AKT and 14-3-3 Regulate Notch4 Nuclear Localization

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.

study demonstrated direct interaction of p53 and Mdm2 with Notch4-ICD, leading to Notch-4 degradation by an ubiquitinmediated pathway 25 . Conversely, Mdm2 has been reported to activate Notch1-ICD 26 . Elongin C complexes were also shown to regulate Notch4-ICD degradation in connection with diabetic kidney fibrosis 27 . Several signaling pathways, including the PI3K-AKT pathway, have been shown to crosstalk with Notch family members (Notch1 and 3), however, the mechanistic aspects of this crosstalk have not been defined 28,29 .
Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that these sites are phosphorylated by AKT in vitro and in vivo. In turn, these phosphorylations generate binding sites for the regulatory 14-3-3 scaffold proteins. The Notch4-ICD-14-3-3 binding in cells is regulated by growth factors and is dependent on the PI3K-AKT pathway. This regulatory network controls the nuclear localization of Notch4-ICD by excluding the phosphorylated Notch4-ICD from the nucleus and thus potentially restricting its transcriptional activity. The identified regulatory mechanism is specific for Notch4 since the other Notch family members do not contain any AKT phosphorylation sites. Our results detail a new regulatory mechanism specific for controlling Notch4 ICD localization, defining a potential regulation of Notch4 function by growth factors and other signals that activate AKT.

Results
Notch4-ICD contains AKT consensus phosphorylation sites and binds AKT. The four mammalian Notch receptors show significant variations in their extracellular and intracellular domains 2 . To explore potential differences in their regulation by phosphory-lation, we analyzed the four mammalian Notch receptors for the presence of phosphorylation motifs using the Motif Scan program (http://scansite.mit.edu) 30 that predicts protein phosphorylation sites and protein interaction motifs. This analysis revealed the presence of four consensus AKT phosphorylation sites in human Notch4-ICD: S1495, S1847, S1865 and S1917 ( Fig 1A). These sites are not conserved in the other Notch receptors, suggesting a specific mechanism for Notch4 regulation by AKT. Interestingly, these four sites are fully conserved in primates, but are only partially conserved in other mammals, including rodents and larger mammals (Supplementary Fig 1 and data not shown), pointing to a somewhat distinctive mechanism for the regulation of human and primate Notch4 proteins.
To determine the physiological relevance of the identified sites, we examined the interaction of AKT and Notch4-ICD in vivo using cotransfection experiments and GST-pull-downs (Fig 1B and C). We found extensive Notch4-ICD co-purification with GST-AKT but not with GST control (Fig 1B, compare lanes 1-6 with 7-12), demonstrating specific binding and indicating on physical interaction between AKT and Notch4-ICD. Interestingly, in contrast to several other AKT targets, such as the FoxO transcription factors that require AKT activation for binding 31,32 , AKT binding to Notch4 was not dependent on AKT activity (Fig 1C, BEZ-235  AKT phosphorylates Notch4-ICD in vitro and in vivo. To determine whether AKT can directly phosphorylate Notch4-ICD, we performed in vitro kinase assays using purified AKT and either wildtype Notch4-ICD or mutant forms substituted with alanines at the predicted AKT phosphorylation sites (Fig 2A). These experiments showed that AKT can directly phosphorylate Notch4-ICD and that all four identified sites contribute to the phosphorylation in vitro. AKT did not phosphorylate a mutant form lacking all four sites (Fig 2A, compare lanes 3 and 11), indicating that the identified sites are the only Notch4-ICD sites phosphorylated by AKT in vitro.
In order to study the regulation of Notch4-ICD phosphorylation on the identified sites in vivo, we developed phosphospecific antibodies for each of the phosphorylation sites ( Fig 2B, C and D). We were successful in obtaining site-specific antibodies with minimal cross-reaction with unphosphorylated Notch4 or among the four AKT phosphorylation sites (Fig 2B, C and D). Using these antibodies, we found that all the four predicted Notch4-ICD sites are phosphorylated in vivo. Importantly, reactivity with these antibodies required cell stimulation with growth factors (e.g., EGF) pointing to the dependency of the phosphorylation on the PI3K-AKT pathway (Fig 2 B, C and D, compare lanes 2 and 3).
