Arachidonic Acid Binds 14-3-3ζ, Releases 14-3-3ζ from Phosphorylated BAD and Induces Aggregation of 14-3-3ζ

Polyunsaturated fatty acids, like arachidonic acid, can bind proteins and affect their function. The 14-3-3 proteins bind phosphorylated sites on a diverse array of client proteins and, in this way, are involved in many intracellular signaling pathways. In this study, we used a novel approach to discover that 14-3-3ζ is able to directly bind arachidonic acid. Furthermore, arachidonic acid, at physiological concentrations, reduced the binding of 14-3-3ζ to phosphorylated BAD, an interaction that is important in regulating apoptosis. In addition, high concentrations of arachidonic acid caused the polymerization of 14-3-3ζ, an event observed in neurodegenerative disorders. Taken together, these results indicate that arachidonic acid directly interacts with 14-3-3ζ and that this interaction may be important in both normal and pathological cellular events. If so, then factors that mediate the release, metabolism and reacylation of arachidonic acid into membranes represent key points of regulation.     

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of σ, all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD₁₄). Long-term treatment of mpkCCD₁₄ cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3β and -ζ, whereas levels of 14-3-3η and -θ were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD₁₄ cells uncovered an AQP2/14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3ζ and -θ. Use of phosphatase inhibitors in mpkCCD₁₄ cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3ζ in mpkCCD₁₄ cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3θ and -ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation.     

Homocysteine Induces Apoptosis of Rat Hippocampal Neurons by Inhibiting 14-3-3ε Expression and Activating Calcineurin

A high level of plasma homocysteine (Hcy) increases the risk for neurodegenerative diseases. One such disorder is Alzheimer’s disease, which involves marked neuronal apoptosis of unknown etiology. This study shows that Hcy inhibits expression of 14-3-3ε and activates calcineurin in rat hippocampal neurons in a dose-dependent manner. Calcineurin-mediated Bad dephosphorylation, which is blocked by calcineurin inhibition and Ca²⁺ chelation, causes mitochondrial translocation of Bad and apoptosis; this step in the apoptotic pathway is synergistically blocked by calcineurin inhibition and overexpression of 14-3-3ε. These findings demonstrated that calcineurin activation and downregulation of 14-3-3ε may be one of the mechanisms of Hcy-induced apoptosis of hippocampal neurons.     

Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.     

Brap2 Regulates Temporal Control of NF-κB Localization Mediated by Inflammatory Response

Nuclear factor-kappaB (NF-κB) is critical for the expression of multiple genes involved in inflammatory responses and cellular survival. NF-κB is normally sequestered in the cytoplasm through interaction with an inhibitor of NF-κB (IκB), but inflammatory stimulation induces proteasomal degradation of IκB, followed by NF-κB nuclear translocation. The degradation of IκB is mediated by a SCF (Skp1-Cullin1-F-box protein)-type ubiquitin ligase complex that is post-translationaly modified by a ubiquitin-like molecule Nedd8. In this study, we report that BRCA1-associated protein 2 (Brap2) is a novel Nedd8-binding protein that interacts with SCF complex, and is involved in NF-κB translocation following TNF-α stimulation. We also found a putative neddylation site in Brap2 associated with NF-κB activity. Our findings suggest that Brap2 is a novel modulator that associates with SCF complex and controls TNF-α-induced NF-κB nuclear translocation.     

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.     

Dissection of Binding between a Phosphorylated Tyrosine Hydroxylase Peptide and 14-3-3ζ: A Complex Story Elucidated by NMR

Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3ζ, by ³¹P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3ζ binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3ζ one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3ζ dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3ζ dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system.     

