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Activating transcription factor 2 (ATF2), a member of the bZIP transcription factor family, is involved in multiple physiological and developmental processes, yet its role in the innate immunity remains unclear. In this study, two isoforms (named as MnATF2a and MnATF2b) of ATF2 gene were identified in Macrobrachium nipponense and were produced by exon skipping. The full length of MnATF2a is 2328 bp with an open reading frame of 2079 bp that encode 692 amino acids. MnATF2a has 237 bp nucleotides more than MnATF2b and the extra 237 bp is a complete exon. MnATF2a and MnATF2b proteins contain the same conserved and typical bZIP domain at the C-terminus. MnATF2a has 79 amino acids more than MnATF2b. MnATF2a and MnATF2b are widely distributed in a variety of immune tissues. After Vibrio parahaemolyticus and Staphylococcus aureus infection, the expression levels of MnATF2a and MnATF2b were significant up-regulated in the gills and stomach at 12 h. RNA interference analysis showed that knockdown of the total MnATF2 gene significantly inhibits the transcription of tumor necrosis factor (TNF) and promotes the expression of crustins (including Cru3, Cru4, and Cru7). Further study showed that knockdown of MnTNF evidently increase the expression of Cru3, Cru4, and Cru7. Our research indicates that ATF2 negatively regulate the expression of AMPs by regulating the transcription of TNF in M. nipponense. This study provides valuable information about the function of ATF2 family in the innate immunity in crustacean., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Candida albicans is the most common fungal pathogen of mucosal infections and invasive diseases in immuno-compromised humans. The abilities of yeast-hyphal growth and white-opaque switching affect C. albicans physiology and virulence. Here, we showed that C. albicans Aft2 regulator was required for embedded filamentous growth and opaque cell-type formation. Under low-temperature matrix embedded conditions, Aft2 functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Moreover, deletion of AFT2 significantly reduced opaque cell-type formation under N-acetylglucosamine (GlcNAc) inducing conditions. Ectopic expression of CZF1 slightly increased the white-opaque switching frequency in the aft2Δ/Δ mutant, but did not completely restore to wild-type levels, suggesting that Czf1 at least partially bypassed the essential requirement for Aft2 in response to opaque-inducing cues. In addition, multiple environmental cues altered AFT2 mRNA and protein levels, such as low temperature, physical environment and GlcNAc. Although the absence of Czf1 or Efg1 also increased the expression level of AFT2 gene, deletion of CZF1 remarkably reduced the stability of Aft2 protein. Furthermore, C. albicans Aft2 physically interacted with Czf1 under all tested conditions, whereas the interaction between Aft2 and Efg1 was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate filamentous growth by antagonizing Efg1-mediated repression under matrix-embedded conditions., (© 2015 Wiley Periodicals, Inc.)

Activating transcription factor 2 (ATF2) and its homolog ATF7 are phosphorylated at Thr-69/Thr-71 and at Thr-51/Thr-53, respectively, by stress-activated MAPKs regulating their transcriptional functions in G1 and S phases. However, little is known about the role of ATF2 and ATF7 in G2/M phase. Here, we show that Cdk1-cyclin B1 phosphorylates ATF2 at Thr-69/Thr-71 and ATF7 at Thr-51/Thr-53 from early prophase to anaphase in the absence of any stress stimulation. Knockdown of ATF2 or ATF7 decreases the rate of cell proliferation and the number of cells in M-phase. In particular, the knockdown of ATF7 severely inhibits cell proliferation and G2/M progression. The inducible expression of a mitotically nonphosphorylatable version of ATF7 inhibits G2/M progression despite the presence of endogenous ATF7. We also show that mitotic phosphorylation of ATF7 promotes the activation of Aurora kinases, which are key enzymes for early mitotic events. These results suggest that the Cdk1-mediated phosphorylation of ATF7 facilitates G2/M progression, at least in part, by enabling Aurora signaling.

JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, which are involved in the transcriptional activation of EBV lytic genes. This study sought to elucidate the mechanism by which Rta activates transcription of the Zta-encoding gene, BZLF1, through the ZII element in the gene promoter. In a DNA affinity precipitation assay, ATF2 was found to associate with an Rta-interacting protein, MCAF1, at the ZII element. The interaction between Rta, MCAF1, and ATF2 at the same site in the ZII region was further verified in vivo by chromatin immunoprecipitation assay. The complex appears to be crucial for the activation of BZLF1 transcription, as the overexpression of two ATF2-dominant negative mutants, or the introduction of MCAF1 siRNA into 293T cells, were both found to substantially reduce Rta-mediated transcription levels of BZLF1. Moreover, this study also found that the Rta-MCAF1-ATF2 complex binds to a typical AP-1 binding sequence on the promoter of BMRF2, a key viral gene for EBV infection. Mutation of this sequence decreased Rta-mediated promoter activity significantly. Taken together, these results indicate a critical role for MCAF1 in AP-1-dependent Rta activation of BZLF1 transcription.

Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.

The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His(6)-tagged proteins and purified using urea refolding and Ni(2+)-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency., (Copyright © 2012 Elsevier Inc. All rights reserved.)

c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

ATF2-Jun, IRF3, and HMGI recognize a composite regulatory element within the interferon-β enhancer (IFNb). Cooperative ATF2-Jun-IRF3 complex formation at IFNb has been proposed to require a fixed orientation of ATF2-Jun binding. Our results show that ATF2-Jun heterodimers bound IFNb in both orientations alone and in association with IRF3 and HMGI. Two sets of symmetrically located amino acid residues in ATF2 and Jun facilitated the interactions between heterodimers bound in opposite orientations and IRF3 at IFNb. IRF3 and HMGI bound IFNb in association with both orientations of ATF2-Jun heterodimers with the same cooperativity. ATF2-Jun heterodimers that bound IFNb in opposite orientations in vitro had different effects on interferon-β gene transcription when they were co-expressed with IRF3 in cultured cells. These heterodimers had different transcriptional activities at different endogenous genes. Different regions of ATF2 and Jun mediated their orientation-dependent transcriptional activities at different genes. These studies revealed that cooperative DNA binding does not require a unique nucleoprotein complex configuration, and that transcription factor complexes that bind the same enhancer in different configurations can have different transcriptional activities.

An increasing number of transcription factors have been shown to elicit oncogenic and tumor suppressor activities, depending on the tissue and cell context. Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. Recent work has shown that the opposing functions of ATF2 are associated with its subcellular localization. In the nucleus, ATF2 contributes to global transcription and the DNA damage response, in addition to specific transcriptional activities that are related to cell development, proliferation and death. ATF2 can also translocate to the cytosol, primarily following exposure to severe genotoxic stress, where it impairs mitochondrial membrane potential and promotes mitochondrial-based cell death. Notably, phosphorylation of ATF2 by the epsilon isoform of protein kinase C (PKCε) is the master switch that controls its subcellular localization and function. Here, we summarize our current understanding of the regulation and function of ATF2 in both subcellular compartments. This mechanism of control of a non-genetically modified transcription factor represents a novel paradigm for 'oncogene addiction'.

The varied selectivity of bZIP transcription factors stems from the fact that they are dimers consisting of two not necessarily identical subunits held together by a leucine zipper dimerization domain. Determining their stability is therefore important for understanding the mechanism of formation of these transcription factors. The most widely used approach for this problem consists of observing temperature-induced dissociation of the bZIPs by any means sensitive to their structural changes, particularly optical methods. In calculating thermodynamic characteristics of this process from such data it is usually assumed that it represents a two-state transition. However, scanning micro-calorimetric study of the temperature-induced unfolding/dissociation of the three bZIPs formed by the ATF-2 and c-Jun proteins, i.e. the two homodimers (ATF-2/ATF-2) and (c-Jun/c-Jun) and the heterodimer (ATF-2/c-Jun), showed that this process does not represent a two-state transition, as found previously with the GCN4 homodimeric bZIP protein. This raises doubt about all indirect estimates of bZIP thermodynamic characteristics based on analysis of their optically-observed temperature-induced changes., (2010 Elsevier B.V. All rights reserved.)

