A role for 14-3-3 tau in E2F1 stabilization and DNA damage-induced apoptosis

Genotoxic stress triggers apoptosis through multiple signaling pathways. Recent studies have demonstrated a specific induction of E2F1 accumulation and a role for E2F1 in apoptosis upon DNA damage. Induction of E2F1 is mediated by phosphorylation events that are dependent on DNA damage-responsive protein kinases, such as ATM. How ATM phosphorylation leads to E2F1 stabilization is unknown. We now show that 14-3-3 tau, a phosphoserine-binding protein, mediates E2F1 stabilization. 14-3-3 tau interacts with ATM-phosphorylated E2F1 during DNA damage and inhibits E2F1 ubiquitination. Depletion of 14-3-3 tau or E2F1, but not E2F2 or E2F3, blocks adriamycin-induced apoptosis. 14-3-3 tau is also required for expression and induction of E2F1 apoptotic targets, such as p73, Apaf-1, and caspases, during DNA damage. Together, these data demonstrate a novel function for 14-3-3 tau in the regulation of E2F1 protein stability and apoptosis during DNA damage.     

Inhibition of Aβ(25-35)-induced cell apoptosis by low-power-laser-irradiation (LPLI) through promoting Akt-dependent YAP cytoplasmic translocation

Deposition of amyloid-β-peptide (Aβ) in the brain is considered a pathological hallmark of Alzheimer's disease (AD). Our previous studies show that Yes-associated protein (YAP) is involved in the regulation of apoptosis induced by Aβ_25-35 through YAP nuclear translocation and its pro-apoptotic function is mediated by its interaction with p73. In the present study, we first found that Low-power laser irradiation (LPLI) promoted YAP cytoplasmic translocation and inhibited Aβ_25-35-induced YAP nuclear translocation. Moreover, the cytoplasmic translocation was in an Akt-dependent manner. Activated Akt by LPLI phosphorylated YAP on ser127 (S127) and resulted in decreasing the interaction between YAP and p73, and in suppressing the proapoptotic gene bax expression following Aβ_25-35 treatment. Inhibition of Akt expression by siRNA significantly abolished the effect of LPLI. More importantly, LPLI could inhibit Aβ_25-35-induced cell apoptosis through activation of Akt/YAP/p73 signaling pathway. Therefore, our findings first suggest that YAP may be a therapeutic target and these results directly point to a potential therapeutic strategy for the treatment of AD through Akt/YAP/p73 signaling pathway with LPLI.     

YAP accelerates Aβ<Subscript>25–35</Subscript>-induced apoptosis through upregulation of Bax expression by interaction with p73

Accumulation of amyloid-β-peptide (Aβ) in the brain is considered as a pathological hallmark of Alzheimer’s disease (AD). Previous studies show that p73 is vital for mediating the pathogenic process of AD. Yes-associated protein (YAP) has been shown to positively regulate p73 in promoting apoptosis induced by anti-cancer agents. However, the functional role of YAP and potential relationship between YAP and p73 in AD are unknown. In the present study, we found that YAP accelerated apoptosis in response to Aβ_25–35 and the nuclear translocation of YAP was involved in cellular signals that regulated the apoptosis. Aβ_25–35 induced YAP translocation from cytoplasm to nucleus accompanied with the increased phosphorylation on Y357, resulting in the enhancement of interaction between YAP and p73. Moreover, inhibition of YAP expression by small hairpin RNA (shRNA) suppressed apoptosis induced by Aβ_25–35. More importantly, p73-mediated induction of Bax expression and activation were in a YAP-dependent manner. Overexpression of YAP accelerated Bax translocation, upregulated Bax expression and promoted caspase-3 activation. Taken together, our findings first demonstrated that YAP accelerated Aβ-induced apoptosis through nucleus translocation, leading to the induction of Bax expression and activation. Our results provided a potential therapeutic strategy for the treatment of AD through inhibiting YAP/p73/Bax pathway.     

