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S Y Fuchs

Publications and source records attributed to S Y Fuchs.

34 records · Page 2Linked to original sources
PubMed1999

Ubiquitination and degradation of ATF2 are dimerization dependent.

Ubiquitination and proteasome-dependent degradation are key determinants of the half-lives of many transcription factors. Homo- or heterodimerization of basic region-leucine zipper (bZIP) transcription factors is required for their transcriptional activities. Here we show that activating transcription factor 2 (ATF2) heterodimerization with specific bZIP proteins is an important determinant of the ubiquitination and proteasome-dependent degradation of ATF2. Depletion of c-Jun as one of the ATF2 heterodimer partners from the targeting proteins decreased the efficiency of ATF2 ubiquitination in vitro, whereas the addition of exogenously purified c-Jun restored it. Similarly, overexpression of c-Jun in 293T human embryo kidney cells increased ATF2 ubiquitination in vivo and reduced its half-life in a dose-dependent manner. Mutations of ATF2 that disrupt its dimerization inhibited ATF2 ubiquitination in vitro and in vivo. Conversely, removal of residues 150 to 248, as in a constitutively active ATF2 spliced form, enhanced ATF2 dimerization and transactivation, which coincided with increased ubiquitination and decreased stability. Our findings indicate the increased sensitivity of transcriptionally active dimers of ATF2 to ubiquitination and proteasome-dependent degradation. Based on these observations, we conclude that increased targeting of a transcriptionally active ATF2 form indicates the mechanism by which the magnitude and the duration of the cellular stress response are regulated.

Activating Transcription Factor 2↗
PubMed1998

Mdm2 association with p53 targets its ubiquitination.

Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.

3T3 Cells↗
PubMed1998

Stress-activated kinases regulate protein stability.

Proteasome inhibitors have been used to demonstrate that many proteins of the signal transduction pathways are regulated by degradation via the ubiquitin-proteasome pathway. The key question is what events target specific proteins for ubiquitination at one time and prevent ubiquitination at other times? In this review, we develop the notion that there is a direct relationship between the phosphorylation/dephosphorylation cascade of the signal transduction pathways and the targeting of the regulatory proteins for ubiquitination. We present examples where phosphorylation appears to alter the interaction between the targeting systems and the substrate by modifying the targeting system, the substrate, or both. These interacting systems are seen in the response of p53, c-jun and ATF-2 in cells subjected to stress or DNA damage and to the normal regulated response in a variety of pathways including the IkappaB-NFkappaB and JAK-STAT pathways. The interweaving of the two post-translational networks, phosphorylation and ubiquitination, provides a powerful insight into global regulatory control pathways.

Animals↗
PubMed1998

MEKK1/JNK signaling stabilizes and activates p53.

Activation of the tumor suppressor p53 by stress and damage stimuli often correlates with induction of stress kinases, Jun-NH2 kinase (JNK). As JNK association with p53 plays an important role in p53 stability, in the present study we have elucidated the relationship between the JNK-signaling pathway and p53 stability and activity. Expression of a constitutively active form of JNKK upstream kinase, mitogen-activated protein kinase kinase kinase (DeltaMEKK1), increased the level of the exogenously transfected form of p53 in p53 null (10.1) cells as well as of endogenous p53 in MCF7 breast cancer cells. Increased p53 level by forced expression of DeltaMEKK1 coincided with a decrease in p53 ubiquitination in vivo and with prolonged p53 half-life. Computerized modeling of the JNK-binding site (amino acids 97-116; p7 region) enabled us to design mutations of exposed residues within this region. Respective mutations (p53(101-5-8)) and deletion (p53(Deltap7)) forms of p53 did not exhibit the same increase in p53 levels upon DeltaMEKK1 expression. In vitro phosphorylation of p53 by JNK abolished Mdm2 binding and targeting of p53 ubiquitination. Similarly, DeltaMEKK1 expression increased p53 phosphorylation by immunopurified JNK and dissociated p53-Mdm2 complexes. Transcriptional activity of p53, as measured via mdm2 promoter-driven luciferase, exhibited a substantial increase in DeltaMEKK1-expressing cells. Cotransfection of p53 and DeltaMEKK1 into p53 null cells potentiated p53-dependent apoptosis, suggesting that MEKK1 effectors contribute to the ability of p53 to mediate programmed cell death. Our results point to the role of MEKK1-JNK signaling in p53 stability, transcriptional activities, and apoptotic capacity as part of the cellular response to stress.