Growth factor stimulation induces c-Jun-dependent survival of primary endothelial cells. However, the mechanism of c-Jun anti-apoptotic activity has not been identified. We here demonstrate that in response to growth factor treatment, primary human endothelial cells as well as mouse fibroblasts respond with an increased expression of c-Jun that forms a complex with ATF2. This complex activates the expression of the anti-apoptotic protein Bcl-X(L). By site-directed mutagenesis experiments, we identified two AP-1-binding sites located within the proximal promoter of the Bcl-X gene. Site-directed mutagenesis demonstrated that these AP-1 sites are required for the transcriptional activation of the promoter. Chromatin immunoprecipitation experiments show that in response to growth factor treatment, the heterodimer c-Jun.ATF2 binds to these functional AP-1 sites. Silencing of either c-Jun or ATF2 demonstrated that both nuclear factors are required for the activation of the proximal Bcl-X promoter. Taken together, our experiments provide evidence that growth factor-independent signaling pathways converge in the formation of an active c-Jun.AFT2 dimer, which induces the expression of the anti-apoptotic factor Bcl-X(L) that mediates a pro-survival response.

Background: The success of anti-TNF biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate TNF production in the quest for an orally-available small molecule inhibitor. p38 is known to strongly regulate TNF production via MK2. The failure of several p38 inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. Recent work has described a 'substrate-selective' p38 inhibitor that is able to preferentially block the activity of p38 against one substrate (MK2) versus another (ATF2). Using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p38 substrates, MK2 and ATF2., Results: We found that in a dual (MK2 and ATF2) substrate assay, MK2-p38 interaction reduced the activity of p38 against ATF2. We further constructed a detailed kinetic mechanistic model of p38 phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called 'substrate-selective' p38 inhibitors in the dual substrate assay. Importantly, it was found that excess MK2 results in a stoichiometric effect in which the formation of p38-MK2-inhibitor complex prevents the phosphorylation of ATF2, despite the preference of the compound for the p38-MK2 complex over the p38-ATF2 complex. MK2 and p38 protein expression levels were quantified in U937, Thp-1 and PBMCs and found that [MK2] > [p38]., Conclusion: Our integrated mechanistic modeling and experimental validation provides an example of how systems biology approaches can be applied to drug discovery and provide a basis for decision-making with limited chemical matter. We find that, given our current understanding, it is unlikely that 'substrate-selective' inhibitors of p38 will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of MK2 relative to p38.

The dimer formed by the ATF-2 and c-Jun transcription factors is one of the main components of the human interferon-beta enhanceosome. Although these two transcription factors are able to form two homodimers and one heterodimer, it is mainly the heterodimer that participates in the formation of this enhanceosome, binding specifically to the positive regulatory domain IV (PRDIV) site of the enhancer DNA. To understand this surprising advantage of the heterodimer, we investigated the association of these transcription factors using fragments containing the basic DNA-recognition segment and the basic leucine zipper domain (bZIP). It was found that the probability of forming the hetero-bZIP significantly exceeds the probability of forming homo-bZIPs, and that the hetero-bZIP interacts more strongly with the PRDIV site of the interferon-beta enhancer, especially in the orientation that places the folded ATF-2 basic segment in the upstream half of this asymmetric site. The effect of salt on the formation of the ATF-2/c-Jun dimer and on its ability to bind the target PRDIV site showed that electrostatic interactions between the charged groups of these proteins and with DNA play an essential role in the formation of the asymmetric ATF-2/c-Jun/PRDIV complex., (2009 Elsevier Ltd. All rights reserved.)