Prostacyclin and PPARα Agonists Control Vascular Smooth Muscle Cell Apoptosis and Phenotypic Switch through Distinct 14-3-3 Isoforms

We hypothesized that prostacyclin (PGI₂) protects vascular smooth muscle cell (VSMC) against apoptosis and phenotypic switch through peroxisome proliferator-activated receptor-α (PPARα) activation and 14-3-3 upregulation. Here we showed that transfection of rat aortic VSMC, A-10, with PGI₂-producing vectors, Ad-COPI, resulted in attenuated H₂O₂-induced apoptosis accompanied by a selective increase in 14-3-3β and 14-3-3θ expression. Carbaprostacyclin (cPGI₂) and Wy14,643 exerted a similar effect. The effects of PGI₂ were abrogated by MK886, a PPARα antagonist, but not GSK3787, a PPARδ antagonist. PPARα transfection upregulated 14-3-3β and θ expression and attenuated H₂O₂-induced apoptosis. H₂O₂-induced 14-3-3β but not 14-3-3θ degradation was blocked by a caspase 3 inhibitor. Furthermore, 14-3-3β but not 14-3-3θ overexpression reduced, while 14-3-3β siRNA aggravated apoptosis. VSMC contractile proteins and serum response factor (SRF) were reduced in H₂O₂-treated A-10 cells which were concurrently prevented by caspase 3 inhibitor. By contrast, PGI₂ prevented H₂O₂-induced SM22α and Calponin-1 degradation without influencing SRF. cPGI₂ and Wy14,643 also effectively blocked VSMC phenotypic switch induced by growth factors (GFs). GFs suppressed 14-3-3β, θ, ε and η isoforms and cPGI₂ prevented the decline of β, θ and η, but not ε. 14-3-3θ siRNA abrogated the protective effect of cPGI₂ on SM22α and Calponin-1 while 14-3-3 θ or 14-3-3β overexpression partially restored SM22α. These results indicated that PGI₂ protects VSMCs via PPARα by upregulating 14-3-3β and 14-3-3θ. 14-3-3β upregulation confers resistance to apoptosis whereas 14-3-3θ and β upregulation protects SM22α and Calponin-1 from degradation.     

Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis

Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C₂-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis.     

Systematic Dissection of the Sequence Determinants of Gene 3’ End Mediated Expression Control

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.     

Site-specific Phosphorylation of Kindlin-3 Protein Regulates Its Capacity to Control Cellular Responses Mediated by Integrin αIIbβ3

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin α_IIbβ₃, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), α_IIbβ₃ became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β₃ integrin and kindlin-3 in focal adhesions, and enhanced β₃ integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr⁴⁸² or Ser⁴⁸⁴ was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476–485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β₃ activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.     

Estrogen Attenuates P2X7‐R—Mediated Apoptosis of Uterine Cervical Cells by Blocking Calcium Influx

Estrogen blocks apoptosis of human ectocervical epithelial cells by modulating P2X₇/Ca^2 + influx. The effect involves decreased Ca^2 +‐influx and cytosolic‐calcium‐increase via ATP‐activated P2X₇ pores. This mechanism may have physiological significance in the human cervix, in‐vivo, and the results suggest a physiological role for estrogen in the cervix as an anti‐apoptotic factor.     

Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr³⁹³/Ser³⁹⁵) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.     

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

Serine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.

Structured summary

MINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)MINT-7233285, MINT-7233297, MINT-7233310: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-κB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-κB activation by H-Ala-D-γGlu-DAP (γTriDAP). GEF-H1 is essential for NF-κB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens.     

Activation of GSK-3β and Caspase-3 Occurs in Nigral Dopamine Neurons during the Development of Apoptosis Activated by a Striatal Injection of 6-Hydroxydopamine

The 6-Hydroxydopamine (6-OHDA) rat model of Parkinson''s disease is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising therapeutic interventions. This work evaluated whether a single striatal injection of 6-OHDA causes progressive apoptosis of dopamine (DA) neurons and activation of glycogen synthase kinase 3β (GSK-3β) and caspase-3 in the substantia nigra compacta (SNc). The loss of DA neurons was shown by three neuron markers; tyrosine hydroxylase (TH), NeuN, and β-III tubulin. Apoptosis activation was determined using Apostain and immunostaining against cleaved caspase-3 and GSK-3β pY216. We also explored the possibility that cleaved caspase-3 is produced by microglia and astrocytes. Our results showed that the 6-OHDA caused loss of nigral TH(+) cells, progressing mainly in rostrocaudal and lateromedial directions. In the neostriatum, a severe loss of TH(+) terminals occurred from day 3 after lesion. The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc. Apostain immunoreactivity was observed from days 3 to 21 postlesion. Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion. The cleaved caspase-3 also collocated with microglia and astrocytes indicating its participation in glial activation. Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.