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.     

Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester.

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.     

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.     

Direct interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta translocation and function.

Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.     

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.     

Melatonin activates PKC-alpha but not PKC-epsilon in N1E-115 cells.

Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.     

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.     

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.     

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.     

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.     

Purification and characterization of the zeta isoform of protein kinase C from bovine kidney.

The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.     

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.     

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.     

A key role for a 145-kDa cytosolic protein in the stimulation of Ca(2+)-dependent secretion by protein kinase C.

Cytoplasmic Ca2+ is a major regulator of exocytosis in secretory cells; however, the Ca(2+)-dependent mechanisms that trigger secretion have not been elucidated. Protein kinase C (PKC) has been proposed to be an important Ca(2+)-dependent component of this regulation; however, the effects of this enzyme on the exocytotic apparatus have not been identified. We developed a PKC-deficient, semi-intact PC12 cell system in which direct stimulatory effects of purified PKC on Ca(2+)-dependent norepinephrine secretion were studied. The reconstitution of optimal Ca(2+)-activated norepinephrine secretion by semi-intact PC12 cells required the addition of MgATP and cytosolic proteins. PKC-deficient cytosol exhibited reduced reconstituting activity that was fully restored by the addition of purified PKC. The restoration of Ca(2+)-dependent norepinephrine secretion by PKC required the presence of other proteins in the cytosol, in particular, a high molecular weight protein. The high molecular weight protein was identified as p145, a recently characterized 145-kDa brain protein. The addition of PKC enhanced phosphorylation of p145 under conditions of fully reconstituted Ca(2+)-activated norepinephrine secretion. The results indicate that 1) PKC is neither necessary nor sufficient for Ca(2+)-activated secretion, whereas other cytosolic proteins are required; and 2) the stimulation of Ca(2+)-activated secretion by PKC is dependent upon cytosolic proteins such as p145 and may be largely mediated through the phosphorylation of p145.     

Uncoupling of ATP-induced inositol phosphate formation and Ca(2+) mobilization by phorbol ester in canine cultured tracheal epithelial cells

The regulation of the increase in inositol phosphates (IPs) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca(2+) mobilization. The concentrations of PMA that gave half-maximal (EC(50)) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca(2+)](i) were 5-10 and 4-12 nM, respectively. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -theta, and -zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, and -theta from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-alpha, -betaI, -betaII, -delta, -epsilon, -gamma, and -theta induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca(2+) mobilization in TECs.     

Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains

We have previously demonstrated that hexanoyl-D-erythro-sphingosine (C(6)-ceramide), an anti-mitogenic cell-permeable lipid metabolite, limited vascular smooth muscle growth by abrogating trauma-induced Akt activity in a stretch injury model of neointimal hyperplasia. Furthermore, ceramide selectively and directly activated protein kinase C zeta (PKC zeta) to suppress Akt-dependent mitogenesis. To further analyze the interaction between ceramide and PKC zeta, the ability of ceramide to localize within highly structured lipid microdomains (rafts) and activate PKC zeta was investigated. Using rat aorta vascular smooth muscle cells (A7r5), we now demonstrate that C(6)-ceramide treatment results in an increased localization and phosphorylation of PKC zeta within caveolin-enriched lipid microdomians to inactivate Akt. In addition, ceramide specifically reduced the association of PKC zeta with 14-3-3, a scaffold protein localized to less structured regions within membranes. Pharmacological disruption of highly structured lipid microdomains resulted in abrogation of ceramide-activated, PKC zeta-dependent Akt inactivation, whereas molecular strategies suggest that ceramide-dependent PKC zeta phosphorylation of Akt3 at Ser(34) was necessary for ceramide-induced vascular smooth muscle cell growth arrest. Taken together, these data demonstrate that structured membrane microdomains are necessary for ceramide-induced activation of PKC zeta and resultant diminished Akt activity, leading to vascular smooth muscle cell growth arrest.     

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.