Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis

The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).     

Conserved role for 14-3-3epsilon downstream of type I TGFbeta receptors

Schistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor beta (TGFbeta) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S. mansoni 14-3-3epsilon. The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3epsilon also binds to TbetaRI, the human type I TGFbeta receptor. 14-3-3epsilon enhances TGFbeta-mediated signaling by TbetaRI and is the first TbetaRI-interacting non-Smad protein identified that positively regulates this receptor. The interaction of 14-3-3epsilon with schistosome and human TbetaRI suggests a conserved, but previously unappreciated, role for this protein in TGFbeta signaling pathways.     

Dynamic expression and cellular localization of the drosophila 14-3-3epsilon during embryonic development.

The 14-3-3 protein family is known to interact with various proteins involved in signaling pathways. We report here the expression pattern of the Drosophila 14-3-3 (d14-3-3epsilon) protein during embryonic development. In syncytial blastoderm when the nuclei divided rapidly, d14-3-3epsilon localized in the nuclei. During cellularization d14-3-3epsilon gradually became membrane-bound. During gastrulation, an enhanced staining in the perinuclear region was observed in various tissues. Co-labeling with dp-ERK which recognized the activated form of MAPK suggested that d14-3-3epsilon was expressed prior to MAPK activation. During neuronal differentiation, the d14-3-3epsilon protein remained at a high level in the neuronal cytoplasm.     

Cloning of Schistosoma japonicum 14-3-3 epsilon (Sj14-3-3 epsilon), a new member of the 14-3-3 family of proteins from schistosomes

A new member of the 14-3-3 protein family from Schistosoma japonicum has been identified. Phylogenetic analysis showed that this member belongs to the epsilon subfamily of the 14-3-3 proteins, and it is therefore named Sj14-3-3 epsilon. Consistent with the findings for the previously reported S. japonicum 14-3-3 protein (Sj14-3-3), Southern analysis suggested the presence of more than one gene, and/or introns or allelic polymorphism in this epsilon isoform. By RT-PCR, Sj14-3-3 epsilon was shown to be stage-specifically transcribed, being abundant in adults, present in sporocysts but absent in cercariae. Furthermore, mRNA of the epsilon isoform seemed to be much less abundant in the sporocyst stage, compared with Sj14-3-3. This suggests varying requirements of the different 14-3-3 isoforms at different stages of the life cycle.     

14-3-3 epsilon modulates the stimulated secretion of endopeptidase 24.15

Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine-scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser(644) by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 microm). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 microm) from 10%[1.4 +/- 0.24 AFU/(min 10(6) cells)] to 19%[2.54 +/- 0.24 AFU/(min 10(6) cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.     

Regulation of poly(A) polymerase by 14-3-3epsilon

Poly(A) polymerase (PAP) is a key enzyme responsible for the addition of the poly(A) at the 3' end of pre-mRNA. The C-terminal region of mammalian PAP carries target sites for protein-protein interaction with the 25 kDa subunit of cleavage factor I and with splicing factors U1A and U2AF65. We used a yeast two-hybrid screen to identify 14-3-3epsilon as an additional protein binding to the C-terminal region of PAP. Interaction between PAP and 14-3-3epsilon was confirmed by both in vitro and in vivo binding assays. This interaction is dependent on PAP phosphorylation. Deletion analysis of PAP suggests that PAP contains multiple binding sites for 14-3-3epsilon. The binding of 14-3-3epsilon to PAP inhibits the polyadenylation activity of PAP in vitro, and overexpression of 14-3-3epsilon leads to a shorter poly(A) mRNA tail in vivo. In addition, the interaction between PAP and 14-3-3epsilon redistributes PAP within the cell by increasing its cytoplasmic localization. These data suggest that 14-3-3epsilon is involved in regulating both the activity and the nuclear/ cytoplasmic partitioning of PAP through the phosphorylation-dependent interaction.     

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.     

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.     

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1′ insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal α-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.     

Inhibitory interaction of the plasma membrane Na+/Ca2+ exchangers with the 14-3-3 proteins

The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.     

Interaction of HCV core protein with 14-3-3epsilon protein releases Bax to activate apoptosis

Through protein-protein binding assays, we found that HCV core protein interacted with 14-3-3epsilon protein. Interestingly, the expression of HCV core protein induced apoptosis in 293T cells. The apoptosis induced by core expression is accompanied by translocation of Bax from cytosol to mitochondria, disruption of mitochondrial membrane potential, cytochrome c release, and activation of caspase-9 and caspase-3. Furthermore, over-expression of 14-3-3epsilon inhibited the core-induced apoptosis and Bax translocation to mitochondria. These results indicate that HCV core protein induces the Bax-mediated apoptosis by interacting with 14-3-3epsilon protein in 293T cells. As a mechanism of apoptosis induction by HCV core, we propose that the interaction of HCV core with 14-3-3epsilon causes the dissociation of Bax from the Bax/14-3-3epsilon complex in cytosol, and the free Bax protein provokes activation of the mitochondrial apoptotic pathway.     