Apoptosis↗
PubMed1998

JNK targets p53 ubiquitination and degradation in nonstressed cells.

In this study we elucidated the role of nonactive JNK in regulating p53 stability. The amount of p53-JNK complex was inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocked ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than p53(wt). Outcompeting JNK association with p53 increased the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibited p53 accumulation. JNK-p53 and Mdm2-p53 complexes were preferentially found in G0/G1 and S/G2M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells.

3T3 Cells↗
PubMed1998

ATF2 confers radiation resistance to human melanoma cells.

We have previously identified a U.V.-response element (URE; TGACAACA) and its bound proteins, members of the AP1 and ATF transcription factor families, in melanoma cells. Using a mutant form of cylic AMP response element binding (CREB), we found that CREB-associated-URE-bound proteins conferred characteristic melanoma phenotypes, including radiation resistance (Oncogene 12: 2223, 1996). In the present study we sought to determine which of the CREB-associated proteins confers radiation resistance on human melanoma cells. To this end we purified and identified via microsequencing ATF2 as a major URE- bound and CREB-associated protein in MeWo cells--a late stage human melanoma cell line. To determine the contribution of ATF2 to radiation resistance, MeWo cells were transfected with ATF2 cDNA lacking the trans-activation domain (ATF2(delta1-195)). MeWo cells that stably express ATF2(delta1-195) showed weaker transcriptional activities and an altered pattern of homo/hetero dimers. ATF2(delta1-195) clones exhibited up to tenfold lower resistance to irradiation by either U.V. or X-rays. The degree of resistance to radiation in the ATF2(delta1-195)-expressing clones could be increased upon transient transfection with ATF2(wt), but not with phosphorylation-defective mutant ATF2(69,71). Similarly, transfection of ATF2(wt) to WM3211, an early stage human melanoma cells line, increased resistance to radiation. Finally, changes elicited through ATF2(delta1-195) also led to reduced drug resistance, as shown for MMC, araC and cisplatinum. Our results suggest that ATF2 is a regulator of radiation and drug resistance in melanomas, and that tumor targeted ATF2 modulators may be useful sensitizers in the treatment of tumors of this type.

Activating Transcription Factor 2↗
PubMed1998

Selective in vivo stimulation of stress-activated protein kinase in different rat tissues by immobilization stress.

Stress activated protein kinases (SAPK) are key enzymes mediating the cellular response to stressful stimuli. While they are intensively studied in cultured cells, little is known about their physiological role in vivo, or relevance to pathological conditions. Therefore we examined the effect of various times of immobilization on c-Jun N-terminal protein kinase (JNK) activity in several rat stress responsive tissues and in a number of other locations. The abundance and relative distribution of JNK isoforms, the basal levels, time course and relative magnitude of stress induced JNK activity differed among tissues and regions of the brain of the same animal. JNK immunoreactive proteins were most abundant in the brain, especially in the hippocampus, hypothalamus and frontal cortex. Marked activation in response to immobilization stress was observed in adrenal medulla, adrenal cortex, aorta and hippocampus, less pronounced in locus coeruleus. JNK was not affected in superior cervical ganglia, pituitary, hypothalamus, frontal cortex and cerebellum. In adrenal medulla, the activation of JNK by single immobilization stress is correlated with increased transcription of stress-responsive genes, tyrosine hydroxylase and dopamine beta-hydroxylase. These data suggest a potential role of JNK signal transduction pathway in mediating the long term adaptation to stressful stimuli in vivo.

Animals↗
PubMed1997

c-Jun NH2-terminal kinases target the ubiquitination of their associated transcription factors.

Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH2-terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed.

3T3 Cells↗
PubMed1997

The expression of insecticide resistance-related cytochrome P450 forms is regulated by molting hormone in Drosophila melanogaster.

The expression and enzymatic activities of insecticide resistance-related cytochrome P450B are increased by the treatment with 20-hydroxyecdysone (20HE) in D. melanogaster Oregon R flies. We have explored the role of this hormone in the maintenance of P450B basal expression. Arrest of ecdysone synthesis led to a decrease in CYP6A2 mRNA level, as well as in P450B expression and activities. This effect occurred both in insecticide susceptible (ecd1) and resistant (IRED) strains carrying the temperature-sensitive ecd mutation. The role of the 20HE in the regulation of cytochrome P450-mediated insecticide resistance has been proposed.

Animals↗
PubMed1997

Conformation-dependent phosphorylation of p53.

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.

Adenosine Triphosphate↗
PubMed1996

Phosphorylation-dependent targeting of c-Jun ubiquitination by Jun N-kinase.

Ubiquitination of key cellular regulatory proteins marks them for efficient degradation via the proteasome pathway. The delta domain of c-jun is essential for its ubiquitination and also for the activating phosphorylation of neighboring serines by the stress activated jun-N-terminal kinases (JNK). Using an in vitro model system we demonstrate that JNK is among the hydrophobic binding proteins that target c-jun for efficient ubiquitination. Immunodepletion of JNK markedly inhibits c-jun ubiquitination. Conversely, c-jun ubiquitination is increased by adding purified JNK2 or extracts prepared from cells transfected with JNK2. Although c-jun ubiquitination is enhanced by JNK, the phosphorylation of c-jun on Ser73 by JNK protects c-jun from ubiquitination and prolongs its half-life. The dual activity of JNK in targeting c-jun for ubiquitination or in protecting c-jun from entering this pathway via phosphorylation points to the role of JNK in the control of c-jun stability in cells exposed to environmental stress or inflammatory cytokines.

Animals↗
PubMed1996

Elevated binding to URE/PEBP2 during the late stages of NNK and benzo[a]pyrene-induced carcinogenesis in A/J mice.

To provide better understanding about alterations in transcription factor activities during the promotion and progression stages of lung carcinogenesis we have utilized the A/J mice lung tumor model in which two potent lung carcinogens, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP), were co-administered. Nuclear proteins prepared from lung, brain, kidney, liver and colon of the A/J mice, 19 weeks after the last carcinogen administration, as well as from their respective non-treated controls, were tested for their binding to polyoma enhancer binding protein 2 (PEBP2) target sequence and UV-responsive element (URE). PEBP2 represents a newly identified family of transcription factors that was shown to play a role in cellular differentiation and transformation. URE is similar to CRE and AP1 target sequences, to which the members of ATF/AP1 transcriptional factors family bind. We demonstrate here that there is a marked increase in binding to both PEBP2 and URE sequences in lung, liver, kidney and brain of the treated mice. Such binding appears to be dependent on the mode of carcinogen administration as it was better noticed in the intragastric injected group than in the intraperitoneal group. Taken together, our findings suggest that increased binding to the URE and PEBP2 target sequence reflects changes in transcriptional activities which occur at late stages of lung carcinogenesis in a fashion which appear to depend on mode of carcinogen administration.

Animals↗
PubMed1995

Ultraviolet irradiation and c-jun over-expression regulates replication of polyoma sequences in WOP cells through a PEBP2 binding site.

