PI(3,4,5)P3 regulates the interaction between Akt and B23 in the nucleus

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1 BMB reports PI(3,4,5)P3 regulates the interaction between Akt and B23 in the nucleus Il-Sun Kwon 1,3, Kyung-Hoon Lee 2,3, *, Joung Woo Choi 1 & Jee-Yin Ahn 1,3, * 1 Department of Molecular Cell Biology, 2 Department of Anatomy, 3 Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon , Korea Phosphatidylinositol (3,4,5)-triphosphate (PIP 3) is a lipid second messenger that employs a wide range of downstream effector proteins for the regulation of cellular processes, including cell survival, polarization and proliferation. One of the most well characterized cytoplasmic targets of PIP 3, serine/threonine protein kinase B (PKB)/Akt, promotes cell survival by directly interacting with nucleophosmin (NPM)/B23, the nuclear target of PIP 3. Here, we report that nuclear PIP 3 competes with Akt to preferentially bind B23 in the nucleoplasm. Mutation of Arg23 and Arg25 in the PH domain of Akt prevents binding to PIP 3, but does not disrupt the Akt/B23 interaction. However, treatment with phosphatases PTEN or SHIP abrogates the association between Akt and B23, indicating that nuclear PIP 3 regulates the Akt/B23 interaction by controlling the concentration and subcellular dynamics of these two proteins. [BMB reports 2010; 43(2): ] INTRODUCTION Phosphatidylinositol 3,4,5-triphosphate (PIP 3) is a lipid second messenger that employs a wide range of downstream effector proteins for the regulation of cellular processes, including cell survival, polarization and proliferation. PIP 3 is upregulated within cells by phosphoinositide 3-kinase (PI3K) and is degraded by two specific phosphatases, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and SH2 domain-containing inositol 5 -phosphatase (SHIP) (1, 2). PIP 3 regulates cellular processes primarily by binding to downstream effector proteins. One of the most well characterized targets of PIP 3 is serine/threonine protein kinase B (PKB)/Akt. Specifically, PIP 3 binds to the plecstrin homology (PH) domain of Akt, promoting translocation to the membrane. At the plasma *Corresponding author. Jee-Yin Ahn, Tel: ; Fax: ; jyahn@med.skku.ac.kr, Kyung-Hoon Lee, Tel: ; Fax: ; khlee@med.skku.ac.kr Received 24 November 2009, Accepted 4 December 2009 Keywords: Akt, B23, Neuronal cell, Nuclear PIP3, Subcellular dynamics membrane, Akt becomes phosphorylated at two sites, T308 and S473, resulting in full activation (3) and the initiation of survival signaling. Upon stimulation by growth factors, both PI3K and Akt translocate to the nucleus (4-6). We recently demonstrated that nuclear PI3K plays an essential role in promoting cell survival through the regulation of nuclear PIP 3 (7). However, it is not clear whether PIP 3 remains associated with Akt in the nucleus. Compelling evidence suggests that nearly all phosphoinositides along with the enzymes responsible for inositol lipid metabolism are present in the nucleus (8-10). Given the presence of PIP 3 in the nucleus, we used a PIP 3 column and nuclear extracts from nerve growth factor (NGF)-treated neuronal cells to confirm that nucleophosmin (NPM)/B23 associates with PIP 3 in the nucleus through its C-terminus (11). This interaction forms a complex that mediates the anti-apoptotic effects of NGF by inhibiting the DNA fragmentation activity of caspase-activating DNase (CAD). Accordingly, B23 mutants that cannot bind to PIP 3 fail to prevent apoptosis in PC12 cells. More recently, we showed that B23 associates directly with nuclear Akt in response to NGF treatment, thereby enhancing the protein stability of Akt and promoting cell survival (12). A B23-K263 mutant unable to bind to ATP, along with a major sumoylation site mutant (sumoylation is critical for the nucleolar localization and anti-apoptotic functions of Akt; 13), interacted more strongly with Akt than did the non-mutant protein, indicating that B23 must localize to the nucleoplasm in order to interact with Akt (14, 15). In addition, we demonstrated that the Akt/B23 interaction occurs between the C-terminus of B23 and the PH domain of Akt (12). However, the ability of Akt and B23 to bind PIP 3 has not yet been evaluated. Nuclear PIP 3 in neuronal cells not only activates the atypical protein kinase C (PKC)-zeta, but also drives its nuclear translocation in NGF-treated PC12 cells (16). Moreover, the PIP 3 binding protein PIP 3BP is abundant in the brain where it is localized to the nucleus (17), suggesting that PIP 3 may be involved in nervous system functions. In this report, we show that the binding affinity of PIP 3 shifts from Akt to B23 in the nucleus upon NGF stimulation in PC12 cells, which suggests that Akt and PIP 3 may not physically interact in the nucleus. Akt-PH domain mutants that have lost the ability to recognize D-3-phosphate can still interact with BMB reports 127