AKT phosphorylation promotes Notch4-ICD interaction with 14-3-3f. AKT phosphorylation of target proteins often generates binding sites for the 14-3-3 adaptor protein family, and in many cases, 14-3-3 binding rather than the phosphorylation itself is responsible for regulating the function of the target protein [32][33][34][35] . Indeed, the Motif Scan analysis identified several consensus 14-3-3 binding sites in Notch4-ICD, overlapping with the AKT phosphorylation sites at S1495, S1865 and S1917 ( Fig 1A). The site surrounding S1495 showed the strongest 14-3-3 binding consensus from all three sites, suggesting it could serve as the primary 14-3-3 binding site. To examine whether 14-3-3 binds to Notch4-ICD, we used similar GST-pull-down experiments as with AKT, demonstrating in vivo interaction between 14-3-3f and Notch4-ICD ( Fig 3A). The binding was induced by growth factor treatment and was sensitive to PI3K inhibition ( Fig 3A and B), indicating regulation through the PI3K-AKT pathway. The binding was also dependent on the AKT phosphorylation sites, as individual mutation of the AKT phosphorylation sites decreased the binding to 14-3-3 and mutation of all four  sites completely eliminated the binding (Fig 3C). The results also showed that the site surrounding S1495 is the main binding site, since mutation of this site resulted in the most marked reduction in 14-3-3 binding compared to all other single mutations (Fig 3C, compares lanes 10 and 11 with 13-15). Supporting this notion, mutation of proline 1497 diminished 14-3-3 binding (Fig 3C, lane  12), consistent with the dependency of 14-3-3 proteins on proline at the 12 position of the phosphorylated residue for optimal bind-ing 31,36 . 14-3-3 proteins bind their targets as dimers. The binding is facilitated by the presence of two or more binding sites on the target proteins and usually depends on one optimal site for the initial interaction, which then facilitates the binding of the other chain to less optimal sites 34,36 . Our results suggest that the site at S1495 serves as this initial interaction site and that the other, less optimal sites, serve as the secondary interaction points. Interestingly, this site is only present in primates but is not conserved in other mammals  the PI3K inhibitor LY 294002 for 1 h or with 100 ng/ml EGF for 15 min and cells were fractionated for nuclear and cytoplasmic fractions. Equal amount of nuclear and cytoplasmic protein was analyzed by immunoblotting using the indicated antibodies. Note the exclusion of phosphorylated-Notch4-ICD from the nuclear fraction (top panel). (B) 14-3-3-Notch4 association was analyzed in COS-7 cells co-expressing GST-14-3-3f and HA-Notch4-DhN4 (Notch4 form that contains the transmembrane region and parts of the extracellular domain and requires proteolytic cleavage for releasing the ICD domain). Cells were treated as indicated with the c-secretase inhibitor BMS-708163 (GSI, 1 mM for 1 h), EGF (100 ng/ml for 15 min) or their combination. Note that the GSI treatment increased Notch4-14-3-3 association without affecting the AKT activation level. (C) A model for Notch4-ICD regulation by AKT and 14-3-3. Based on our results, we propose that AKT is constitutively associated with Notch4 at the membrane in basal conditions. In this situation, activation of Notch4 by one of its ligands will result in the release of active Notch4-ICD that will translocate to the nucleus and participate in gene regulation. Whether AKT is bound to Notch4-ICD in the nucleus remains to be determined. In growth factor-activated cells or cancer cells with constitutive AKT activation, Notch4-ICD is phosphorylated by AKT and is bound to 14-3-3 at the membrane. Notch4 receptor activation results in release of Notch4-ICD, however, 14-3-3 restricts its translocation to the nucleus and participation in gene regulation.  Supplementary Fig 1 and data not shown), suggesting that the 14-3-3-mediated regulation of Notch4-ICD is somewhat unique to primates.
AKT-phosphorylated Notch4-ICD is restricted to the cytoplasm. 14-3-3 binding can regulate the function of its targets by several mechanisms: it can affect stability and dephosphorylation, increase or decrease enzymatic activity and alter subcellular localization 33 . In the case of FoxO transcription factors, for example, 14-3-3 proteins have been shown to exert all three functions: restricting their nuclear localization, obstructing their binding to DNA and stabilizing the phosphorylated form 31,32,37 . To determine the effects of AKT phosphorylation and the concomitant 14-3-3 binding on Notch4-ICD localization, we fractionated cells into cytoplasmic and nuclear fractions and examined the presence of total and phosphorylated Notch4-ICD in the fractions (Fig 4A). These experiments demonstrated that the phosphorylated Notch4-ICD was present only in the cytoplasm, while total Notch4-ICD was both in the cytoplasm and the nucleus (Fig 4A). These results were similar to our results with FoxO transcription factors, suggesting that the AKT-14-3-3 network acts to restrict the translocation of Notch4-ICD to the nucleus. To examine whether 14-3-3 binding to the Notch4-ICD occurs before or after cleavage of the receptor, we examined the binding of 14-3-3 to Notch4-DhN4, which contains the transmembrane domain and is anchored in the membrane (Fig 4B). We obtained a robust binding of 14-3-3 to this Notch4 form and the binding was stabilized by treating the cells with c-secretase inhibitors that block cleavage of the Notch4-ICD domain (Fig 4B, compare lanes 1 and 2). The results suggest that Notch4 phosphorylation by AKT and the binding of 14-3-3 occur already at the membrane before the cleavage of the ICD domain.

Discussion
The results presented in this study suggest a novel mechanism for the posttranslational regulation of Notch4-ICD (Fig 4C). This regulation involves the binding of inactive AKT to Notch4, possibly at the membrane, and phosphorylation of Notch4-ICD upon AKT activation. This phosphorylation in turn generates binding sites for the regulatory 14-3-3 proteins. Our data suggests that the Notch4-ICD could be pre-bound to 14-3-3 before cleavage and that 14-3-3 binding restricts the phosphorylated Notch4-ICD to the cytoplasm. It remains to be determined whether 14-3-3 binding also restricts the incorporation of Notch4-ICD into active transcriptional complexes or its association with chromatin by masking the RAM domains on Notch4-ICD and whether it has a role in controlling the Notch4-ICD half-life by affecting its degradation. The physiological function of phosphorylated, 14-3-3-bound Notch4-ICD remains to be clarified. It may serve as a reservoir to be mobilized after another signal triggers its dephosphorylation, and/or it may signal through non-canonical, cytoplasmic pathways.
