Role of individual phosphorylation sites for the 14-3-3-protein-dependent activation of yeast neutral trehalase Nth1

Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1.     

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.     

Molecular and cellular characterization of a water-channel protein,AQP-h3, specifically expressed in the frog ventral skin

The Ca2+ content of pancreatic juice is closely regulated by yet unknown mechanisms. One aim of the present study was to find whether rat pancreatic ducts have a Na+/Ca2+ exchanger, as found in some Ca2+ transporting epithelia. Another aim was to establish whether the exchanger is regulated by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1 nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2+ concentrations in pancreatic juice.     

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.     

BHK cells transfected with NCX3 are more resistant to hypoxia followed by reoxygenation than those transfected with NCX1 and NCX2: Possible relationship with mitochondrial membrane potential

The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.     

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers

Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, epsilon and gamma. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer-dimer dissociation constant from its normal value of 120-150 nM.     

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.     

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.     

Up-regulation of Na(+)-Ca2+ exchange activity by interferon-gamma in cultured rat microglia

The Na(+)-Ca2+ exchanger (NCX) plays a role in regulating intracellular Ca2+ concentration, but little is known about NCX in microglia. We examined mRNA expression of NCX isoforms in cultured rat microglia and the effect of interferon-gamma (IFN-gamma) on NCX activity. RT-PCR showed that all of the known NCX isoforms, NCX1-3, are expressed in cultured microglia. Ouabain and monensin increased 45Ca2+ uptake and intracellular Ca2+ concentration in microglia, suggesting the presence of NCX activity in the reverse mode. Treatment with IFN-gamma (100 U/mL) caused a biphasic increase in NCX activity. The transient increase in NCX activity by IFN-gamma for 1 h was blocked by the protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and the tyrosine kinase inhibitor, herbimycin A. The delayed increase in NCX activity by IFN-gamma for 24 h was blocked by the protein synthesis inhibitor cycloheximide and actinomycin D. Treatment with IFN-gamma for 24 h increased NCX mRNA and protein levels. The increase in NCX activity and NCX protein by IFN-gamma for 24 h was blocked by staurosporine, GF109203X, herbimycin A and the extracellular signal-regulated kinase inhibitor, PD98059. These findings suggest that NCX is up-regulated by IFN-gamma in a biphasic manner in microglia. Moreover, PKC and tyrosine kinase are involved in the up-regulation of NCX and the extracellular signal-regulated protein kinase is also involved in the delayed increase in NCX activity.     

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

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

An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.     

Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-(32)P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.     

Cellular and subcellular localization of Na+-Ca2+ exchanger protein isoforms, NCX1, NCX2, and NCX3 in cerebral cortex and hippocampus of adult rat

Na(+)-Ca(2+) exchanger (NCX) controls cytosolic Ca(2+) and Na(+) concentrations ([Ca(2+)](i) and [Na(+)](i)) in eukaryotic cells. Here we investigated by immunocytochemistry the cellular and subcellular localization of the three known NCX isoforms, NCX1, NCX2 and NCX3, in adult rat neocortex and hippocampus. NCX1-3 were widely expressed in both brain areas: NCX1 immunoreactivity (ir) was exclusively associated to neuropilar puncta, while NCX2-3 were also detected in neuronal somata and dendrites. NCX1-3 ir was often identified around blood vessels. In both neocortex and hippocampus, all NCX isoforms were prominently expressed in dendrites and dendritic spines contacted by asymmetric axon terminals, whereas they were poorly expressed in presynaptic boutons. In addition, NCX1-3 ir was detected in astrocytes, notably in distal processes ensheathing excitatory synapses. All NCXs were expressed in perivascular astrocytic endfeet and endothelial cells. The robust expression of NCX1-3 in heterogeneous cell types in the brain in situ emphasizes their role in handling Ca(2+) and Na(+) in both excitable and non-excitable cells. Perisynaptic localization of NCX1-3 in dendrites and spines indicates that all isoforms are favourably located for buffering [Ca(2+)](i) in excitatory postsynaptic sites. NCX1-3 expressed in perisynaptic glial processes may participate in shaping astrocytic [Ca(2+)](i) transients evoked by ongoing synaptic activity.     

Synthesis and structure-activity relationships of 6-{4-[(3-fluorobenzyl)oxy]phenoxy}nicotinamide derivatives as a novel class of NCX inhibitors: a QSAR study

The sodium-calcium exchanger (NCX) transports Na+ and Ca2+ ions, and controls the Ca2+ concentration in myocytes. Calcium overload is induced via activation of reverse NCX, and is responsible for reperfusion injury in heart failure. Hence, NCX is an attractive target for prevention and treatment of reperfusion arrhythmias, myocardial contracture, and necrosis. We have synthesized a series of 6-{4-[(3-fluorobenzyl)oxy]phenoxy}nicotinamide derivatives, and evaluated their inhibitory activity against the reverse and forward modes of NCX. N-(3-Aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy}nicotinamide (8) was shown to be a potent inhibitor of reverse NCX activity, with an IC50 value of 0.24 microM. A QSAR study showed that inhibition of reverse NCX activity by 6-{4-[(3-fluorobenzyl)oxy]phenoxy}nicotinamide derivatives is multiply dependent on the hydrophobicity (pi) and the shape (B(iv)) of the substituent at the 3-position of the phenyl ring.     

Fluorescent Na+-Ca+ exchangers: electrophysiological and optical characterization

The cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1) influences cardiac contractility by extruding Ca2+ from myocytes. As a Ca2+ efflux mechanism, the exchanger plays a prominent role in Ca2+ homeostasis. To track NCX1 and study changes in conformation, NCX1 was tagged with derivatives of green fluorescent protein. Cyan (CFP) and yellow (YFP) fluorescent proteins were used for both visualization of the protein in HEK cells and fluorescent resonance energy transfer (FRET). CFP or YFP was inserted at position 266, 371, 467, or 548 of the large intracellular loop of NCX1 located between transmembrane segments 5 and 6. These constructs were tested for functional activity and visualized for cell surface expression. All constructs were targeted to the plasma membrane. Transport properties were assessed by both 45Ca2+ uptake and electrophysiological measurements. The fluorescent-tagged exchangers had similar biophysical properties to the wild type NCX1. Unexpectedly, all constructs retain their sensitivity to regulation by cytoplasmic Na+ and Ca2+ ions. FRET analysis indicates the proximity of NCX1 to plasma membrane phosphatidylinositol 4,5-bisphosphate. These results indicate that insertion of CFP or YFP into the large intracellular loop of NCX1 protein does not impair exchanger properties. These constructs will be useful to further characterize the biological properties of the exchanger in intact cells.     

Structural and dynamic aspects of Ca2+ and Mg2+ binding of the regulatory domains of the Na+/Ca2+ exchanger

Intracellular Ca2+ regulates the activity of the NCX (Na+/Ca2+ exchanger) through binding to the cytosolic CBD (Ca2+-binding domain) 1 and CBD2. In vitro studies of the structure and dynamics of CBD1 and CBD2, as well as studies of their kinetics and thermodynamics of Ca2+ binding, greatly enhanced our understanding of NCX regulation. We describe the fold of the CBDs in relation to other known structures and review Ca2+ binding of the different CBD variants from a structural perspective. We also report on new findings concerning Mg2+ binding to the CBDs and finally we discuss recent results on CBD1-CBD2 interdomain interactions.