2 B23. However, a high concentration of PIP 3 abrogates this interaction in vitro, as does pretreatment with PTEN or SHIP, suggesting that precise control of the spatial and temporal dynamics of PIP 3 is critical for its biological function. RESULTS Nuclear PIP 3 binds preferentially to B23 rather than Akt PI3K is a critical regulator of numerous growth factor signaling pathways. In general, individual growth factors invoke specific downstream signaling pathways, leading to significant differences in PI dynamics. Therefore, to monitor PIP 3 dynamics of different subcellular fractions upon NGF signaling, we employed PIP 3-agarose conjugated beads along with nuclear extracts of HA-Akt and myc-b23 cotransfected PC12 cells. The binding affinity of PIP 3 to Akt decreased gradually upon NGF stimulation, whereas phosphorylation of Akt was delayed for min in the cytoplasm. In the nucleus, Akt did not interact with PIP 3 upon NGF stimulation, although phosphorylation of nuclear Akt was gradually increased and sustained for over 60 min. These results suggest that the amount of accessible PIP 3 may be low due to sequestration of PIP 3 by other cellular components. Accordingly, immunoblotting revealed that binding between PIP 3 and B23 occurred exclusively in the nucleus upon NGF stimulation. Presumably, PIP 3 binds preferentially to B23 and exerts its anti-apoptotic effect. Similar levels of HA-Akt and Myc-B23 were detected, and the purity of the nuclear fractions was verified (Fig. 1A). To further evaluate the interaction between PIP 3 and Akt in different cellular compartments, we performed PIP 3-agarose conjugated bead pull-down assays with HEK293 cells infected with adenovirus expressing Akt-NLS-Myc or HA-myristoylated- Akt. Akt experienced slightly increased nuclear targeting as well as binding to PIP 3 upon NGF stimulation, but its binding affinity was much lower than that of myristoylated-akt (Fig. 1B). Consistent with these findings, PIP 3 pull-down assay with PC12 cells stably expressing constitutively active nuclear Akt (NLS-Akt-T308DS472D) showed that the phosphorylation status of nuclear Akt does not alter PIP 3 binding (data not shown). Thus, our data indicate that the activation of Akt as well as the maintenance of Akt phosphorylation do not require PIP 3 binding in the nucleus. The PH domain of Akt reportedly binds two major PI3K products, PIP 3 and PI(3,4)P2 (18, 19) with similar affinity. We also demonstrated that B23 binds to the PH domain of Akt (12). Therefore, it was hypothesized that PIP 3 regulates the interaction between Akt and B23. To evaluate this, we generated a GFP-Akt-PH domain mutant incapable of binding 3 - phospholipids, GFP-Akt-PH (R23, 25A), and cotransfected this mutant construct with myc-b23 into HEK293 cells. Unexpectedly, mutagenesis of the Akt lipid binding pocket did not impair the interaction between Akt and B23. This is despite the failure of EGF stimulation to enhance the association between the Akt-PH mutant (R23, 25A) and B23 compared to Akt-WT (Fig. 1C). Thus, the PIP 3 binding ability of Akt is unnecessary for interaction with B23, a result consistent with our observation that PIP 3 barely interacts with Akt in the nuclear compartment. PIP 3 regulates the interaction between Akt and B23 We previously determined that the positively charged C-terminus of B23 is required for interaction with negatively charged PIP 3 molecules (11). In addition, we also demonstrated that the 56 C-terminal amino acids of B23 are sufficient for binding to Akt. Therefore, we next examined whether PIP 3 disrupts the interaction between the C-terminus of B23 and Akt. We used purified recombinant GST-B23 and Akt from NGF-treated HA- Akt-overexpressing PC12 cells, followed by chasing with increasing amounts of free PIP 3. Akt displayed the strongest bin- Fig. 1. Nuclear PIP 3 binds preferentially to B23 rather than Akt. (A) A lipid binding assay was performed using cytosolic and nuclear protein extracts following treatment of cells with NGF at the indicated times. The products then were immunoprobed with anti-akt or anti-b23 antibodies. Anti-PARP and antitubulin were used as markers of nuclear protein purity. (B) PC12 cells were infected with mock, myri-akt and NLS- Akt virus. After 24 hours, cells were treated with NGF for 30 min and a lipid binding assay was performed. Cells infected with virus containing NLS-Akt or myri-akt were probed with anti-akt antibody. (C) PC12 cells were co transfected with myc-b23 and GFP-Akt or GFP-Akt- R23, 25A. Cell lysates were immunoprecipitated with anti-gfp antibody and probed with anti-myc antibody. 128 BMB reports

