14-3-3σ and p21 synergize to determine DNA damage response following Chk2 inhibition

DNA damage checkpoints are critical for preventing tumorigenesis and regulating the response of cells to genotoxic agents. It is believed that the coordinated actions of a number of effectors underlie proper checkpoint function. The kinase Chk2, p21, and 14-3-3σ have each been shown to be independent effectors of the G2 DNA damage checkpoint. However, the relative roles of these proteins remain unclear. To help elucidate this question, we have perturbed each of these 3 genes in combination in human cells. We show that Chk2 depletion causes markedly increased sensitivity to DNA damage in p21-/-, 14-3-3σ-/- cells but not in cells lacking only one or none of these genes. This greater sensitivity was due to an increase in apoptosis following DNA damage and not due to exacerbation of G2 checkpoint defects. Pharmacologic inhibition of Chk2 in p21-/-, 14-3-3σ-/- cells also resulted in greater sensitivity to DNA damage. Our data indicates that p21 and 14-3-3σ synergize as molecular determinants of sensitivity to DNA damage following Chk2 inhibition, and Chk2 modulates the biological rheostat that determines whether a cancer cell undergoes arrest versus death after treatment with a chemotherapeutic agent. These findings have implications for the targeting of Chk2 in human cancers.     

DNA Damage and Replication Checkpoints in Fission Yeast Require Nuclear Exclusion of the Cdc25 Phosphatase via 14-3-3 Binding

In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.     

Functions of Saccharomyces cerevisiae 14-3-3 proteins in response to DNA damage and to DNA replication stress

Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Delta mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polalpha-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Delta and bmh1-170 bmh2Delta mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability.     

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.     

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.     

锌指蛋白Miz1磷酸化对其泛素化作用的影响研究

蛋白的磷酸化与其泛素化作用有着广泛而密切的联系。有研究报道,在DNA损伤的情况下,蛋白激酶Akt能磷酸化转录因子Miz1,参与细胞周期停滞的调节;同时,Miz1因子还可在TNFα诱导下被E3泛素连接酶Mule泛素化而降解,从而解除其对JNK信号通路的阻遏,致使JNK信号通路激活。对鼠源野生型Miz1因子(WT Miz1)的AKT磷酸化保守位点进行定点突变,得到磷酸化的突变因子S419AMiz1,并进行了免疫印记和细胞体内泛素化分析。结果显示:Miz1的磷酸化非但不是其泛素化所必需的因素,反而会对其泛素化起到一定的抑制作用。     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

14-3-3 proteins integrate E2F activity with the DNA damage response

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.     

The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax

In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.     

HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation

Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.     

Phosphorylation-dependent binding of 14-3-3 proteins controls TRESK regulation

The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel) is reversibly activated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3eta with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3eta accelerated the return of the K(+) current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3eta was also effective, when it was tethered to the channel by a flexible polyglutamine-containing chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The gamma and eta isoforms of 14-3-3 controlled TRESK regulation, whereas the beta, zeta, epsilon, sigma, and tau isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3eta. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K(+) channel family.     

14-3-3 proteins block apoptosis and differentially regulate MAPK cascades

14-3-3 family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control pathways. In this work, dominant-negative mutant forms of 14-3-3 were used to disrupt 14-3-3 function in cultured cells and in transgenic animals. Transfection of cultured fibroblasts with the R56A and R60A double mutant form of 14-3-3zeta (DN-14-3-3zeta) inhibited serum-stimulated ERK MAPK activation, but increased the basal activation of JNK1 and p38 MAPK. Fibroblasts transfected with DN-14-3-3zeta exhibited markedly increased apoptosis in response to UVC irradiation that was blocked by pre-treatment with a p38 MAPK inhibitor, SB202190. Targeted expression of DN-14-3-3eta to murine postnatal cardiac tissue increased the basal activation of JNK1 and p38 MAPK, and affected the ability of mice to compensate for pressure overload, which resulted in increased mortality, dilated cardiomyopathy and massive cardiomyocyte apoptosis. These results demonstrate that a primary function of mammalian 14-3-3 proteins is to inhibit apoptosis.     