Mouse fibroblast cells (WOP) express permissive factors which support polyoma DNA replication. However, electroporation into WOP cells of a mammalian expression vector that encodes the c-jun cDNA results in repression of polyoma DNA replication in a dose-dependent manner. In previous studies we have shown that UV-irradiation is capable of mediating a similar effect on polyoma DNA replication. When c-jun over-expression was combined with ultraviolet (UV)-irradiation, polyoma DNA replication decreased further. The repression of replication mediated by c-jun appears to be mediated by factor(s) that bind to PEBP4/2 target sequences as oligomers bearing the PEBP2/4 target site were capable of restoring polyoma DNA replication when added to UV-treated or c-jun over-expressing cells. The binding to the PEBP2/4 is partially dependent on the availability of AP-1 proteins, since an AP-1 target sequence can efficiently compete one of the three complexes formed with the PEBP2 target site. PEPB2 sequences do not, however, affect binding to the AP1 site. The effect of PEBP2 on polyoma replication is not dependent on the adjacent AP-1 site since PEBP2 could restore replication of polyomavirus which is mutated at the AP-1 sequence. A similar replication pattern was noted in a deletion mutant of polyoma which lacks PEBP4, yet, contains an intact PEBP2 binding sequence, suggesting that PEBP2 is the principle target for mediating repression of polyoma DNA replication.

Animals↗
PubMed1995

jun-NH2-terminal kinase activation mediated by UV-induced DNA lesions in melanoma and fibroblast cells.

jun-NH2-terminal kinase (JNK) belongs to a family of protein kinases that phosphorylates c-Jun, ATF2, and Elk1 in response to various forms of stress including UV irradiation and heat shock. Although in previous studies we have demonstrated the importance of membrane components for JNK activation by UV irradiation, here we have elucidated the role of DNA damage in this response. We show that in vitro-irradiated or sonicated DNA that is added to proteins prepared from UV-treated cells can further induce JNK activation in a dose-dependent manner. When compared with UV-B (300 nm), UV-C (254 nm), which is better absorbed by the DNA, is significantly more potent in activating JNK. Furthermore, when wavelengths lower than 300 nm were filtered out, UV-B was no longer able to activate JNK. With the aid of melanoma and fibroblast cells, which exhibit different resistances to irradiation and require different UV doses to generate the same number of DNA lesions, we demonstrate that above a threshold level of 0.45 lesions and up to 0.75 lesions per 1875 bp, the degree of JNK activation correlates with the amount of lesions induced by UV-C irradiation. Finally, to explore the role of nuclear and mitochondrial DNA (mtDNA) in mediating JNK activation after UV irradiation, we have used cells that lacks mtDNA. Although the lack of mtDNA did not impair the ability of UV to activate JNK, when enucleated, these cells had lost the ability to activate JNK in response to UV irradiation. Overall, our results suggest that DNA damage in the nuclear compartment is an essential component that acts in concert with membrane-anchored proteins to mediate c-Jun phosphorylation by JNK.

3T3 Cells↗
PubMed1994

Inducibility of various cytochrome P450 isozymes by phenobarbital and some other xenobiotics in Drosophila melanogaster.

The inducibility of cytochrome P450 isozymes has been investigated in the Drosophila melanogaster insecticide susceptible (Oregon R) and insecticide resistant (91R) strains. Both the level and induction kinetics of 7-ethoxycoumarin O-deethylase activity were stimulated by phenobarbital (PB) to a lower extent than that of aryl hydrocarbon hydroxylase in the Oregon R strain. The basal level of the cytochrome P450-linked activities in insecticide resistant flies was higher than that noted in susceptible ones. However, treatment with PB has increased levels of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities more in susceptible flies than in resistant ones. In contrast to PB, the polycyclic aromatic hydrocarbon benzo[a]pyrene induced both activities in 91R flies to a greater extent than in Oregon R ones. The potent PB-like inducer in mice but not in rats 1.4-bis[2-(dichloropyridyloxy)]-benzene failed to induce the cytochrome P450 system in D. melanogaster, when triphenyldioxane (PB-like inducer in rats but not in mice) markedly affected this system in a PB-like manner. The SDS-PAGE followed by immunoblotting analysis using monoclonal antibodies 13-2e and 8-1d have shown that the level of the 56,000 and 54,000 Da insecticide resistance-related forms has increased in the susceptible strain by PB and some other PB like inducers. The relationship between these isozymes appearance and 7-ethoxycoumarin O-deethylase activity has been discussed.

7-Alkoxycoumarin O-Dealkylase↗