3 ding affinity for B23 when PIP 3 was not available. However, the binding affinity of Akt for B23 was decreased gradually as the amount of PIP 3 was increased, and the association between Akt and B23 was completely abolished by high concentrations of PIP 3 (Fig. 2A, top panel). This implies that excess amounts of PIP 3 can disrupt the interaction between Akt and B23. Hence, the PIP 3 concentration must be precisely regulated in order to maintain proper cellular behavior. Next, an equal amount of Akt was employed in all samples and the purity of GST-B23 was confirmed (Fig. 2A, middle and bottom panels). An in vitro competition assay involving pretreatment with increasing concentrations of free PIP 3 revealed that the Akt/B23 complex in nuclear extracts of HA-Akt and myc-b23 cotransfected 293 cells decreased significantly upon increasing amounts of free PIP 3 (Fig. 2B). These findings indicate that PIP 3 interacts with B23 at a similar region, if not the same, as that which mediates the Akt/B23 association. Moreover, PIP 3 competes with Akt for binding to the C-terminus of B23. The tumor suppressor PTEN inhibits PI3K signaling by hydrolyzing PI(3,4,5)P3 to PI(4,5)P2 (20), whereas SHIP converts PI(3,4,5)P3 to PI(3,4)P2 (21). Both of these phosphatases are present in the nucleus (22, 23). To further verify whether or not nuclear PIP 3 regulates the interaction between Akt and B23, we pretreated nuclear extracts from GFP-B23 and HA-Akt cotransfected PC12 cells with PTEN or SHIP following NGF stimulation. In the absence of PTEN or SHIP treatment, NGF strongly stimulated the interaction between Akt and B23. In contrast, this interaction was completely abolished in the presence of either PTEN or SHIP, suggesting that nuclear PIP 3 is required for the association between Akt and B23 (Fig. 2C). PIP 3/Akt/B23 are colocalized to the nucleoplasm While Akt is translocated from the cytoplasm to the nucleus upon NGF stimulation, B23 is shuttled from the nucleolus to the nucleoplasm. To determine whether the localization of PIP 3 is responsible for its interaction with target proteins, we transfected GFP-Akt or GFP-B23 into PC12 cells and performed immunofluorescent staining using anti-pip 3 antibody. As expected, Akt was found to reside predominantly in the cytoplasm whereupon it moves to the nucleus upon NGF treatment. Furthermore, Akt remained in the nucleus for 90 min, indicating that Akt and PIP 3 colocalization occurs throughout the nucleus, excluding the nucleolus, upon NGF treatment. Consistent with our previous observations, B23 was concentrated in the nucleolus in the absence of NGF, but became dispersed throughout the nucleoplasm following 30 min of NGF stimulation and relocalized to the nucleolus after 90 min. The intensity of PIP 3 increased notably following NGF treatment, due most likely to synthesis by NGF-dependent signaling pathways. Furthermore, PIP 3 was distributed throughout the cell with the exception of the nucleolus, a pattern similar to that observed for Akt (Fig. 3). B23 and PIP 3 only transiently colocalized to the nucleoplasm following the redistribution of B23 due to NGF stimulation. DIC images supported our present understanding of B23 dynamics within the nucleus. Thus, both PIP 3 and Akt interact with B23 in the nucleoplasm, further supporting the idea that PIP 3 and Akt are competitors for B23 binding. DISCUSSION The link between nuclear PIP 3 and cell survival signaling has become the focus of intense investigation. In this study, we determined that nuclear PIP 3 prefers B23 to Akt as its nuclear binding partner, even though Akt is a well-known cytoplasmic target of PIP 3. Interestingly, although Akt barely interacted with PIP 3 in the nucleus, Akt activation was maintained for a substantial period of time in the nuclear compartment. Therefore, it remains unclear how nuclear Akt activity is generated and maintained in the absence of accessible PIP 3. A previous study has suggested that Akt translocates from the cytoplasm to the nucleus following phosphorylation at the plasma membrane Fig. 2. PIP 3 is involved in the interaction between Akt and B23. (A) Purified GST-B23 proteins were incubated with NGF-treated, HA-Akt-transfected PC12 cell lysates along with the indicated amounts of free PIP 3. After GST pull-down, samples were probed with anti-ha antibody. Coomassie blue staining was performed to monitor GST protein purification. (B) PC12 cells were transfected with myc-b23-wt and HA-Akt. After 24 hours, cell lysates were incubated with the indicated amounts of free PIP 3, and the reaction mixtures were precipitated and probed as indicated. Myc-B23 and HA- Akt expression was determined using anti-myc and anti-ha antibodies, respectively. (C) GFP-B23 and HA-Akt were cotransfected into PC12 cells and PTENor SHIP-pretreated nuclear extracts were immunoprecipitated and immunoblotted as indicated. BMB reports 129

