of GST-Ebp1 but not GST alone restores the inhibitory activity (upper left panel, lanes 5 and 6). The inhibitory activity is also abrogated by anti-phospho-Ebp1 antibody (upper right panel). Western blotting analysis of Ebp1 and its phosphorylated counterpart in the supernatant from control IgG, anti-Ebp1, anti-phospho-Ebp1 antibodies-depleted nuclear extract (lower panels). (B) Titration of the inhibitory activity of GST-Ebp1. DNA fragmentation reveals that 3 μg of GST-Ebp1 is sufficient to inhibit CAD, however, the same amount of GST alone fails. (C) Overexpression of Ebp1 prevents DNA fragmentation. PC12 cells were stably transfected with inducible form of Ebp1 and cultured in medium with or without tetracycline for 24 h, and followed by 250 nM staurosporine treatment for 24 h. Induction of Ebp1 prevents DNA fragmentation even in the absence of NGF (left panel). Compared with Ebp1-induced cells, DFF45 is markedly cleaved in uninduced cells (middle panel). The expression of induced myc-Ebp1 was verified (right panel). (D) Knocking down of Ebp1 enhances DNA fragmentation in PC12 cells. PC12 cells were treated with penetratin 1-conjugated sense or antisense oligonucleotides for 6 h. Apoptosis was introduced by addition of staurosporine in the presence of NGF. Knockdown of Ebp1 triggers DNA fragmentation compared to sense control. The protein expression of Ebp1 was clearly decreased by antisense oligonucleotide compared to sense. However, as a control, α-tubulin was not changed (left panels). Quantitative analysis of apoptotic rates with DAPI staining in Ebp1-depleted PC12 cells (right panel). (E) Knockdown of Ebp1 enhances apoptosis in primary cultured neurons. Hippocampal neurons were treated with penetratin 1-conjugated sense or antisense oligonucleotides. Apoptosis was introduced by addition of 300 μM glutamate for 16 h. Apoptotic percentage was determined from total 500 cells in different fields, and calculated as means (±s.d.) of three independent experiments (^*P<0.005, Student's t-test) (left panel). The fragmented genomic DNA was extracted and analyzed on 2% agarose gel (right panel). (F) Ebp1 is required for preventing apoptosis in NGF-treated PC12 cells. PC12 cells were cultured in medium containing 50 ng/ml NGF for 0, 1, 3 and 5 days, respectively, and infected with control adenovirus or adenovirus expressing shRNA for 16 h, then induced apoptosis by NGF withdrawal for 24 h. Depletion of Ebp1 enhances DNA fragmentation. The DNA cleavage activities increase with the duration of NGF treatment (upper panel). PARP cleavage and Ebp1 knock down were monitored (lower panels).