Our findings have potentially strong implications to understanding normal developmental processes that involve Notch4 function as well as cancer pathogenesis by providing a novel link between insulin and growth factor-activated AKT signaling and the transcriptional regulator Notch4. AKT is activated in 50% of breast and prostate cancers and promotes tumorigenesis by teaming with 14-3-3 to regulate key proteins involved in cellular transformation, such as FoxO transcription factors, the pro-apoptotic protein BAD and the cell cycle regulator p27Kip1 among others. To explore whether this crosstalk may operate in human breast cancer, we examined the well-characterized Nottingham-Tenovus breast cancer cohort TMA (n 5 1079) by immunohistochemistry for pAKT (S473) and Notch4 (unpublished observation). Interestingly, pAKT correlated inversely with high nuclear Notch4 (Pearson Chi square p 5 0.023) and directly with cytoplasmic Notch4 (p 5 0.036). This suggests that the mechanism we have uncovered may operate in breast cancer.
Notch inhibitors, as well as PI3K and AKT inhibitors, are in clinical trials, but their optimal use, as for other targeted drugs, will require mechanism-based combinations. Though there is strong evidence that Notch receptors cooperate with the PI3K-AKT pathway in breast cancer, the mechanism of this crosstalk and thus the potential therapeutic implications of combining their inhibitors is not well defined. Our data suggests that AKT inhibition may result in release of Notch4-ICD from 14-3-3 and increased nuclear Notch4 signaling. The possible role of this pathway in the non-canonical oncogenic activity of Notch4-ICD remains to be determined. Interestingly, mammary mouse tumors caused by expression of Notch4-ICD display high levels of pAKT and pMEK, and cell lines derived from these tumors require PI3K and ERK activities for anchorage-independent growth 38 . However, whether murine Notch4 is also regulated by AKT and/or 14-3-3 through partially conserved sites ( Supplementary Fig  1) remains to be determined.
Based on our data, it is also possible that Notch4-ICD may act as an AKT scaffold, facilitating AKT signaling and potentially transporting AKT into the nucleus. Evidence also exists that Notch4 is essential for the maintenance of the elusive breast cancer stem cells. Thus, the identification of Notch4 as a direct AKT target could provide a breakthrough in understanding Notch4 regulation and highlight a rationale for combining Notch4 and AKT targeting drugs.
Cell culture and transfection. HEK-293T and COS-7 cells were maintained in highglucose DMEM media (Invitrogen), supplemented with 10% Fetal Bovine Serum in 5% CO 2 . Transient transfections were performed using FuGENE HD (Roche) according to the manufacturer's instructions.
Cell extraction, fractionation and western blot analysis. Cellular protein was extracted in lysis buffer containing 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1% Triton, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 50 mM b-glycerolphosphate, 2 mM Na 2 VO 4 and protease inhibitors and cleared by centrifugation. The NE-PER fractionation kit (Pierce) was used for nuclear/cytoplasmic fractionation according to the manufacturer's instructions. Protein extracts were separated using SDS-PAGE, transferred to 0.45 uM Immobilon-P PVDF membrane (Millipore) and immunoblotted using the indicated antibodies followed by ECL. The blots were analyzed using ChemiDoc digital imaging system (BioRad).
Immunoprecipitation and protein binding assays. Cells expressing the indicated vectors were lysed and proteins were extracted as detailed above. Equal amounts of protein (1-2 mg/point) were incubated with protein A/G agarose beads (Santa Cruz Biotechnology) coupled with the appropriate antibody or with GSH sepharose beads (Amersham/GE Healthcare) for GST purification for 90 minutes followed by 2X washes with lysis buffer, 1X wash with lysis buffer containing 0.5 M LiCl and 2X washes with buffer containing 40 mM Tris-Cl, pH 7.5, 0.1 mM EDTA and 5 mM MgCl2. For the last wash, the beads were transferred to a new tube and the proteins were eluted using SDS-PAGE sample buffer at 95uC for 5 minutes. Total cell extracts from each sample were saved to examine protein expression and pathway activation/ inhibition.
In vitro kinase assay. HA-FoxO3, FLAG-Notch4-ICD and the indicated Notch4 mutants were purified by HA or FLAG-immunoprecipitation from HEK-293T cells expressing the corresponding vectors and the agarose beads were incubated with 200 ng active GST-AKT that was affinity-purified from HEK-293T cells using GSH purification. The kinase assays were performed in a kinase buffer containing 40 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM MgCl2, 2 mM dithiothreitol and 100 mM ATP supplemented with [c-32 P]ATP (10 mCi/reaction) at 30uC for 20 min. HA-FoxO3 served in the experiments as a positive AKT substrate control. www.nature.com/scientificreports