4 Fig. 3. PIP 3/Akt/B23 is colocalized to the nucleoplasm. Naïve PC12 cells were cotransfected with GFP-Akt or GFP-B23. After stimulation with NGF, immunofluorescence staining was performed using an anti-pip 3 antibody. (6). Accordingly, our data suggest that Akt is activated in the cytoplasm upon NGF stimulation whereupon it maintains its phosphorylation status during nuclear translocation, as opposed to being activated in situ by nuclear PIP 3. Even with the enzymes responsible for PIP 3 production and degradation in the nucleus having been identified, it is not yet clear whether the presence of PIP 3 in the nucleus is due to synthesis or translocation. Using fluorescence resonance energy transfer (FRET) and nuclear-targeted proteins, Zhang and colleagues found that no substantial amounts of PIP 3 accumulated in the nucleus upon PDGF or IGF-1 stimulation. However, it is possible that different growth factors, such as neurotrophins, may differentially regulate the pool of PIP 3 in neuronal cells. Detachment of Akt from PIP 3 in the nucleus does not appear to impair the activity of nuclear Akt. We recently reported that nuclear Akt interacts with a longer isoform of the ErbB3 binding protein 1 (Ebp1), p48, as well as suppresses CAD activity, thereby inhibiting apoptosis in neuronal cells (24). Furthermore, nuclear Akt specifically regulates B23 protein stability through direct interaction, leading to neuronal cell survival (12). Nuclear Akt also promotes cell survival by phosphorylating acinus, which induces apoptotic chromatin condensation and inhibits proteolytic cleavage (25). There is no doubt that other nuclear targets of Akt will emerge based on the fact that the consensus sequence for Akt phosphorylation has been identified in more than 400 different proteins (26). However, the question remains as to how Akt activity is turned off in the nucleus. It will be of great interest to find whether or not nuclear Akt activity is regulated by nuclear-localized PP2A or whether other phosphatases are involved. B23 is an abundant nuclear protein that mediates neuronal cell survival by binding to PIP 3 and whose stability is increased by binding to Akt. In agreement with our observation that the GFP-Akt-PH (R23, 25A) mutant can still bind to B23 (Fig. 1), we found that the GFP-Akt-PH (R23, 25A) mutant was predominantly localized to the nucleus whereupon it redistributes to the cytoplasm upon EGF stimulation. In contrast, the major sumoylation site mutant, B23-K263R, bound to Akt (12) and not PIP 3, regardless of growth factor stimulation (11, 14). Taken together, these observations indicate that the subcellular localization of Akt is the most important factor for determining binding to B23. The interaction between Akt and B23 was significantly inhibited by high concentrations of PIP 3, which binds predominantly to B23 (Fig. 2). Under these circumstances, PIP 3-dependent signaling is active in the nucleus and plays an essential role in neuronal cell survival. Nevertheless, destabilization of PIP 3 by PTEN or SHIP also dramatically destabilized the Akt/B23 interaction in the nuclear fraction, supporting the notion that precise control of the spatial and temporal dynamics of PIP 3 is critical for the proper physiological functioning of its target proteins. Due to the instability of B23 in the nucleoplasm, we predict that Akt/B23 interactions could be stronger in the nucleoplasm. However, we also found that PIP 3 and B23 may interact. Conceivably, Akt might coordinate the anti-apoptotic effects of B23 in cells with PIP 3 increasing the stability of B23 in the nucleoplasm. MATERIALS AND METHODS Cell culture and transfection PC12 cells were maintained in DMEM medium (Dulbecco s modified Eagle s medium, Cellgro, Herndon, VA, USA) supplemented with 10% fetal bovine serum, 5% horse serum, and 100 units of penicillin-streptomycin. PC12 cells were transfected by electroporation (Digital Bio Technology, Korea). All transfection procedures were performed according to the manufacturer s recommended protocols. Chemicals and reagents Anti-Akt (pan), anti-p-akt (S473), anti-gst and anti-parp antibodies were obtained from Cell Signaling (Danvers, MA, 130 BMB reports

5 USA). Anti-tubulin, anti-gfp, anti-ha and anti-c-myc (9E10) antibodies were acquired from Santa Cruz (Santa Cruz, CA, USA). Anti-PIP 3 antibody and PIP 3-conjugated-agarose beads were purchased from Echelon Bioscience, Inc (Salt Lake City, UT, USA). Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG were acquired from Molecular Probes (Eugene, OR, USA). Mouse anti-npm polyclonal antibody was generated in our lab. All chemicals not included above were obtained from Sigma (St. Louis, MO, USA). Subcellular fractionation and coimmunoprecipitation The cytosolic and nuclear fractions from transfected PC12 cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA), according to the manufacturer s protocol. Cytosolic and nuclear B23 or Akt from NGF-treated PC12 cells was immunoprecipitated using PIP 3-conjugated agarose beads. The precipitated proteins were analyzed as indicated. 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