Phosphorylation Of Activating Transcription Factor-2 Research Articles

High glucose stimulates angiotensinogen gene expression and cell hypertrophy via activation of the hexosamine biosynthesis pathway in rat kidney proximal tubular cells.

The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG) gene expression and immortalized renal proximal tubular cell (IRPTC) hypertrophy. IRPTC were cultured in monolayer. ANG, renin, and beta-actin mRNA expression were determined by specific RT-PCR assays. Phosphorylation of p38 MAPK, activating transcription factor-2 (ATF-2), and cAMP-responsive element-binding protein (CREB) was determined by Western blot analysis. Cell hypertrophy was assessed by flow cytometry, intracellular p27kip1 protein levels, and [3H]leucine incorporation into proteins. Glucosamine stimulated ANG and renin mRNA expression and enhanced p38 MAPK, ATF-2, and CREB phosphorylation in normal glucose (5 mm) medium. Azaserine and 6-diazo-5-oxo-l-norleucine (inhibitors of glutamine: fructose-6-phosphate amino transferase enzyme) blocked the stimulatory effect of high glucose, but not that of glucosamine, on ANG gene expression in IRPTCs. SB 203580 (a specific p38 MAPK inhibitor) attenuated glucosamine action on ANG gene expression as well as p38 MAPK and ATF-2 phosphorylation, but not that of CREB. GF 109203X and calphostin C (inhibitors of protein kinase C) blocked the effect of glucosamine on ANG gene expression and CREB phosphorylation, but had no impact on p38 MAPK and ATF-2 phosphorylation. Finally, both glucosamine and high glucose induced IRPTC hypertrophy. The hypertrophic effect of glucosamine was blocked in the presence of GF 109203X, but not azaserine and SB 203580. In contrast, the hypertrophic effect of high glucose was blocked in the presence of azaserine and GF 109203X, but not SB203580. Our studies demonstrate that the stimulatory effect of high glucose on ANG gene expression and IRPTC hypertrophy may be mediated at least in part via activation of hexosamine biosynthesis pathway signaling.

The Activation of c-Jun NH2-terminal Kinase (JNK) by DNA-damaging Agents Serves to Promote Drug Resistance via Activating Transcription Factor 2 (ATF2)-dependent Enhanced DNA Repair

The activating transcription factor 2 (ATF2) is a member of the ATF/cAMP-response element-binding protein family of basic-leucine zipper proteins involved in cellular stress response. The transcription potential of ATF2 is enhanced markedly by NH2-terminal phosphorylation by c-Jun NH2-terminal kinase (JNK) and mediates stress responses including DNA-damaging events. We have observed that four DNA-damaging agents (cisplatin, actinomycin D, MMS, and etoposide), but not the cisplatin isomer, transplatin, which does not readily damage DNA, strongly activate JNK, p38, and extracellular signal-regulated kinase (ERK), and strongly increase phosphorylation and ATF2-dependent transcriptional activity. Selective inhibition studies with PD98059, SB202190, SP600125, and the dominant negative JNK indicate that activation of JNK but not p38 kinase or ERK kinase is required for the phosphorylation and transcriptional activation of ATF2. Stable expression of ATF2 in human breast carcinoma BT474 cells increases transcriptional activity and confers resistance to the four DNA-damaging agents, but not to transplatin. Conversely, stable expression of a dominant negative ATF2 (dnATF2) quantitatively blocks phosphorylation of endogenous ATF2 leading to a marked decrease in transcriptional activity by endogenous ATF2 and a markedly increased sensitivity to the four agents as judged by decreased cell viability. Similarly, application of SB202190 at 50 micro m or SP600125 inhibited JNK activity, blocked transactivation, and sensitized parental cells to the four DNA-damaging drugs. Moreover, the wild type ATF2-expressing clones exhibited rapid DNA repair after treatment with the four DNA-damaging agents but not transplatin. Conversely, expression of dnATF2 quantitatively blocks DNA repair. These results indicate that JNK-dependent phosphorylation of ATF2 plays an important role in the drug resistance phenotype likely by mediating enhanced DNA repair by a p53-independent mechanism. JNK may be a rational target for sensitizing tumor cells to DNA-damaging chemotherapy agents.

