Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsy

14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3gamma and -zeta were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3gamma levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3zeta, while cytoplasm-localized 14-3-3beta, -epsilon and -eta remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3epsilon and -zeta levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures.     

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.     

14-3-3 zeta mediates integrin-induced activation of Cdc42 and Rac. Platelet glycoprotein Ib-IX regulates integrin-induced signaling by sequestering 14-3-3 zeta

Integrin-induced cytoskeletal reorganizations are initiated by Cdc42 and Rac1 but little is known about mechanisms by which integrins activate these Rho GTPases. 14-3-3 proteins are adaptors implicated in binding and regulating the function and subcellular location of numerous signaling molecules. In platelets, the 14-3-3 zeta isoform interacts with the glycoprotein (GP) Ibalpha subunit of the adhesion receptor GP Ib-IX. In this study, we show that integrin-induced activation of Cdc42, activation of Rac, cytoskeletal reorganizations, and cell spreading were inhibited in Chinese hamster ovary cells expressing full-length GP Ibalpha compared with GP Ibalpha lacking the 14-3-3 zeta binding site. Activation of Rho GTPases and cytoskeletal reorganizations were restored by expression of 14-3-3 zeta. Spreading in cells expressing truncated GP Ibalpha was inhibited by co-expressing a chimeric receptor containing interleukin 2 receptor alpha and GP Ibalpha cytoplasmic domain. These results identify a previously unrecognized function of 14-3-3 zeta, that of mediating integrin-induced signaling. They show that 14-3-3 zeta mediates Cdc42 and Rac activation. They also reveal a novel function of platelet GP Ib-IX, that of regulating integrin-induced cytoskeletal reorganizations by sequestering 14-3-3 zeta. Signaling across integrins initiates changes in cell behavior such as spreading, migration, differentiation, apoptosis, or cell division. Thus, introduction of the 14-3-3 zeta binding domain of GP Ibalpha into target cells might provide a method for regulating integrin-induced pathways in a variety of pathological conditions.     

14-3-3zeta is an effector of tau protein phosphorylation

Neurofibrillary tangles associated with Alzheimer's disease are composed mainly of paired helical filaments that are formed by the aggregation of abnormally phosphorylated microtubule-associated protein tau. 14-3-3, a highly conserved protein family that exists as seven isoforms and regulates diverse cellular processes is present in neurofibrillary tangles (Layfield, R., Fergusson, J., Aitken, A., Lowe, J., Landon, M., Mayer, R. J. (1996) Neurosci. Lett. 209, 57-60). The role of 14-3-3 in Alzheimer's disease pathogenesis is not known. In this study, we found that the 14-3-3zeta isoform is associated with tau in brain extract and profoundly stimulates cAMP-dependent protein kinase catalyzed in vitro phosphorylation on Ser(262)/Ser(356) located within the microtubule-binding region of tau. 14-3-3zeta binds to both phosphorylated and nonphosphorylated tau, and the binding site is located within the microtubule-binding region of tau. From brain extract, 14-3-3zeta co-purifies with microtubules, and tubulin blocks 14-3-3zeta-tau binding. Among four 14-3-3 isoforms tested, beta and zeta but not gamma and epsilon associate with tau. Our data suggest that 14-3-3zeta is a tau protein effector and may be involved in the abnormal tau phosphorylation occurring during Alzheimer's disease ontogeny.     

Selective association of protein kinase C with 14-3-3 zeta in neuronally differentiated PC12 Cells. Stimulatory and inhibitory effect of 14-3-3 zeta in vivo

The 14-3-3 protein is a family of highly conserved acidic proteins found in a wide range of eukaryotes from yeast to mammals. 14-3-3 acts as an adapter protein and interacts with signaling molecules including protein kinase C (PKC). Although 14-3-3 zeta was originally characterized as an endogenous PKC inhibitor, it was reported to activate PKC in vitro, but the in vivo regulation of PKC by 14-3-3 is still not well understood. To examine the regulation of PKC by 14-3-3 in the cell, we have generated a sub-cell line, PC12-B3, that stably expresses FLAG epitope-tagged 14-3-3 zeta isoform in PC12 cells. Here we show that PKC-alpha and PKC-epsilon become associated with 14-3-3 zeta when the cells are neuronally differentiated by nerve growth factor. We found that the immunoprecipitate by anti-FLAG antibody contains constitutive and autonomous Ca(2+)-independent non-classical PKC activity. In contrast, the FLAG immunoprecipitate has no Ca(2+)-dependent classical PKC activity despite the fact that PKC-alpha is present in the FLAG immunoprecipitate from differentiated PC12-B3 cells. Our results show that the association with 14-3-3 zeta has distinct effects on classical PKC and non-classical PKC activity.     

A novel binding assay for detection of 14-3-3 protein in cerebrospinal fluid from Creutzfeldt-Jakob patients.

Here we describe a simple and fast microplate assay for 14-3-3 protein, which is based on the binding of 14-3-3 protein to a chemically synthesized peptide containing the phosphorylated recognition sequence of this protein. The peptide is covalently bound to maleimide-activated microplates through reaction of the sulfhydryl group of a N-terminal cysteine residue linked to the peptide. The peptide- 14-3-.3 complexes formed are detected using--as a second recognition principle--an anti-14-3-3 protein antibody. This assay was used for measurement of 14-3-3 protein in brain (bovine) and in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease (CJD) and control cases with diseases other than CJD. This novel assay allowed also the detection of low 14-3-3 protein levels present in cerebrospinal fluid, without further concentration, in contrast to conventional immunoblotting or immunoassay procedures.     

