Biomedical subjects

Z Ronai

Publications and source records attributed to Z Ronai.

At least 73 records · Page 4Linked to original sources

PubMed1999

Identification of a glutathione-S-transferase effector domain for inhibition of jun kinase, by molecular dynamics.

We have recently found that the glutathione-S-transferase pi-isozyme (GST-pi), a cellular detoxification enzyme, potently and selectively inhibits activation of jun protein by its upstream kinase, jun kinase (JNK). This newly identified regulatory activity of GST-pi is strongly inhibited by a group of agents that inhibit its enzymatic activity. Since loss of enzymatic activity in general does not correlate with loss of regulatory activity, it is likely that inhibitor binding induces changes in the structure of one or more domains of GST that block its interaction with JNK. To identify regions of GST that change conformation on the binding of inhibitors, we have performed molecular dynamics calculations on GST-pi to compute its average structure in the presence and absence of the inhibitor, glutathione sulfonate. Superposition of the two average structures reveals that several regions change local structure depending upon whether the inhibitor is bound or not bound. Two of these regions, residues 36-50 and 194-201, are highly exposed. We have synthesized peptides corresponding to these two segments and find that the 194-201 sequence strongly inhibits the ability of GST-pi to block the in vitro phosphorylation of jun by JNK. These results suggest that this region of GST-pi is critical to its functioning as a newly discovered regulator of signal transduction.

Amino Acid Sequence↗

PubMed1999

Environmental factors as regulators and effectors of multistep carcinogenesis.

This review highlights current knowledge of environmental factors in carcinogenesis and their cellular targets. The hypothesis that environmental factors influence carcinogenesis is widely supported by both epidemiological and experimental studies. The fact that only a small fraction of cancers can be attributed to germline mutations in cancer-related genes further buttresses the importance of environmental factors in carcinogenesis. Furthermore, penetrance of germline mutations may be modified by either environmental or other genetic factors. Examples of environmental factors that have been associated with increased cancer risk in the human population include chemical and physical mutagens (e.g. cigarette smoke, heterocyclic amines, asbestos and UV irradiation), infection by certain viral or bacterial pathogens, and dietary non-genotoxic constituents (e.g. macro- and micronutrients). Among molecular targets of environmental influences on carcinogenesis are somatic mutation (genetic change) and aberrant DNA methylation (epigenetic change) at the genomic level and post-translational modifications at the protein level. At both levels, changes elicited affect either the stability or the activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. Together, via multiple genetic and epigenetic lesions, environmental factors modulate important changes in the pathway of cellular carcinogenesis.

Biomarkers↗

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

CREB and its associated proteins act as survival factors for human melanoma cells.

cAMP response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), members of the CREB/ATF family, have been implicated in cAMP- and calcium-induced transcriptional activation. We have previously demonstrated that quenching of CREB-associated proteins in metastatic melanoma cells by a dominant-negative CREB (KCREB) that is mutated within its DNA-binding domain decreased their radiation resistance, and their tumorigenic and metastatic potential in nude mice. As the induction of apoptosis by diverse exogenous signals is dependent on the elevation of intracellular Ca2+, the purpose of this study was to determine the role of CREB and its associated proteins in apoptosis using KCREB. We used thapsigargin (Tg), which inhibits endoplasmic reticulum-dependent Ca2+-ATPase and thereby increases cytosolic Ca2+, to induce apoptosis. MeWo human melanoma cells were transfected with the KCREB expression vector and subsequently analyzed for their susceptibility to Tg-induced apoptosis. Here we demonstrate that expression of KCREB in MeWo cells rendered them susceptible to Tg-induced apoptosis. Tg treatment induced phosphorylation of CREB and possibly ATF-1 transcription factors. Treatment with Tg induced CRE-dependent transcription in parental cells, whereas this activation was reduced in the KCREB-transfected cells. In addition, CAT activity driven by the CRE-dependent promoter was inhibited in parental MeWo cells cotransfected with increasing concentrations of KCREB in a dose-dependent manner. We did not observe any changes in Bcl-2 or Bcl-2-related proteins (Bcl-x, Bax, and Bad) in control or KCREB-transfected cells before or after treatment with Tg. Collectively, these data indicate that CREB and its associated proteins act as survival factors for human melanoma cells, and hence contribute to the acquisition of the malignant phenotype.