A Single c-Jun N-terminal Kinase Isoform (JNK3-p54) Is an Effector in Both Neuronal Differentiation and Cell Death

The c-Jun N-terminal kinases (JNKs) mediate degeneration and apoptosis in the brain. Particularly, JNK3 is considered to be a degenerative enzyme with c-Jun as a relevant substrate. The contribution of individual JNK isoforms, however, to pathological as well as to physiological processes remains to be defined. To analyze the effects of a single JNK isoform on neuronal cell death and differentiation, we transfected PC12 cells, which normally express only JNK1 and JNK2, with JNK3-p54. Transfected JNK3 significantly enhanced cell death after UV irradiation (0.5-6 J/cm(2)) and paclitaxel/taxol treatment (1-10 microm). In contrast, in the context of nerve growth factor-induced (10 or 50 ng/ml) differentiation of PC12 cells, JNK3 expression significantly increased the number and length of neurites. This functional dichotomy of JNK3 was mirrored by differential activation and induction of nuclear JNK substrates; although activating transcription factor-2 phosphorylation was enhanced by death signaling in response to UV and taxol, c-Jun protein expression and N-terminal phosphorylation were increased during nerve growth factor-induced differentiation. The absence of significant JNK activation or target phosphorylation in response to H(2)O(2) (60 microm) further supports the hypothesis that JNK isoforms are not merely injury- or stress-specific kinases but also have context-specific physiological functions.

Activating Transcription Factor-2 Mediates Transcriptional Regulation of Gluconeogenic Gene PEPCK by Retinoic Acid

All-trans-retinoic acid (RA) is known to increase the rate of transcription of the PEPCK gene upon engagement of the RA receptor (RAR). RA also mediates induction of specific gene transcription via several signaling pathways as a nongenomic effect. Here we show that RA upregulation of PEPCK promoter activity requires the cAMP response element (CRE)-1 in addition to the RA-response element and that activating transcription factor-2 (ATF-2) binds the CRE element to mediate this effect. Furthermore, we show that RA treatment potentiates ATF-2-dependent transactivation by inducing specific phosphorylation of ATF-2 by p38β kinase. ATF-2 activation by RA blocked the inhibitory intramolecular interaction of ATF-2 amino and carboxyl terminal domains in a p38β kinase-dependent manner. Consistent with these results, RA treatment increased the DNA binding activity of ATF-2 on the PEPCK CRE-1 sequence. Taken together, the data suggest that RA activates the p38β kinase pathway leading to phosphorylation and activation of ATF-2, thereby enhancing PEPCK gene transcription and glucose production.

Activating transcription factor-2 mediates transcriptional regulation of gluconeogenic gene PEPCK by retinoic acid.

All-trans-retinoic acid (RA) is known to increase the rate of transcription of the PEPCK gene upon engagement of the RA receptor (RAR). RA also mediates induction of specific gene transcription via several signaling pathways as a nongenomic effect. Here we show that RA upregulation of PEPCK promoter activity requires the cAMP response element (CRE)-1 in addition to the RA-response element and that activating transcription factor-2 (ATF-2) binds the CRE element to mediate this effect. Furthermore, we show that RA treatment potentiates ATF-2-dependent transactivation by inducing specific phosphorylation of ATF-2 by p38beta kinase. ATF-2 activation by RA blocked the inhibitory intramolecular interaction of ATF-2 amino and carboxyl terminal domains in a p38beta kinase-dependent manner. Consistent with these results, RA treatment increased the DNA binding activity of ATF-2 on the PEPCK CRE-1 sequence. Taken together, the data suggest that RA activates the p38beta kinase pathway leading to phosphorylation and activation of ATF-2, thereby enhancing PEPCK gene transcription and glucose production.

P38 and Activating Transcription Factor-2 Involvement in Osteoblast Osmotic Response to Elevated Extracellular Glucose

Poorly controlled or untreated type I diabetes mellitus is characterized by hyperglycemia and is associated with decreased bone mass and osteoporosis. We have demonstrated that osteoblasts are sensitive to hyperglycemia-associated osmotic stress and respond to elevated extracellular glucose or mannitol by increasing c-jun and collagen I expression. To determine whether MAPKs are involved in this response, MC3T3-E1 osteoblasts were treated with 16.5 mm glucose, mannitol, or contrast dye for 1 h. Immunoblotting of phosphorylated p38 demonstrated activation of p38 MAPK by hyperosmotic stress in vitro and in vivo. Activation peaked at 20 min, remained detectable after 24 h, and was protein kinase C-independent. Activating transcription factor-2 (ATF-2) activation followed the same pattern as phospho-p38. Transactivation of cAMP response element (CRE)- and c-jun promoter (containing a CRE-like element)-reporter constructs increased following hyperosmotic treatment. SB 203580 (a p38 MAPK inhibitor) blocked ATF-2 phosphorylation, CRE transactivation, and c-jun promoter activation. Hyperosmotic activation of collagen I promoter activity was also inhibited by SB 203580, consistent with the involvement of c-jun in collagen I up-regulation. Therefore, we propose that hyperglycemia-induced increases in p38 MAPK activity and ATF-2 phosphorylation contribute to CRE activation and modulation of c-jun and collagen I expression in osteoblasts.

