Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase
2006; Springer Nature; Volume: 25; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7601111
ISSN1460-2075
AutoresJee‐Yin Ahn, Xia Liu, Zhixue Liu, Lorena Pereira, Dongmei Cheng, Junmin Peng, Paul A. Wade, Anne W. Hamburger, Keqiang Ye,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle27 April 2006free access Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase Jee-Yin Ahn Jee-Yin Ahn Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USAPresent address: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-76, Korea Search for more papers by this author Xia Liu Xia Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Zhixue Liu Zhixue Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Lorena Pereira Lorena Pereira Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dongmei Cheng Dongmei Cheng Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Junmin Peng Junmin Peng Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Paul A Wade Paul A Wade Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Anne W Hamburger Anne W Hamburger Greene Baum Cancer Center, Department of Pathology, University of Maryland, Baltimore, MD, USA Search for more papers by this author Keqiang Ye Corresponding Author Keqiang Ye Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Jee-Yin Ahn Jee-Yin Ahn Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USAPresent address: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-76, Korea Search for more papers by this author Xia Liu Xia Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Zhixue Liu Zhixue Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Lorena Pereira Lorena Pereira Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dongmei Cheng Dongmei Cheng Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Junmin Peng Junmin Peng Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Paul A Wade Paul A Wade Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Anne W Hamburger Anne W Hamburger Greene Baum Cancer Center, Department of Pathology, University of Maryland, Baltimore, MD, USA Search for more papers by this author Keqiang Ye Corresponding Author Keqiang Ye Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Author Information Jee-Yin Ahn1, Xia Liu1, Zhixue Liu1, Lorena Pereira1, Dongmei Cheng2, Junmin Peng2, Paul A Wade1, Anne W Hamburger3 and Keqiang Ye 1 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 2Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA 3Greene Baum Cancer Center, Department of Pathology, University of Maryland, Baltimore, MD, USA *Corresponding author. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA. Tel: +1 404 712 2814; Fax: +1 404 712 2979; E-mail: [email protected] The EMBO Journal (2006)25:2083-2095https://doi.org/10.1038/sj.emboj.7601111 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Nerve growth factor (NGF) regulates survival of several types of neurons by provoking a variety of signaling cascades including PI 3-kinase/Akt pathway, which plays an essential role in this process (Brunet et al, 2001). PI 3-kinase/Akt signaling blocks cell death both by impinging on the cytoplasmic apoptotic machinery and by mediating the expression of genes involved in cell death and survival (Brunet et al, 1999; Brazil et al, 2002). Although PI 3-kinase/Akt cascade is important for the NGF-dependent survival of sympathetic neurons (Kaplan and Miller, 2000), it is not the only required signaling pathway. Under certain culture conditions, PI 3-kinase/Akt appears to be dispensable for cell survival (Philpott et al, 1997; Tsui-Pierchala et al, 2000). Recently, it has been shown that PKC signaling is also involved in the survival of sympathetic neurons. Johnson and his collaborators demonstrated that inhibition of either PKC or PI 3-kinase alone caused only a modest attrition of neurons in the presence of NGF. In contrast, the simultaneous inhibition of both PKC and PI 3-kinase induced massive apoptotic death of NGF-treated sympathetic neurons (Pierchala et al, 2004). Thus, NGF promotes the survival of sympathetic neurons through the cooperative function of the PKC and PI 3-kinase pathways. However, the downstream effectors mediating this effect remain obscure. NGF treatment rapidly increases the enzymatic activity of