14-3-3 is involved in p75 neurotrophin receptor-mediated signal transduction

The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.     

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca(2+)/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser(19), but PKA also phosphorylated Ser(104), as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser(19), Ser(99), Ser(104) and Ser(306). On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser(19). This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins gamma, epsilon and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.     

Inhibitory interaction of the 14-3-3{epsilon} protein with isoform 4 of the plasma membrane Ca(2+)-ATPase pump

The isoform-specific interaction of plasma membrane Ca(2+)-ATPase (PMCA) pumps with partner proteins has been explored using a yeast two-hybrid technique. The 90 N-terminal residues of two pump isoforms (PMCA2 and PMCA4), which have a low degree of sequence homology, have been used as baits. Screening of 5 x 10(6) clones of a human brain cDNA library yielded approximately 100 LEU2- and galactoside-positive clones for both pumps. A clone obtained with the PMCA4 bait specified the epsilon-isoform of the 14-3-3 protein, whereas no 14-3-3epsilon clone was obtained with the PMCA2 bait. The 14-3-3epsilon protein immunoprecipitated with PMCA4 (not with PMCA2) when expressed in HeLa cells. Overexpression of 14-3-3epsilon in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca(2+) was impaired; stimulation with histamine, an inositol 1,4,5-trisphosphate-producing agonist, generated higher cytosolic [Ca(2+)] transients, higher post-transient plateaus of the cytosolic [Ca(2+)], and higher Ca(2+) levels in the endoplasmic reticulum lumen and in the subplasmalemmal domain. Thus, the interaction with 14-3-3epsilon inhibited PMCA4. Silencing of the 14-3-3epsilon gene by RNA interference significantly reduced the expression of 14-3-3epsilon, substantially decreasing the height of the histamine-induced cytosolic [Ca(2+)] transient and of the post-transient cytosolic [Ca(2+)] plateau.     

Association of 14-3-3 proteins to beta1-adrenergic receptors modulates Kv11.1 K+ channel activity in recombinant systems

We identify a new mechanism for the beta(1)-adrenergic receptor (beta(1)AR)-mediated regulation of human ether-a-go-go-related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3epsilon proteins is competed by wild type beta(1)AR by means of a novel interaction between this receptor and 14-3-3epsilon. The association between beta(1)AR and 14-3-3epsilon is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The beta(1)AR/14-3-3epsilon association is direct, since it can be recapitulated using purified 14-3-3epsilon and beta(1)AR fusion proteins and is abolished in cells expressing beta(1)AR phosphorylation-deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that beta(1)AR phosphorylation-deficient mutants do not recruit 14-3-3epsilon away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type beta(1)AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both beta(1)AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness.     

Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon

Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.     

Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response.

Schistosoma mansoni receptor kinase 1 (SmRK1) is a divergent member of the TGF beta receptor family. Intracellular proteins that associate with these receptors are likely to play an important role in signaling. 14-3-3 epsilon is a previously described cytoplasmic protein, which associates with both SmRK1 and the human type I TGF beta receptor (T beta RI); overexpression of 14-3-3 epsilon leads to enhanced TGF beta-mediated signaling by T beta RI. We now describe the identification of S. mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha), through its interaction with SmRK1 in a yeast two-hybrid assay. S. mansoni eIF2 alpha also interacts with human TGF beta receptors. Strongest association was demonstrated with kinase inactive receptors, particularly the type II TGF beta receptor (T beta RII). Both T beta RI and T beta RII phosphorylate eIF2 alpha in vitro, at sites other than the previously described eIF2 alpha phosphorylation sites. EIF2 alpha also modulates signaling by TGF beta receptors; however, in contrast to 14-3-3 epsilon, eIF2 alpha overexpression inhibits the TGF beta-driven response. These data suggest a novel function for eIF2 alpha in the TGF beta signaling pathway. In addition, we have demonstrated an independent interaction between eIF2 alpha and 14-3-3 epsilon. Coexpression of 14-3-3 epsilon with eIF2 alpha leads to the abrogation of the inhibitory effect of eIF2 alpha on TGF beta-mediated signaling. The interaction of these two regulatory proteins with each other and with the TGF beta receptors and their relative expression levels are likely to be important in fine-tuning the regulation of TGF beta signal transduction.     

Triptolide inhibits colon cancer cell proliferation and induces cleavage and translocation of 14-3-3 epsilon

Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii. In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo. Using a proteomic approach, we found that 14-3-3 epsilon, a cell cycle- and apoptosis-related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14-3-3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14-3-3 epsilon.