Survival-promoting functions of 14-3-3 proteins.

The 14-3-3 proteins are a family of phosphoserine/phosphothreonine-binding molecules that control the function of a wide array of cellular proteins. We suggest that one function of 14-3-3 is to support cell survival. 14-3-3 proteins promote survival in part by antagonizing the activity of associated proapoptotic proteins, including Bad and apoptosis signal-regulating kinase 1 (ASK1). Indeed, expression of 14-3-3 inhibitor peptides in cells is sufficient to induce apoptosis. Interestingly, these 14-3-3 antagonist peptides can sensitize cells for effective killing by anticancer agents such as cisplatin. Thus, 14-3-3 may be part of the cellular machinery that maintains cell survival, and targeting 14-3-3-ligand interactions may be a useful strategy to enhance the efficacy of conventional anticancer agents.

Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis.

14-3-3 proteins are a family of homologous eukaryotic molecules with seven distinct isoforms in mammalian cells. Isoforms of 14-3-3 proteins interact with diverse ligands and are involved in the regulation of mitogenesis, cell cycle progression, and apoptosis. However, whether different 14-3-3 isoforms are responsible for distinct functions remains elusive. Here we report that multiple isoforms of 14-3-3 proteins were capable of binding to several ligands, Bad, Raf-1, and Cbl. In a functional assay of 14-3-3 isoforms, all mammalian 14-3-3 isoforms could inhibit Bad-induced apoptosis. Thus, 14-3-3 function in regulating one of its ligands, Bad, is conserved among mammalian isoforms. We addressed whether 14-3-3 isoforms are differentially expressed in tissues, which may in part determine isoform-specific interactions. In situ hybridization revealed that 14-3-3zeta was present in most tissues tested, but sigma was preferentially expressed in epithelial cells. Thus, isoforms of 14-3-3 can interact and control the function of selected protein ligands, and differential tissue distribution of 14-3-3 isoforms may contribute to their specific interactions and subsequent downstream signaling events.

14-3-3 inhibits Bad-induced cell death through interaction with serine-136.

14-3-3 proteins are a family of multifunctional phosphoserine binding molecules that can serve as effectors of survival signaling. Understanding the molecular basis for the prosurvival effect of 14-3-3 may lead to the development of agents useful in the treatment of disorders involving dysregulated apoptosis. One target of 14-3-3 is the proapoptotic Bcl-2 family member Bad. Serine phosphorylation of Bad is associated with 14-3-3 binding and inhibition of Bad-induced cell death, but the relative contributions of the three known phosphorylation sites to 14-3-3 binding have not been established. Here we demonstrate that S136 of Bad is vital for 14-3-3 interaction, but S112 seems to be dispensable. 14-3-3/Bad interaction was strictly dependent on the presence of phosphorylated S136 in vitro, in yeast, and in mammalian cells. However, mutation of S112 did not affect 14-3-3 binding. The death caused by wild-type and S112A Bad, but not that caused by S136A Bad, could be almost completely abrogated by 14-3-3. These data support a critical role for 14-3-3 in regulating Bad proapoptotic activity. The effect of 14-3-3 on Bad is controlled largely by phosphorylation of S136, whereas S112 may represent a 14-3-3-independent pathway.

14-3-3 proteins mediate an essential anti-apoptotic signal.

The 14-3-3 proteins are a family of highly conserved eukaryotic regulatory molecules that play important roles in many biological processes including cell cycle control and regulation of cell death. They are able to carry out these effects through binding and modulating the activity of a host of signaling proteins. The ability of 14-3-3 to inhibit Bad and other proapoptotic proteins argues that 14-3-3 can support cell survival. To examine this issue in a global sense, a specific inhibitor of 14-3-3/ligand interactions, difopein, was used. Difopein expression led to induction of apoptosis. Studies using various components of survival and death signaling pathways were consistent with a vital role for 14-3-3/ligand interactions in signal transduction from upstream pro-survival kinases to the core apoptotic machinery. Because these kinases often become activated during oncogenesis, the effect of difopein on cell death induced by antineoplastic drugs was examined. It was found that difopein enhances the ability of cisplatin to kill cells. These data support the model that 14-3-3, through binding to Bad and other ligands, is critical for cell survival signaling. Inhibition of 14-3-3 may represent a useful therapeutic target for treatment of cancer and other diseases involving inappropriate cell survival.