nuclear Akt, and PI 3-kinase inhibitor LY294002 pretreatment blocks Akt nuclear translocation (Borgatti et al, 2003). Moreover, Akt upstream kinase, PDK1, also localizes in both the cytoplasmic and nuclear compartments. Treatment with the nuclear export inhibitor, Leptomycin B (LMB), leads to constitutive nuclear localization of PDK1 (Lim et al, 2003). Presumably, Akt is phosphorylated and activated by PDK1 in the cytoplasm, which leads to its subsequent nuclear translocation. On the other hand, PDK1 may also directly phosphorylate Akt in the nucleus (Kikani et al, 2005). Recently, we demonstrated that nuclear PI 3-kinase and its upstream regulator PIKE mediate the antiapoptotic activity of NGF in isolated nuclei (Ahn et al, 2004). Nuclei from NGF-treated PC12 cells are resistant to DNA fragmentation initiated by the activated cell-free apoptosome, consisting of HEK293 cell cytosol supplemented with purified active caspase 3 (Liu et al, 1997). We showed that nuclear PI(3,4,5)P3 mimics the antiapoptotic action of NGF, while nuclear Akt appears necessary but not sufficient for this effect. Ebp1, a ubiquitously expressed protein, localizes in both the nucleus and the cytoplasm and binds ErbB3 receptor in human serum-starved breast cancer cell lines (Yoo et al, 2000). Ebp1 is the human homolog of a previously identified cell cycle-regulated mouse protein p38-2G4 (Radomski and Jost, 1995). ErbB3/4 ligand Heregulin (HRG) treatment of serum-starved AU565 breast cancer cells results in dissociation of Ebp1 from ErbB-3 and translocation from the cytoplasm into the nucleus (Yoo et al, 2000). Ebp1 binds tumor suppressor retinoblastoma protein (Rb), leading to inhibition of the E2F1-regulated transcription (Xia et al, 2001; Zhang et al, 2003; Zhang and Hamburger, 2005). Recently, it has been shown that Ebp1 localizes to the nucleolus, and EBP1 overexpression inhibits proliferation of human fibroblasts. This effect is linked to its nucleolar localization (Squatrito et al, 2004). Here, we report that PKC-dependent phosphorylation of Ebp1 stimulates binding to phosphorylated nuclear Akt in NGF-treated PC12 cells. The resulting complex interacts with CAD and inhibits its DNA fragmentation activity. NGF elicits Ebp1 and Akt complex formation in a PKC- and PI 3-kinase-dependent manner. Depletion of Ebp1 or disruption of its association with Akt abolishes its antiapoptotic activity. Therefore, a complex formed by active nuclear Akt and Ebp1 plays an integral role in the antiapoptotic actions of NGF in the nucleus. Results Ebp1 co-purifies with DNA fragmentation inhibitory activity Nuclei from NGF-treated PC12 cells are resistant to DNA fragmentation initiated by the active cell-free apoptosome, consisting of purified His-DFF45/40 (DNA fragmentation factor) and active caspase 3 (Ahn et al, 2004), indicating that NGF activates nuclear pathways inhibiting CAD. To examine whether the nuclear fraction contains an activity that inhibits CAD, we preincubated various amounts of nuclear extracts from NGF-treated PC12 cells with the cell-free apoptotic solution and determined DNA cleavage with the control nuclei. In total, 3 μg of nuclear extract markedly suppresses DNA fragmentation (Figure 1A), suggesting the existence of CAD inhibitor(s) in NGF-treated nuclear extract. No inhibitory activity is detected with the same amount of cytosolic extract (data not shown). To identify the proteins accounting for the DNA fragmentation inhibitory activity, we employed classical biochemical purification columns using in vitro cell-free DNA fragmentation assay. NGF-treated PC12 cell nuclear extract is subjected to sequential column fractionations. The purification scheme is summarized in Figure 1B. Fraction #28 from Mono Q column displays potent inhibitory activity (Figure 1C). The fractions at about 300 mM NaCl are analyzed by SDS–PAGE and silver staining. Four proteins with molecular weights of 45, 50, 55, 65 kDa are present in this fraction with peak of inhibitory activity. Protein sequence analysis reveal their identities: Hexosaminidase A, β-3-glycosyltransferase, Ring finger Protein 149 and Ebp1 (Figure 1D). Among these proteins, only Ebp1 always co-purifies with CAD-inhibitory activity during the purification protocol (data not shown). Moreover, immunoblotting with anti-phospho-Ebp1 360 antibody reveals that Ebp1 distribution correlates with the inhibitory activities in the fractions, indicating that Ebp1 is responsible for DNA fragmentation inhibitory effect. 