P38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK(1)-FBPase(+) cells.

LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.

Hepatocyte growth factor/scatter factor activates proliferation in melanoma cells through p38 MAPK, ATF-2 and cyclin D1.

Members of the mitogen-activated protein kinase (MAPK) superfamily, including p38 kinase and SAPK/JNK, play a central role in mediating cellular response to environmental stress, growth factors and cytokines. Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine capable of eliciting mitogenic, motogenic and morphogenetic activities in responsive cells, and has been implicated in tumor development and metastasis. Binding of HGF/SF to its tyrosine kinase receptor c-Met stimulates multiple signal transduction pathways, leading to the activation of numerous transcription factors. We here report that HGF/SF can induce cyclin D1 expression in mouse melanoma cells, and that this up-regulation is mediated in part by the activating transcription factor-2 (ATF-2). HGF/SF-mediated phosphorylation of ATF-2 was reduced in the presence of either the p38 kinase-specific inhibitor SB203580, a dominant negative p38 mutant, the SAPK/JNK inhibitor JNK-interacting protein-1 (JIP-1), or the phosphatidylinositol 3-kinase (PI3K)-specific inhibitor LY294002. Activation of p38 kinase by HGF/SF was partially blocked by the PI3K-specific inhibitor as well. The upstream kinases for p38, MKK3/6, did not become activated following HGF/SF exposure, and ATF-2 activation was undiminished by transient transfection of a dominant negative MKK6 mutant. However, transcriptional up-regulation of cyclin D1 by HGF/SF was partially inhibited by the p38 kinase-specific inhibitor, and cyclin D1 protein induction was partially blocked by a dominant negative ATF-2 mutant. Notably, the p38 kinase-specific inhibitor was able to block melanoma cell proliferation but not motility. We conclude that the ATF-2 transcription factor becomes activated by HGF/SF through p38 MAPK and SAPK/JNK. Moreover, the p38-ATF-2 pathway can help mediate proliferation signals in tumor cells through transcriptional activation of key cell cycle regulators.

Crosstalk between Myc and activating transcription factor 2 (ATF2): Myc prolongs the half-life and induces phosphorylation of ATF2.

Myc is a key regulator of cell growth, differentiation and apoptosis, and affects cell fate decisions by activating as well as by inhibiting the expression of cellular genes. Myc is a member of the basic region-helix-loop-helix-leucine zipper (b-HLH-Zip) class of transcription factors, which heterodimerizes with the Max protein and recognizes a consensus Myc binding motif. Stimulation of gene expression by Myc is thought to be mediated by direct binding of Myc-Max heterodimers to specific target genes. So far, only a few genes have been identified as direct binding targets of Myc, raising the possibility that Myc affects gene expression also by indirect mechanisms. In this work we present evidence that v-Myc encoded by the avian retrovirus MC29 stimulates activating transcription factor 2 (ATF2)-dependent transcription. Analysis of the effect of Myc on ATF2 shows that v-Myc prolongs the half-life of ATF2 and induces the phosphorylation of N-terminal sites of ATF2 (Thr-69 and Thr-71) which have previously been identified as the target sites of stress-activated protein kinases and implicated in the regulation of ATF2 activity. Taken together, our results suggest that v-Myc can affect gene expression indirectly by modulating the activity of ATF2.