The proapoptotic protein Bad binds the amphipathic groove of 14-3-3zeta.

Through interaction with a multitude of target proteins, 14-3-3 proteins participate in the regulation of diverse cellular processes including apoptosis. These 14-3-3-interacting proteins include a proapoptotic Bcl-2 homolog, Bad (Bcl-2/Bcl-XL-associated death promoter). To understand how 14-3-3 interacts with Bad and modulates its function, we have identified structural elements of 14-3-3 necessary for 14-3-3/Bad association. 14-3-3 contains a conserved amphipathic groove that is required for binding to several of its ligands. We used peptides of known binding specificity as competitors to demonstrate that Bad interacts with 14-3-3zeta via its amphipathic groove. More detailed analysis revealed that several conserved residues in the groove, including Lys-49, Val-176, and Leu-220, were critical for Bad interaction. These results were applied to investigations of the ability of 14-3-3 to prevent Bad-induced cell death. When co-expressed with Akt, wild-type 14-3-3 could reduce the ability of Bad to cause death, however 14-3-3zetaK49E, which cannot bind Bad, failed to inhibit Bad. It seems that the amphipathic groove of 14-3-3 represents a general binding site for multiple ligands, raising issues related to competition of ligands for 14-3-3.

Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation.

BAD is a proapoptotic member of the BCL-2 family of proteins, which play a major role in regulating apoptosis in cytokine-dependent hematopoietic cells. The function of BAD is regulated by reversible phosphorylation. Deprivation of survival factors induces BAD dephosphorylation, resulting in apoptosis. Serine-threonine phosphatase activity dephosphorylated BAD in interleukin-3-dependent FL5.12 lymphoid cells. Inhibition of PP2A activity by treatment of cells with PP2A-selective inhibitors, okadaic acid and fostriecin, prevented BAD dephosphorylation in these cells. Conversely, BAD dephosphorylation was not inhibited by the PP1-selective inhibitor tautomycin. In cell-free extracts, BAD phosphatase activity was also inhibited by the PP2A-selective inhibitors okadaic acid and fostriecin, but not by the PP1-specific protein inhibitor I-2. Dissociation of 14-3-3 from BAD was a prerequisite for BAD dephosphorylation in vitro, suggesting a mechanism by which 14-3-3 can regulate the activation of the proapoptotic function of BAD in vivo. Significantly, the inhibition of BAD phosphatase activity rescued cell death induced by survival factor withdrawal in FL5.12 cells expressing wild-type BAD but not phosphorylation-defective mutant BAD. These data indicate that PP2A, or a PP2A-like enzyme, dephosphorylates BAD and, in conjunction with 14-3-3, modulates cytokine-mediated survival.

14-3-3 proteins: structure, function, and regulation.

The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. A striking feature of the 14-3-3 proteins is their ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. This plethora of interacting proteins allows 14-3-3 to play important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. In this review, we examine the structural basis for 14-3-3-ligand interactions, proposed functions of 14-3-3 in various signaling pathways, and emerging views of mechanisms that regulate 14-3-3 actions.

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa.

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.

14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove.

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.

Subjects with polycystic ovaries without hyperandrogenaemia exhibit similar disturbances in insulin and lipid profiles as those with polycystic ovary syndrome.