14-3-3 gamma is required to enforce both the incomplete S phase and G2 DNA damage checkpoints

Checkpoint pathways inhibit mitotic progression by inducing the phosphorylation of serine 216 in cdc25C resulting in the generation of a 14-3-3 binding site on cdc25C. Two 14-3-3 isoforms, 14-3-3ε and 14-3-3γ form a complex with cdc25C and inhibit cdc25C function. To examine the contribution of 14-3-3γ to checkpoint regulation, the expression of 14-3-3γ was inhibited in HCT116 cells using vector based RNA interference. A transient reduction in the expression of 14-3-3γ in HCT116 cells resulted in an override of both the incomplete S phase and the G2 DNA damage checkpoint. A 14-3-3γ knockdown clone also showed an override of both checkpoint pathways. These phenotypes were reversed upon expression of a shRNA resistant 14-3-3γ cDNA. Override of the G2 DNA damage checkpoint pathway was accompanied by a decrease in the levels of inhibitory phosphorylation on cdc25C and cdk1. However, there was no difference in the γ-H2AX foci formation and levels of phospho-chk1 and phospho-chk2, suggesting that activation of the DNA damage checkpoint response and subsequent activation of the checkpoint kinases Chk1 and Chk2 was not perturbed. These results suggest that the override of checkpoint observed in 14-3-3γ knockdown cells is due to failure to inhibit cdc25C function.     

Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345

Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to DNA damage and replication interference by blocking the activation of Cdk1/cyclin B. Chk1 is phosphorylated on Ser-317 and Ser-345 following a checkpoint signal, a process that is regulated by Atr, and by the sensor complexes containing Rad17 and Hus1. We show that Chk1 is associated with chromatin in cycling cells and that the chromatin-associated Chk1 is phosphorylated in the absence of exogenous DNA damage. The UV-induced Ser-345-phosphorylated forms of Chk1 that appear minutes after treatment are predominantly associated with chromatin. The Ser-345 site is in a 14-3-3 consensus binding motif and is required for nuclear retention of Chk1 following an hydroxyurea-induced checkpoint signal; nonetheless, Ser-345 or Ser-317 are not required for the chromatin association of Chk1. Hus1, a member of the proliferating cell nuclear antigen-like damage recognition complex plays a role in the phosphorylation of Chk1 on Ser-345, however, Hus1 is not required for phosphorylation on Ser-317 or for Chk1 localization to chromatin. These results indicate that there is more than one step in Chk1 activation and that the regulation of this checkpoint signaling is achieved at least in part through phosphorylation of Ser-345, which serves to localize Chk1 in the nucleus presumably by blocking Crm1-dependent nuclear export.     

14-3-3 proteins as signaling integration points for cell cycle control and apoptosis

14-3-3 proteins play critical roles in the regulation of cell fate through phospho-dependent binding to a large number of intracellular proteins that are targeted by various classes of protein kinases. 14-3-3 proteins play particularly important roles in coordinating progression of cells through the cell cycle, regulating their response to DNA damage, and influencing life-death decisions following internal injury or external cytokine-mediated cues. This review focuses on 14-3-3-dependent pathways that control cell cycle arrest and recovery, and the influence of 14-3-3 on the apoptotic machinery at multiple levels of regulation. Recognition of 14-3-3 proteins as signaling integrators that connect protein kinase signaling pathways to resulting cellular phenotypes, and their exquisite control through feedforward and feedback loops, identifies new drug targets for human disease, and highlights the emerging importance of using systems-based approaches to understand signal transduction events at the network biology level.     

IKKalpha shields 14-3-3sigma, a G(2)/M cell cycle checkpoint gene, from hypermethylation, preventing its silencing

We recently reported that a large proportion of aggressive squamous cell carcinomas of humans and mice express markedly reduced IKKalpha. However, the role of IKKalpha in maintaining genomic stability is unknown. Here we reported that IKKalpha-deficient keratinocytes had a defect in the G(2)/M cell-cycle arrest in response to DNA damage due to downregulated 14-3-3sigma, a cell cycle checkpoint protein. Trimethylated histone H3 lysine 9 (H3-K9) was found to associate with the histone trimethyltransferase Suv39h1 and DNA methyltransferase Dnmt3a in the methylated 14-3-3sigma locus. Reintroduction of IKKalpha restored the expression of 14-3-3sigma. IKKalpha was found to associate with H3 in 14-3-3sigma, which prevented access of Suv39h1 to H3, thereby preventing hypermethylation of 14-3-3sigma. IKKalpha mutants that failed to bind to H3 did not restore the expression of 14-3-3sigma. Thus, IKKalpha protects the 14-3-3sigma locus from hypermethylation, which serves as a mechanism of maintaining genomic stability in keratinocytes.