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. Download figure Download PowerPoint Ebp1 is required for the antiapoptotic activity of NGF Immunodepletion of Ebp1 from NGF-treated PC12 nuclear extract abolishes the capacity of NGF-treated nuclear extracts to inhibit Caspase-3/CAD-triggered DNA fragmentation, while mock depletion has no effect (Figure 2A, upper left panel lanes 3 and 4). Adding back purified GST-Ebp1, but not GST alone, restores the inhibitory activity (lanes 5 and 6). Moreover, immunodepletion with phospho-Ebp1 antibody but not preimmune antiserum or protein A/G beads markedly diminishes the inhibitory activity (Figure 2A, right panel). Both Ebp1 and phosphorylated Ebp1 are substantially removed by immunodepletion (Figure 2A, lower panels). The nuclei from PC12 cells, which were treated with or without NGF, act as positive and negative controls (lanes 1 and 2). Dose–response experiments reveal inhibition of DNA fragmentation following addition of purified GST-Ebp1; in contrast, GST control fails at the same concentration (Figure 2B). Thus, Ebp1 is required for the antiapoptotic activity of NGF in this cell-free assay. The amounts of recombinant Ebp1 necessary to inhibit DNA fragmentation are relatively high, suggesting that it might need phosphorylation or binding partners to reveal the inhibitory effect under physiological conditions. 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). Download figure Download PowerPoint To investigate whether Ebp1 could prevent DNA fragmentation in intact cells, we generated a stably transfected PC12 cell line with an inducible form of Ebp1. The pronounced DNA fragmentation elicited by staurosporine in uninduced control cells is markedly decreased in Ebp1-overexpressed cells (Figure 2C, left panel). The cleavage of DFF45, a well-characterized marker of apoptosis, couples to the DNA fragmentation activity (Figure 2C, right panel). Depletion of Ebp1 in PC12 cells by Penetratin 1-conjugated antisense oligonucleotide elicits evident fragmented DNA compared to sense control (Figure 2D, left panel), suggesting that Ebp1 plays a critical role in preventing DNA fragmentation in apoptosis. Quantitative analysis reveals that about 50% cells in apoptosis in antisense-treated samples compared with 15% in sense control. The expression level of Ebp1 is substantially decreased by antisense but not sense control (Figure 2D, lower left panels). We also extended these studies into primary culture of hippocampal neurons. We introduced Penetratin-conjugated antisense oligonucleotide into serum-starved neurons for 6 h. The neuronal preparations were treated with glutamate, and apoptosis was evaluated by DNA fragmentation and nuclear morphological alteration. Depleting Ebp1 with antisense oligonucleotide clearly enhances neuronal cell death compared to sense control (Figure 2E). To further determine whether Ebp1 is required for preventing DNA fragmentation, we incubated PC12 cells with NGF for various times, infected the cells with control adenovirus or adenovirus expressing shRNA of Ebp1, and induced apoptosis through NGF withdrawal. Compared with control adenovirus, knocking down of Ebp1 elicits strong DNA degradation. The amounts of fragmented DNA increase with the duration of NGF treatment, and the strongest DNA fragmentation occurs in cells treated with NGF for 3–5 days (Figure 2F, top panel). PARP cleavage correlates with DNA fragmentation activities. The expression level of Ebp1 is substantially decreased by its shRNA but not control. As a control, α-tubulin expression level is not changed (Figure 2F, lower panels). Collectively, these data support the notion that Ebp1 is required for the antiapoptotic actions. Ebp1 interacts with active Akt To determine potential partners for Ebp1 in protection of DNA from fragmentation, we performed co-immunoprecipitation experiments. Immunoblotting analysis of the coprecipitated proteins from NGF-treated nuclear extract reveals that Akt specifically associates with Ebp1 (Figure 3A). To examine whether Akt phosphorylation status plays any role in its binding to Ebp1, we cotransfected