Cytoplasmic Localization of Human cdc25C during Interphase Requires an Intact 14-3-3 Binding Site

cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (amino acids 201 to 258) in cdc25C that was required for the cytoplasmic localization of cdc25C. This region contained a specific binding site for 14-3-3 proteins, and mutations in cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic localization of cdc25C during interphase. cdc25C proteins that do not contain a binding site for 14-3-3 proteins showed a pancellular localization and an increased ability to induce premature chromosome condensation. The cytoplasmic localization of cdc25C was not altered by gamma irradiation or treatment with the nuclear export inhibitor leptomycin B. These results suggest that 14-3-3 proteins may negatively regulate cdc25C function by sequestering cdc25C in the cytoplasm.     

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.     

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.     

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.     

Vpr protein of human immunodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex formation with Cdc25C: implications for cell cycle arrest

Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G(2)/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3sigma. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G(2)/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.     

14-3-3 cruciform-binding proteins as regulators of eukaryotic DNA replication

Cruciforms are secondary DNA structures, serving as recognition signals at or near eukaryotic (yeast and mammalian) origins of DNA replication. The cruciform-binding protein is a member of the 14-3-3 protein family and binds to origins of DNA replication in a cell cycle-dependent manner. Five 14-3-3 protein isoforms (beta, gamma, epsilon, zeta and sigma) have been identified as having cruciform binding activity.     

A novel pocket in 14-3-3? is required to mediate specific complex formation with cdc25C and to inhibit cell cycle progression upon activation of checkpoint pathways

Mitotic progression requires the activity of the dual specificity phosphatase, cdc25C. Cdc25C function is inhibited by complex formation with two 14-3-3 isoforms, 14-3-3? and 14-3-3γ. To understand the molecular basis of specific complex formation between 14-3-3 proteins and their ligands, chimeric 14-3-3 proteins were tested for their ability to form a complex with cdc25C in vivo. Specific complex formation between cdc25C and 14-3-3? in vivo requires a phenylalanine residue at position 135 (F135) in 14-3-3?. Mutation of this residue to the corresponding residue present in other 14-3-3 isoforms (F135V) leads to reduced binding to cdc25C and a decrease in the ability to inhibit cdc25C function in vivo. Similarly, F135V failed to rescue the incomplete S phase and the G2 DNA damage checkpoint defects observed in cells lacking 14-3-3?. A comparative analysis of the 14-3-3 structures present in the database suggested that the F135 in 14-3-3? was required to maintain the integrity of a pocket that might be involved in secondary interactions with cdc25C. These results suggest that the specificity of the 14-3-3 ligand interaction may be dependent on structural motifs present in the individual 14-3-3 isoforms.     

Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein,its heterodimerization profile with endogenous 14-3-3 isoforms,and effect on mammalian DNA replication in vitro

The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication.     

Inactivation of 14-3-3sigma influences telomere behavior and ionizing radiation-induced chromosomal instability

Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G(2) and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3sigma gene has been reported to be a checkpoint control gene, since it promotes G(2) arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3sigma(-/-) cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G(1) checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G(2) after irradiation with gamma rays, corroborating the role of the 14-3-3sigma protein in G(2)/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G(2) checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3sigma(-/-) cells are defective in maintaining G(2) arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G(2)/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.     

14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.     

Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers

We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, beta, gamma, epsilon, eta, sigma, tau, and zeta of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of tau isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of beta, epsilon, gamma and eta and a low level of zeta and tau, but not sigma isoform. However, the levels of 14-3-3 epsilon, gamma and eta were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.     

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.     