The activator protein 1 (AP-1) transcription factor c-Jun is crucial for neuronal apoptosis. However, c-Jun dimerization partners and the regulation of these proteins in neuronal apoptosis remain unknown. Here we report that c-Jun-mediated neuronal apoptosis requires the concomitant activation of activating transcription factor-2 (ATF2) and downregulation of c-Fos. Furthermore, we have observed that c-Jun predominantly heterodimerizes with ATF2 and that the c-Jun/ATF2 complex promotes apoptosis by triggering ATF activity. Inhibition of c-Jun/ATF2 heterodimerization using dominant negative mutants, small hairpin RNAs, or decoy oligonucleotides was able to rescue neurons from apoptosis, whereas constitutively active ATF2 and c-Jun mutants were found to synergistically stimulate apoptosis. Bimolecular fluorescence complementation analysis confirmed that, in living neurons, c-Fos downregulation facilitates c-Jun/ATF2 heterodimerization. A chromatin immunoprecipitation assay also revealed that c-Fos expression prevents the binding of c-Jun/ATF2 heterodimers to conserved ATF sites. Moreover, the presence of c-Fos is able to suppress the expression of c-Jun/ATF2-mediated target genes and, therefore, apoptosis. Taken together, our findings provide evidence that potassium deprivation-induced neuronal apoptosis is mediated by concurrent upregulation of c-Jun/ATF2 heterodimerization and downregulation of c-Fos expression. This paradigm demonstrates opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis.

c-jun-N-terminal kinase 1alpha1 (JNK1alpha1) is a serine/threonine kinase of the mitogen-activated protein (MAP) kinase family that phosphorylates protein transcription factors after activation by a variety of environmental stressors. In this study, the kinetic mechanism for JNK1alpha1 phosphorylation of activating transcription factor 2 (ATF2) was determined utilizing steady-state kinetics in the presence and absence of both ATF2 and ATP competitive inhibitors. Data from initial velocity studies were consistent with a sequential mechanism for JNK1alpha1. AMP-PCP exhibited competitive inhibition versus ATP and pure noncompetitive inhibition versus ATF2. JIP-1 peptide (RPKRPTTLNLF) was competitive versus ATF2 and mixed noncompetitive versus ATP. These data suggest that JNK1alpha1 proceeded via a random sequential kinetic mechanism with non-interacting ATF2 and ATP substrate sites.

c-jun-N-Terminal kinase 3alpha1 (JNK3alpha1) is a mitogen-activated protein (MAP) kinase family member expressed primarily in the brain that phosphorylates protein transcription factors including c-jun and activating transcription factor 2 (ATF2) upon activation by a variety of stress-based stimuli. In this study, the kinetic mechanism for JNK3alpha1 was determined via initial velocity patterns in the presence and absence of both ATP and ATF2 competitive inhibitors. Peptide inhibitors from both ATF2 (peptide 1) and JNK-interacting protein 1 (JIP-1) (peptide 3), derived from the homologous delta-domain JNK docking sequence, inhibited JNK3alpha1 activity in a competitive fashion versus ATF2 while being pure noncompetitive toward ATP. In contrast, peptides derived from the phosphoacceptor activation domain on ATF2 (peptides 4 and 5) were recognized neither as substrates nor as inhibitors of JNK3alpha1. AMP-PCP and compound 6, a small molecule analinopyrimidine, exhibited pure noncompetitive inhibition versus ATF2 and competitive inhibition versus ATP. Peptide inhibitors based on the delta-domain sites of JIP ( 3) and ATF2 ( 1) were not recognized by p38, also of the MAPK family, which may give insight into finding more selective inhibitors for the JNK family of kinases. Collectively these data showed that JNK3alpha1 proceeded by a random sequential kinetic mechanism and that the ATP and ATF2 substrate sites were non-interacting. Moreover, these results established the 11-mer JIP peptide ( 3) as a potent ( K i = 25 +/- 6 nM) competitive inhibitor versus ATF2 in JNK3alpha1.