Regulation of MDMX nuclear import and degradation by Chk2 and 14-3-3

The MDM2 homolog MDMX is an important regulator of p53 during mouse embryonic development. DNA damage promotes MDMX phosphorylation, nuclear translocation, and degradation by MDM2. Here we show that MDMX copurifies with 14-3-3, and DNA damage stimulates MDMX binding to 14-3-3. Chk2-mediated phosphorylation of MDMX on S367 is important for stimulating 14-3-3 binding, MDMX nuclear import by a cryptic nuclear import signal, and degradation by MDM2. Mutation of MDMX S367 inhibits ubiquitination and degradation by MDM2, and prevents MDMX nuclear import. Expression of 14-3-3 stimulates the degradation of phosphorylated MDMX. Chk2 and 14-3-3 cooperatively stimulate MDMX ubiquitination and overcome the inhibition of p53 by MDMX. These results suggest that MDMX-14-3-3 interaction plays a role in p53 response to DNA damage by regulating MDMX localization and stability.     

Phosphorylation of MDMX mediated by Akt leads to stabilization and induces 14-3-3 binding

The critical tumor suppressor p53 is mutated or functionally inactivated in nearly all cancers. We have shown previously that the MDM2-MDMX complex functions as an integral unit in targeting p53 for degradation. Here we identify the small protein 14-3-3 as a binding partner of MDMX, which binds at the C terminus (Ser367) in a phosphorylation-dependent manner. Importantly, we demonstrate that the serine/threonine kinase Akt mediates phosphorylation of MDMX at Ser367. This phosphorylation leads to stabilization of MDMX and consequent stabilization of MDM2. Previous studies have shown that Akt phosphorylates and stabilizes MDM2. Our data suggest that stabilization of MDMX by Akt may be an alternative mechanism by which Akt up-regulates MDM2 protein levels and exerts its oncogenic effects on p53 in tumor cells.     

14-3-3Gamma inhibition of MDMX-mediated p21 turnover independent of p53

The stability of p21, a cyclin-dependent kinase inhibitor, is highly regulated by various protein molecules through the cell cycle and in response to extracellular signals. One of the p21 regulators is MDMX, which can directly bind to p21 and mediate its proteasomal degradation in an ubiquitination-independent fashion. The fact that 14-3-3γ binds to the MDMX domain adjacent to p21 binding suggests that this 14-3-3γ may affect MDMX-mediated p21 proteasomal turnover. Indeed, we found that overexpression of 14-3-3γ increased the level of both endogenous and exogenous p21 in p53-null cells by extending its half-life, leading to p21-dependent G1 arrest. Also, 14-3-3γ excluded p21 from binding to MDMX in a dose-dependent manner as determined by co-immunroprecipitation in vitro using purified proteins and in cells. In response to DNA damage, the level of the 14-3-3γ-MDMX complex increased whereas that of the MDMX-p21 complex declined as detected by co-immunoprecipitation assays, leading to the induction of p21 in p53-null cells. Knockdown of 14-3-3γ inversely alleviated the induction of p21 levels by DNA damage. Hence, our study as presented here unravels a new role for 14-3-3γ in protecting p21 from MDMX-mediated proteasomal turnover, which may partially account for DNA damage-induced elevation of p21 levels independent of p53.     

JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis

Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP''s function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP''s ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP''s function.     

Phosphorylation of p27Kip1 at threonine 198 by p90 ribosomal protein S6 kinases promotes its binding to 14-3-3 and cytoplasmic localization