Regulation of Activating Transcription Factor-2 in Early Stage of the Adipocyte Differentiation Program

p38β mitogen-activated protein kinase activity is required for the differentiation of 3T3-L1 fibroblasts into adipocytes. Activating transcription factor-2 (ATF-2) is efficiently phosphorylated and activated by p38β kinase. These findings led us to examine a regulatory role of ATF-2 in adipocyte differentiation. The induction of ATF-2 protein precedes the expression of the transcription factors, peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein (C/EBP) α. Consistent with early activation of p38β kinase, the phosphorylation of ATF-2 was also detected in early stage of adipocyte differentiation. ATF-2 regulated gene transcription of PPARγ, which was synergistically enhanced by p38β kinase and C/EBPβ proteins expression. Ectopic expression of ATF-2 in 3T3-L1 cells induced the endogenous PPARγ protein levels. These results suggest that ATF-2 plays a role in a primary regulator of adipocyte differentiation with C/EBPβ through promoting adipogenesis-inducing transcription factors including PPARγ and becomes associated earlier in the differentiation program as mitotic clonal expansion proceeds and the cells become initially differentiated.

Stability of the ATF2 Transcription Factor Is Regulated by Phosphorylation and Dephosphorylation

Trans-activation of the activating transcription factor-2 (ATF2) in response to cellular stress requires the N-terminal phosphorylation of ATF2 by stress-activated protein kinases (SAPK). In this study, we investigated the role of ATF2 phosphorylation in the maintenance of ATF2 stability. Activation of SAPK by forced expression of DeltaMEKK1 increased overall ATF2 ubiquitination, presumably because of the enhanced dimerization of ATF2. Treatment of DeltaMEKK1-expressing cells with okadaic acid led to the increase in N-terminal phosphorylation, protection from ubiquitination, and accumulation of exogenously expressed ATF2, indicating the role of protein phosphatases in balancing the effects of stress kinases. Analysis of ubiquitination and degradation of the constitutively dimerized ATF2 mutant (ATF2(Delta150-248)) showed that activation of JNK or p38 kinase renders ATF2 resistant to ubiquitination and degradation. This effect is mediated by JNK/p38-dependent phosphorylation of ATF2 at Thr-69 and Thr-71, because the phosphorylation-deficient mutant (ATF2(Delta150-248-T69A,T71A)) was not protected from ubiquitination and degradation by the activation of SAPK. Treatment of cells with okadaic acid elevated the tumor necrosis factor alpha-induced ATF2 level and the extent of its specific N-terminal phosphorylation. Cycloheximide, which activates SAPK, while inhibiting protein synthesis, stabilized endogenous ATF2. However, treatment of cells with the high dose of SB203580, which inhibits JNK and p38 kinase, resulted in efficient degradation of ATF2 in cells exposed to cycloheximide. This degradation was abrogated by co-treatment with the proteasome inhibitor MG132. Our findings suggest that N-terminal phosphorylation of ATF2 dimers protect ATF2 from ubiquitination and degradation. We propose the hypothesis that the balance between SAPK and protein phosphatases affects the duration and magnitude of ATF2 transcriptional output because of the effect on substrate recognition for ubiquitination and degradation.

Activating Transcription Factor 2 Is Necessary for Maximal Activity and Serum Induction of the Cyclin A Promoter in Chondrocytes

Endochondral bone growth is regulated through the proliferation and differentiation of growth plate chondrocytes. Mice deficient for the activating transcription factor 2 (ATF-2) gene show reduced proliferation of chondrocytes. Here we demonstrate that the cyclin A gene is a target of ATF-2 in chondrocytes. Serum stimulation of chondrogenic rat chondrosarcoma cells induces cyclin A expression. A cyclic AMP response element (CRE) is necessary for optimal activity and serum inducibility of the cyclin A promoter and confers regulation by ATF-2. Phosphorylation and activity of ATF-2 are enhanced dramatically upon serum stimulation of rat chondrosarcoma cells. Mutation of the CRE or overexpression of dominant-negative ATF-2 inhibits serum induction of the cyclin A promoter. Chondrocytes from ATF-2-deficient mice display reduced and delayed induction of cyclin A upon serum stimulation. The ATF-2-related transcription factor CRE-binding protein contributes to the activity of the cyclin A CRE in chondrocytes, whereas c-Jun and c-Fos regulate the promoter independently of the CRE. Our data suggest that the reduction in cyclin A levels in chondrocytes from ATF-2-deficient mice contributes to their phenotype of reduced chondrocyte proliferation and dwarfism.

Association of Activating Transcription Factor 2 (ATF2) with the Ubiquitin-conjugating Enzyme hUBC9: IMPLICATION OF THE UBIQUITIN/PROTEASOME PATHWAY IN REGULATION OF ATF2 IN T CELLS

Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.