An essential phosphorylation-site domain of human cdc25C interacts with both 14-3-3 and cyclins

Human cdc25C is a dual-specificity phosphatase involved in the regulation of cell cycle progression in both unperturbed cells and in cells subject to DNA damage or replication checkpoints. In this study, we describe the structure-function relationship of an essential domain of human cdc25C that interacts with 14-3-3 proteins. We show that this domain is a bi-functional interactive motif that interacts with cyclins primarily through their P-box motif in addition to 14-3-3 proteins. Characterization of the structural features of this domain by NMR and circular dichroism reveals two distinct alpha helical moieties interconnected by a loop carrying the 14-3-3 binding site. Moreover, the helical folding is induced upon binding to 14-3-3, suggestive of a conformational regulation of this domain of cdc25C through interactions with partner proteins in vivo. Combining our structural and biochemical data, we propose a detailed model of the molecular mechanism of cdc25C regulation by differential association with 14-3-3 and cdc2-cyclin B.     

Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.     

A structural basis for 14-3-3sigma functional specificity

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.     

Schizosaccharomyces pombe Cells Lacking the Amino-Terminal Catalytic Domains of DNA Polymerase Epsilon Are Viable but Require the DNA Damage Checkpoint Control

In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol epsilon) is encoded by cdc20(+) and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol epsilon that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol epsilon (cdc20(DeltaN-term)) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability of cdc20(DeltaN-term) cells is dependent on expression of rad3(+), hus1(+), and chk1(+), three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol epsilon, cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly for cdc20(DeltaN-term) cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol epsilon is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.     

Downregulation of 14-3-3 Proteins in a Kainic Acid-Induced Neurotoxicity Model

The 14-3-3 proteins are among the most abundant proteins expressed in the brain, comprising about 1% of the total amount of soluble brain proteins. Through phosphoserine- and phosphothreonine-binding motifs, 14-3-3 proteins regulate many signaling proteins and cellular processes including cell death. In the present study, we utilized a well-known kainic acid (KA)-induced excitotoxicity rat model and examined the expression of 14-3-3 and its isoforms in the frontal cortex of KA-treated and control animals. Among the different 14-3-3 isoforms, abundant levels of eta and tau were detected in the frontal cortex, followed by sigma, epsilon, and gamma, while the expression levels of alpha/beta and zeta/delta isoforms were low. Compared to the control animals, KA treatment induced a significant downregulation of the overall 14-3-3 protein level as well as the levels of the abundant isoforms eta, tau, epsilon, and gamma. We also investigated two 14-3-3-interacting proteins that are involved in the cell death process: Bcl-2-associated X (BAX) and extracellular signal-regulated kinase (ERK). Both BAX and phosphorylated ERK showed increased levels following KA treatment. Together, these findings demonstrate an abundance of several 14-3-3 isoforms in the frontal cortex and that KA treatment can cause a downregulation of 14-3-3 expression and an upregulation of 14-3-3-interacting proteins BAX and phospho-ERK. Thus, downregulation of 14-3-3 proteins could be one of the early molecular events associated with excitotoxicity. This could lead to subsequent upregulation of 14-3-3-binding proteins such as BAX and phospho-ERK that contribute to further downstream apoptosis processes, eventually leading to cell death. Maintaining sufficient levels of 14-3-3 expression and function may become a target of therapeutic intervention for excitotoxicity-induced neurodegeneration.     

Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo

The 14-3-3 protein family associates with many proteins involved in intracellular signalling. In many cases, there is a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of 14-3-3 dimer formation may therefore influence which of the interacting proteins could be brought together. We have analysed the pattern of dimer formation for two of the most abundant isoforms of 14-3-3, epsilon ( epsilon ) and gamma (gamma), following their stable expression. This revealed a distinct preference for particular dimer combinations that is largely independent of cellular conditions. gamma 14-3-3 occurred as homodimers and also formed heterodimers, mainly with epsilon 14-3-3 (In PC12 and Cos cells). The epsilon isoform formed heterodimers with 14-3-3 beta, gamma, zeta, and eta, but no homodimers were detected. The two 14-3-3 homologues, BMH1 and BMH2 from Saccharomyces cerevisiae, were mainly heterodimers.     

Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation

The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, beta, epsilon, theta and zeta, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, sigma and gamma, were present only at very low levels. Immunoprecipitation data showed 14-3-3zeta could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3zeta in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G(2)/M-phase arrest. Moreover, 16% 14-3-3zeta antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3zeta is involved in G(2) checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.     

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.