The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in cell cycle regulation. The cyclin-dependent kinase-inhibitory activity of p27Kip1 is regulated by changes in its concentration and its subcellular localization. Several reports suggest that phosphorylation of p27Kip1 at serine 10, threonine 157, and threonine 187 regulate its localization. We have previously identified that carboxyl-terminal threonine 198 (Thr198) in p27Kip1 is a novel phosphorylation site and that Akt is associated with the phosphorylation at the site (Fujita, N., Sato, S., Katayama, K., and Tsuruo, T. (2002) J. Biol. Chem. 277, 28706-28713). We show herein that activation of the Ras/Raf/mitogen-activated protein kinase kinase (MAPK kinase/MEK) pathway also regulates phosphorylation of p27Kip1 at Thr198. MAPKs were not directly associated with p27Kip1 phosphorylation at Thr198, but the p90 ribosomal protein S6 kinases (RSKs) could bind to and directly phosphorylate p27Kip1 at Thr198 in a Ras/Raf/MEK-dependent manner. RSK-dependent phosphorylation promoted the p27Kip1 binding to 14-3-3 and its cytoplasmic localization. To prove the direct relationship between 14-3-3 binding and cytoplasmic localization, we constructed a p27Kip1-R18 fusion protein in which the R18 peptide was fused to the carboxyl-terminal region of p27Kip1. The R18 peptide is known to interact with 14-3-3 independent of phosphorylation. The p27Kip1-R18 distributed mainly in the cytosol, whereas mutant p27Kip1-R18 (p27Kip1-R18-K2) that had no 14-3-3 binding capability existed mainly in the nucleus. These results indicate that RSKs play a crucial role in cell cycle progression through translocation of p27Kip1, in addition to Akt, to the cytoplasm in a phosphorylation and 14-3-3 binding-dependent manner.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.     

Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1

Camptothecin (CPT) and Nutlin-3 caused apoptosis by increasing p53 protein and its activation in intestinal epithelial cells (IEC-6). We studied the effectiveness of these inducers on apoptosis in human colon cancer cells (Caco2) lacking p53 expression. CPT failed to activate caspase-3 and cause apoptosis in these cells. The absence of p53 expression, higher basal Bcl-xL and lower Bax proteins prevented CPT-induced apoptosis. However, the Mdm2 antagonist Nutlin-3 induced apoptosis in a dose dependent manner by activating caspases-9 and -3. Nutlin-3 prevented the activation of AKT via PTEN-mediated inhibition of the PI3K pathway. Nutlin-3 increased the phosphorylation of retinoblastoma protein causing E2F1 release leading to induction of Siva-1. Nutlin-3-mediated degradation of Mdm2 caused the accumulation of p73, which induced the expression of p53 up-regulated modulator of apoptosis (PUMA). E2F1 and p73 knockdown decreased the expression of Siva and PUMA, respectively and abolished Nutlin-3-induced caspase-3 activation. Cycloheximide (CHX) inhibited Nutlin-3-induced Siva, Noxa, and PUMA expression and inhibited apoptosis in IEC-6 and Caco2 cells. These results indicate that translation of mRNAs induced by Nutlin-3 is critical for apoptosis. In summary, apoptosis in Caco2 cells lacking functional p53 occurred following the disruption of Mdm2 binding with p73 and Rb leading to the expression of pro-apoptotic proteins, PUMA, Noxa, and Siva-1.     

Inhibitory effect of 14-3-3 proteins on serum-induced proliferation of cardiac fibroblasts

Proliferation in cardiac fibroblasts (CFs) can be induced by a wide variety of growth factors that recruit multiple signal transduction pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C. As a family of dimeric phophoserine-binding proteins, 14-3-3s are associated with a multitude of proteins that regulate signal transduction, apoptosis and checkpoint control pathways. However, it remains unknown whether the 14-3-3 proteins play an active role in cardiac proliferation and alter their expression patterns in response to growth factors in CFs. R18 peptide, an isoform-independent 14-3-3 inhibitor, was used to disrupt 14-3-3 function by adenovirus-mediated transfer of R18-EYFP (AdR18). Our results demonstrate that the 14-3-3 isoforms gamma, zeta and epsilon were highly expressed in CFs and the expression of 14-3-3 epsilon was elevated following serum stimulation. Inhibition of 14-3-3 proteins by AdR18 potentiated mitogen-induced DNA synthesis in CFs. This potentiation was presumably due to the increased inactivated glycogen synthase kinase-3 beta by Ser9 phosphorylation and nuclear factor of activated T-cell nuclear accumulation. However, AdR18 had no effect on extracellular signal-regulated kinase phosphorylation and reduced p70 S6 kinase (p70S6K) phosphorylation upon mitogenic stimulation. Furthermore, though R18 can block 14-3-3 binding abilities, it did not affect the serum-induced upregulation of 14-3-3 epsilon protein. Collectively, these findings reveal that the expression of 14-3-3 epsilon can be upregulated by serum in CFs and 14-3-3s may exert an inhibitory effect on serum-induced proliferation.