Flag-Ebp1 with various wild-type and mutant HA-Akt constructs. Co-immunoprecipitation demonstrates that the strongest interaction occurs between Ebp1 and constitutively active Akt (T308DS473D), a phosphorylation mimetic mutant, indicating that active Akt selectively binds to Ebp1. Interestingly, its kinase activity is not required for the binding (Figure 3B, top panel). EGF treatment of HEK293 cells, cotransfected with GST-Akt and Flag-Ebp1, substantially increases the association, suggesting that Akt phosphorylation promotes its interaction with Ebp1 (Figure 3C, top panel). Control experiments verify that both endogenous and transfected Akt are phosphorylated on S473 (Figure 3C). 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). Download figure Download PowerPoint To investigate further whether Ebp1 binds to Akt in cells, we conducted co-immunoprecipitation assay with Ebp1 antibody in Myc-NLS-Akt stably transfected PC12 cells, treated with NGF in the presence or absence of PI 3-kinase inhibitor Wortmannin or MEK1 inhibitor PD98059. NGF triggers demonstrable association between Ebp1 and Akt, and Wortmannin pretreatment disrupts the interaction. By contrast, PD98059 pretreatment fails to interrupt it (Figure 3D, upper panel). The phosphorylation status of Akt correlates with its interaction with Ebp1 (Figure 3D, lower panel). Equal amount of Ebp1 is immunoprecipitated (data not shown). Mapping experiment with GST-tagged Akt fragments reveals that Ebp1 evidently binds the N-terminal PH and catalytic domains of Akt (Figure 3E). Truncation assay with GFP-tagged Ebp1 fragments shows that both N- and C-termini of Ebp1 bind to Akt, and deletion of the N-terminal 48 residues increases Ebp1 binding to Akt (Figure 3F). Co-immunoprecipitation assay with Ebp1 antibody in PC12 cells, infected with adenovirus expressing Akt-NLS-Myc (Shiraishi et al, 2004) or myristoylated-Akt (Suhara et al, 2002), demonstrates that both plasma membrane and nuclear Akt interacts with Ebp1 in an NGF-dependent manner, but nuclear Akt binds much more Ebp1 than plasma membrane-bound Akt does (Figure 3G, upper panel). The expression of Myc-NLS-Akt and HA-myristoylated-Akt was verified (Figure 3G, lower panels). Subcellular fractionation and co-immunoprecipitation assay demonstrate that endogenous Ebp1 potently binds nuclear translocated Akt after 10–30 min of NGF stimulation, whereas cytoplasmic Ebp1 weakly associates with Akt at about 10 min (Figure 3H). Thus, NGF specifically regulates the interaction between Ebp1 and active Akt in the nucleus of PC12 cells. Protein kinase C phosphorylates Ebp1 Ebp1 is a target for phosphorylation by PKC in vitro and in vivo, and its C-terminus has been suggested to harbor the phosphorylation site (Lessor and Hamburger, 2001). To determine the PKC phosphorylation residue on Ebp1, we performed an in vitro PKC assay in the presence of [γ-32P]ATP with various purified Ebp1 recombinant proteins (Figure 4A). Both full-length Ebp1 and a C-terminal fragment (292–394) are clearly phosphorylated; by contrast, N-terminal fragments (1–136) and (133–336) are not phosphorylated. Previous in vitro PKC phosphorylation study with Ebp1 peptide suggests that threonine 366 might be a putative phosphorylate site (Lessor and Hamburger, 2001). Surprisingly, Ebp1 T366A mutant is also phosphorylated, indicating that it is not the phosphorylate site by PKC (Figure 4B, upper panel). Kinase assay with a variety of Ebp1 mutants suggests that S360 and S361 residues are responsible for the phosphorylation (Figure 4C, upper panel). Phosphorylation assay with full-length Ebp1 S360A and Ebp1 S361A mutants reveals that serine 360 is the PKC phosphorylation site (Figure 4D, lower panel). Nevertheless, Ebp1 is not significantly phosphorylated by active Akt, suggesting that Ebp1 might not be a physiological substrate of Akt (data not shown). 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). Download figure Download PowerPoint To explore whether Ebp1 S360 can also be phosphorylated by PKC in intact cells, we examined the subcellular fractionated Ebp1 phosphorylation status with its specific anti-phospho-Ebp1 S360 antibody. NGF elicits Ebp1 phosphorylation at 10 min in nuclear fraction and peaks at 30 min. However,
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