Identification and characterization of the interaction between tuberin and 14-3-3zeta

Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.     

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II–IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.     

14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1

The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 ζ and 14-3-3 σ on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-β1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-β1-mediated growth inhibition displayed increased expression of 14-3-3 ζ and decreased expression of 14-3-3 σ compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 σ or 14-3-3 ζ, we showed that 14-3-3 σ is required for TGF-β1-mediated growth inhibition whereas 14-3-3 ζ negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 ζ increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-β1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 ζ phosphorylation sites in Smad3 markedly reduced the 14-3-3 ζ-mediated inhibition of TGF-β1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 ζ in the suppression of TGF-β1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 σ or 14-3-3 ζ contributes to TGF-β1 resistance in cancer cells.     

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.     

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.     

Detection of adiponectin in cerebrospinal fluid in humans

Adiponectin circulates in the body in high concentrations, and 100-fold lower amounts were described in the cerebrospinal fluid (CSF) of mice, whereas in humans, contradictory results have been published. To clarify whether adiponectin is present in human CSF and is derived from the circulation, it was determined in human CSF and plasma of 52 nonselected patients. Adiponectin was detected by immunoblot in CSF and was quantified in CSF and serum by ELISA. CSF adiponectin was positively correlated to systemic levels, and the CSF/serum adiponectin ratio was correlated to the CSF/serum albumin ratio. Furthermore, disturbed function of the blood-brain barrier (BBB) was associated with an elevated CSF/serum adiponectin ratio. Adiponectin mRNA was not found in the brain, indicating that adiponectin crosses the BBB and/or the blood-cerebrospinal fluid barrier (BCB). Rat adiponectin with a COOH-terminal tag was injected into the tail vein of rats and was detected 3 h later in CSF. However, CSF adiponectin in humans and rats was approximately 0.1% of the serum concentration and therefore was below the 0.5% expected in the CSF because of the residual leakage of an undisturbed BBB/BCB. Taken together, data from the present study show that adiponectin in human CSF is far below the level expected by the baseline BBB/BCB permeability, indicating that adiponectin enters the brain much less efficiently than albumin, thus supporting recent data that exclude adiponectin transport to the CSF. Additional studies are needed to reveal whether these low levels of adiponectin in CSF have a physiological function.     

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.     

Effect of mutations mimicking phosphorylation on the structure and properties of human 14-3-3zeta

Effect of mutations mimicking phosphorylation on the structure of human 14-3-3ζ protein was analyzed by different methods. Mutation S58E increased intrinsic Trp fluorescence and binding of bis-ANS to 14-3-3. At low protein concentration mutation S58E increased the probability of dissociation of dimeric 14-3-3 and its susceptibility to proteolysis. Mutation S184E slightly increased Stokes radius and thermal stability of 14-3-3. Mutation T232E induced only small increase of Stokes radius and sedimentation coefficient that probably reflect the changes in the size or shape of 14-3-3. At low protein concentration the triple mutant S58E/S184E/T232E tended to dissociate, whereas at high concentration its properties were comparable with those of the wild type protein. The triple mutant was highly susceptible to proteolysis. Thus, mutation mimicking phosphorylation of Ser58 destabilized, whereas mutation of Ser184 induced stabilization of 14-3-3ζ structure.     

Protein 14-3-3zeta binds to protein phosphatase PP1gamma2 in bovine epididymal spermatozoa

The protein phosphatase PP1gamma2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1gamma2 is phosphorylated and that the amount of phosphorylated PP1gamma2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439-447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1gamma2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species-bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1gamma2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1gamma2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1gamma2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1gamma2 is one of its binding partners.     

Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen-activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)-differentiated PC12 cells, we found that constitutive and autonomous Ca2+-independent PKC activity is associated with 14-3-3 zeta. Because PKC and 14-3-3 zeta are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14-3-3 zeta in the brain and whether the PKC/14-3-3 zeta complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+-dependent classical PKC, Ca2+-independent novel PKC, and Ca2+-independent and diacylglycerol-insensitive atypical PKC, all interact with 14-3-3 zeta in the rodent brain. The pool size of 14-3-3 zeta bound form of PKC is small (1-4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14-3-3 zeta is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14-3-3 zeta could be a mechanism for the persistent PKC activation in the brain.     

The interaction between ADAM 22 and 14-3-3zeta: regulation of cell adhesion and spreading

The ADAM family consists of a number of transmembrane proteins that contain disintegrin-like and metalloproteinase-like domains. Therefore, ADAMs potentially have cell adhesion and protease activities. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in the regulation of various cellular functions. Here we report the identification of a novel interaction between the ADAM 22 cytoplasmic tail and the 14-3-3zeta isoform by a yeast two-hybrid screen. The interaction between the ADAM 22 cytoplasmic tail and 14-3-3zeta was confirmed by an in vitro protein pull-down assay as well as by co-immunoprecipitation, and the binding sites were mapped to the 28 amino acid residues of the C-terminus of the ADAM 22 cytoplasmic tail. Furthermore, we found that overexpression of the ADAM 22 cytoplasmic tail in human SGH44 cells inhibited cell adhesion and spreading and that deletion or mutation of the binding site for 14-3-3zeta within the ADAM 22 cytoplasmic tail abolished the ability of the overexpressed cytoplasmic tail to alter cell adhesion and spreading. Taken together, these results for the first time demonstrate an association between ADAM 22 and a 14-3-3 protein and suggest a potential role for the 14-3-3zeta/ADAM 22 association in the regulation of cell adhesion and related signaling events.