Figure 3

Ebp1 binds active nuclear Akt. (A) Ebp1 binds Akt in the nuclear extract. NGF-treated nuclear extract was incubated with agarose beads-conjugated Ebp1 antibody or control IgG for 2 h at 4°C. The co-precipitated proteins were analyzed with anti-Akt antibody. Nuclear Akt specifically binds Ebp1 but not control IgG. (B) Ebp1 selectively associates with phosphorylated Akt. HA-Akt constructs (kinase-dead K179A; dominant-negative K179AT308AS473A; active but kinase-deficient K179AT308DS473D) were cotransfected with Flag-Ebp1 into HEK293 cells. Co-immunoprecipitation reveals that the strongest interaction occurs to constitutively active Akt (T308DS473D) constructs (top panel). Equal levels of transfected HA- and Flag-constructs were expressed (middle and bottom panels). (C) EGF enhances the interaction between Akt and Ebp1. GST-Akt and Flag-Ebp1 were cotransfected into HEK293 cells, and stimulated with 50 ng/ml EGF for 20 min. The glutathione beads-bound proteins were analyzed with anti-Ebp1 antibody (top panel). Both transfected and endogenous Akt was phosphorylated (bottom panel). (D) Active Akt binds Ebp1. Akt stably transfected PC12 cells were preincubated with or without Wortmannin (20 nM) or PD98059 (20 μM), then treated with NGF for 30 min. The endogenous Ebp1 was immunoprecipitated with anti-Ebp1. Wortmannin but not PD98059 blocks the interaction between Akt and Ebp1 (upper panel). The phosphorylation status of Akt was verified by phospho-Akt-473 antibody (lower panel). (E) Ebp1 binds the N-terminal PH and catalytic domains of Akt. Diagram of Akt constructs is depicted (top panel). GST-Akt full-length and various truncates were cotransfected with Flag-Ebp1 into HEK293 cells. The full-length Akt and catalytic domain faintly interact with Ebp1, whereas C-terminal regulatory domain truncated fragment strongly binds Ebp1 (upper left panel). (F) Both N- and C-termini of Ebp1 bind to Akt. GST-Akt and various GFP-tagged Ebp1 fragments were cotransfected into HEK293 cells, followed by EGF stimulation. Both N-terminal 1–183 and C-terminal 183–394 fragments interact with Akt. Deletion of N-terminal 48 amino acids enhances Ebp1 to bind Akt. (G) Akt binds Ebp1 in an NGF-dependent manner. PC12 cells were infected with adenovirus expressing HA-tagged myristoylated-Akt or nuclear Akt, and treated with NGF for various times. Ebp1 clearly binds to nuclear Akt in a time-dependent manner; however, it just weakly interacts with plasma membrane Akt (upper panel). The expression of both plasma membrane and nuclear Akt was confirmed (lower panels). (H) NGF mediates endogenous Ebp1/Akt association in the cytoplasmic and nuclear fractions. PC12 cells were treated with NGF for various times, and the cytoplasmic and nuclear fractions were prepared. Ebp1 distributes in both fractions. Interestingly, a band with molecular weight at 48 kDa, recognized by Ebp1 antibody, selectively occurs in the nuclear fraction (top panel). The identity and purity of each fraction are confirmed by their specific markers (second and third panels). Co-immunoprecipitation reveals that NGF elicits cytoplasmic Akt and Ebp1 faint interaction at 10 min and decays thereafter. However, nuclear Akt and Ebp1 complex formation peaks at about 30 min (bottom panel).

Figure 4

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).

Figure 5

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.

Figure 6

Akt/Ebp1 complex inhibits DNA fragmentation activity of CAD. (A) Akt/Ebp1 complex potently inhibits active CAD. Various GST-recombinant proteins (1 μg) were treated with purified active Akt or inactive Akt for 30 min. The mixture was introduced to the active CAD in the cell-free apoptosome. Full-length Ebp1 and its N-terminal 1–136 truncate inhibit DNA fragmentation elicited by CAD (top panel) by contrast, none of Ebp1 proteins is able to prevent DNA cleavage in the presence of inactive Akt. (B) Active Akt is required for the inhibitory effect of Ebp1. 1.5 μg of 1:1 ratio of active Akt and Ebp1 mixture evidently suppresses DNA fragmentation, and Akt alone fails even at higher concentration. (C) Ebp1 S360D displays inhibitory effect on DNA fragmentation. GST-recombinant Ebp1 proteins (0.5 μg) were incubated with same amount of active Akt. GST-Ebp1 S360D demonstrates the strongest inhibitory activity, followed by wild-type GST-Ebp1. In contrast, GST-Ebp1 S360A fails to prevent DNA fragmentation (upper panel). Equal amount of extracted DNA was loaded in each lane (lower panel). (D) Knocking down of Ebp1 enhances DNA fragmentation. Myc-NLS-Akt-CA cells were infected with adenovirus expressing shRNA of Ebp1, and treated with staurosporine for 24 h. Ebp1 depletion yields substantial DNA cleavage compared with control (upper left panel). Quantitative analysis of apoptotic rates in Ebp1-depleted cells (right panel). (E) Depletion of Akt abolishes the antiapoptotic effect of Ebp1. Ebp1 stably transfected cells were infected with control adenovirus or adenovirus expressing shRNA-Akt for 36 h, followed by NGF stimulation. The isolated nuclei were analyzed in active CAD apoptotic solution (left panel). Quantitative analysis of apoptotic rates in Akt-depleted cells (right panel). Apoptotic percentage was determined from total 500 cells in different fields, and calculated as means (±s.d.) of three independent experiments (^*P<0.005, Student's t-test). Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 4,6-diamidino-2-phenylindole (DAPI). Only the nuclei possessing multiple condensed and aggregated chromatin were counted. (F) A model for NGF-mediated antiapoptotic action by nuclear Akt and PKC-δ-phosphorylated Ebp1 complex.