Polycystic ovaries (PCO) are detected using ultrasonography in a proportion of women who do not have clinical symptoms of the polycystic ovary syndrome (PCOS). The aim of this study was to compare the metabolic and endocrine differences between women with such ultrasound-detected PCO and women with PCOS, and to relate these changes to clinical presentation with particular reference to cycle irregularity. A group of 118 women showing PCO on vaginal ultrasound scan was divided into those who had no hyperandrogenaemia (n = 21) and those who had increased androgens and a clinical presentation normally associated with PCOS (n = 97). These were compared with a reference group of 26 normal subjects. Glucose tolerance, lipid concentrations and endocrine profiles were compared between groups. Apart from higher concentrations of androgens in the PCOS group, there were no significant differences between the PCO and PCOS groups in either fasting and stimulated insulin and glucose or in concentrations of sex hormone-binding globulin, gonadotrophins and blood lipids or in ovarian volume. Both PCO and PCOS subjects with cycle irregularity had significantly higher concentrations of serum fasting and stimulated insulin independent of androgens and body mass index than those with normal cycles. It was concluded that: (i) PCO and PCOS patients have equivalent disturbances in relation to insulin and glucose metabolism as well as lipid and lipoprotein disturbances compared to reference subjects; (ii) higher serum insulin values are associated with menstrual irregularity in both groups; (iii) ultrasound evidence for PCO predicts similar metabolic sequelae to PCOS and can therefore be used for studies of the genetics and long term risks for this condition.

Metabolic approaches to the subclassification of polycystic ovary syndrome.

OBJECTIVES: To examine the relationship between various hormonal and metabolic variables in a large group of women with unequivocal evidence of polycystic ovarian syndrome (PCOS) to dissect out the metabolic heterogeneity of this condition. DESIGN: Cross-sectional observational study of PCOS (n = 122) and non-PCOS (n = 26) subjects. SETTING: Reproductive medicine unit in a tertiary teaching hospital. PATIENTS: Subjects with presumed PCOS were recruited from the Reproductive Medicine and Gynaecological Clinics and later confirmed as PCOS with recognized criteria. Several other subjects were identified through recruiting reference subjects. The PCOS population consisted of 122 patients. Reference subjects were recruited from partners of male factor infertility patients in the clinics and from the general population (n = 27). INTERVENTIONS: A 75 g 2-hour oral glucose tolerance test was performed on all subjects in their midluteal phase. Blood was taken at fasting and at 30, 60, 90, and 120 minutes. MAIN OUTCOME MEASURES: Age, body mass index (BMI), waist to hip ratio, levels of integrated glucose and insulin, concentrations of maximum insulin, sex hormone-binding globulin, T, triglyceride, apolipoproteins (Apo A1, B), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDLC). RESULTS: Five clusters could be identified. They are characterized as a nonobese group, a moderately obese group, and three very obese groups. The nonobese group (n = 41, BMI = 24.1) exhibited the lowest level of integrated insulin (236.4 mIU/L or microU/mL) and concentration of serum T (5.5 nmol/L). The moderately obese group had the second lowest level of integrated insulin (497.1 mIU/L) whereas the three very obese groups (n = 15, 13, and 5, respectively) had significantly higher but different levels of integrated insulin (group 3: 850.8 mIU/L; group 4: 1,131.5 mIU/L; and group 5: 1,531.9 mIU/L), triglyceride (group 3: 1.39 mmol/L; group 4: 1.76 mmol/L; and group 5: 2.78 mmol/L [1 mmol/L = 88mg/mL]), Apo B (group 3: 1.18 g/L; group 4: 1.08 g/L; and group 5: 1.55 g/L) and LDLC (group 3: 3.81 mmol/L; group 4: 3.05 mmol/L; and group 5: 5.06 mmol/L [1 mmol/L = 38.6 mg/100 mL]). CONCLUSIONS: The metabolic heterogeneity of the PCOS population is reflected at least partly in patients' levels of insulin, lipids, and lipoproteins, dependent and independent of BMI.