Neonatal encephalopathy with seizures (NEWS) is a previously undescribed autosomal recessive disease of standard poodle puppies. Affected puppies are small and weak at birth. Many die in their first week of life. Those surviving past 1 week develop ataxia, a whole-body tremor, and, by 4 to 6 weeks of age, severe generalized clonic-tonic seizures. None have survived to 7 weeks of age. Cerebella from affected puppies were reduced in size and often contained dysplastic foci consisting of clusters of intermixed granule and Purkinje neurons. We used deoxyribonucleic acid samples from related standard poodles to map the NEWS locus to a 2.87-Mb segment of CFA36, which contains the canine ortholog of ATF2. This gene encodes activating transcription factor 2 (ATF-2), which participates in the cellular responses to a wide variety of stimuli. We amplified and sequenced all coding regions of canine ATF2 from a NEWS-affected puppy and identified a T > G transversion that predicts a methionine-to-arginine missense mutation at amino acid position 51. Methionine-51 lies within a hydrophobic docking site for mitogen-activated protein kinases that activate ATF-2 so the arginine substitution is likely to interfere with ATF-2 activation. All 20 NEWS-affected puppies in the standard poodle family were homozygous for the mutant G allele. The 58 clinically normal family members were either G/T heterozygotes or homozygous for the ancestral T allele. There are no previous reports of spontaneous ATF2 mutations in people or animals; however, atf2-knockout mice have cerebellar lesions that are similar to those in puppies with NEWS.

The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.

Yeast Sin1 binds to the Sty1 kinase, a member of the stress-activated kinases (SAPKs), and is required for stress-induced phosphorylation and activation of the transcription factor Atf1, a homolog of the vertebrate-activating transcription factor-2 (ATF-2). Here we report that mammalian Sin1 plays an important role in the SAPK signaling pathway by binding to both ATF-2 and p38. In response to stress, ATF-2, a member of the ATF/cAMP response element-binding protein family, is phosphorylated by p38/Jun NH2-terminal protein kinase and activates the transcription of apoptosis-related genes. In contrast, in response to serum stimulation, ATF-2 is phosphorylated via the Ras effector pathway and leads to the induction of growth-related genes. We found that Sin1 binds directly to both ATF-2 and p38. Sin1 over-expression enhanced osmotic stress-induced phosphorylation of ATF-2 and ATF-2-mediated transcription, whereas knockdown of Sin1 expression by siRNA suppressed these responses. Moreover, a reduction in Sin1 expression suppressed osmotic stress-induced apoptosis and the expression of Gadd45beta, one of the ATF-2 target genes that is correlated with apoptosis. Decreased Sin1 expression, however, did not affect the serum stimulation-induced phosphorylation of ATF-2. Sin1 may contribute to ATF-2 signaling specificity by acting as a nuclear scaffold.

B-ZIP transcription factors dimerization is mediated by a parallel coiled-coil termed the leucine zipper. We have evaluated the dimerization specificity of the seven coiled-coil B-ZIP proteins (ATF6, XBP, LZIP, NFIL3, TEF, CREB, and C/EBPalpha) with themselves and each other. To do this, we designed dominant negative proteins, termed A-ZIPs, that contain the leucine zipper dimerization domain of a B-ZIP protein and an acidic amphipathic N-terminal extension. The A-ZIPs heterodimerize with B-ZIP proteins in a leucine zipper-dependent manner. The acidic N-terminal extension is hypothesized to form an heterodimeric coiled-coil structure with the basic region, essentially zippering the leucine zipper into the basic region. We now present a new acidic extension design that stabilizes heterodimerization with B-ZIP proteins up to 11 kcal mol(-1). We have used these A-ZIP proteins in a competition EMSA to evaluate which A-ZIP can prevent DNA binding of which B-ZIP domain. Inhibition of DNA binding is interpreted to indicate that the A-ZIP is forming a heterodimer with the B-ZIP domain and thus prevents the B-ZIP from binding to DNA. All leucine zippers examined can homodimerize and two pairs (CREB & NFIL3 and ATF6 & XBP) can heterodimerize. We discuss these results with reference to the amino acid sequence of the leucine zipper region. These A-ZIP reagents may be of value in biological systems to inhibit the DNA binding and transcriptional potential of specific B-ZIP families.