Activating Transcription Factor 1↗

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

Rapid activation of JNK1 in UV-B irradiated epidermal keratinocytes.

Jun N-terminal kinase (JNK1) is a member of a family of stress-activated protein kinases which are activated by many forms of stress including UV radiation, resulting in the phosphorylation of c-Jun, ATF-2, Elk-1 and p53. As UV-B radiation is mainly responsible for ultraviolet (UV)-induced skin cancers, we chose to elucidate JNK1 activation in keratinocytes which represent a UV-relevant cell system. We have demonstrated rapid activation of JNK1 in a keratinocyte cell line, C50, in response to multiple doses of UV-B irradiation. JNK1 activation occurred within 1 min, peaked by 10 min and returned to near basal levels within 2 h following the UV-B treatments. Our data provide the first evidence to show that keratinocytes do respond to multiple doses of the physiologically relevant UV-B radiation through rapid activation of the JNK1 pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

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

Dominant-negative CREB inhibits tumor growth and metastasis of human melanoma cells.

The ATF/CREB family of eukaryotic transcription factors contain the bZIP structural motif and mediate their transcriptional activities via heterodimerization with ATF and AP-1 family members. Quenching of CREB-associated proteins by a dominant-negative CREB (KCREB) that is mutated within its DNA-binding domain decreases radiation resistance of human melanoma cells. The purpose of this study was to determine the role of CREB in tumor growth and metastasis of human melanoma using KCREB. Highly metastatic MeWo human melanoma cells were transfected with the KCREB expression vector and subsequently analysed for changes in their tumorigenic and metastatic potential. Expression of KCREB in MeWo human cells decreased their tumorigenic and metastatic potential in nude mice compared with parental and control transfected cells. The KCREB-transfected cells displayed downregulation of 72 kDa collagenase type IV (MMP-2) mRNA expression and activity and decreased invasiveness through Matrigel-coated filters. Moreover, transcriptional activities mediated by the CAT gene driven by the MMP-2 promoter were decreased by 14-45-fold in KCREB-transfected cells. In addition, the cell-surface adhesion molecule MCAM/MUC18 that is involved in metastasis of human melanoma was downregulated in the KCREB-transfected cells. These data indicate that, through their transcriptional activities, CREB and its associated proteins play an important role in the acquisition of the metastatic phenotype of human melanoma cells.

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↗

PubMed1997

Quantitative enriched PCR (QEPCR), a highly sensitive method for detection of K-ras oncogene mutation.

We have developed a rapid and highly sensitive method for the detection of mutant K-ras codon 12 allele in the presence of 10(5) copies of the wild-type alleles. This sensitivity is achieved by selective amplification of mutant K-ras sequences, using a two-stage procedure with modified primers. In the first stage, primers consist of K-ras sequences in the 3' portion and polyomavirus sequence (to minimize homology with human genome) on the 5' portion. The 3' portion also consists of mismatch sequence that generates an MvaI site in normal, but not mutant, K-ras codon 12 alleles. Thus, following the first round of 20 cycles, restriction enzyme cleavage is carried out to selectively digest normal K-ras codon 12 alleles. To enrich mutant alleles, a second amplification is performed using tail primers that recognize the polyoma, but not human sequences. This design ensures that in the second amplification only mutant alleles that were pre-amplified in the first round would serve as template for this reaction. Ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE) of second-stage PCR product that has been digested with MvaI is used to monitor the presence of mutant alleles, detected at sensitivity of 1/10(5). This technique offers high sensitive detection of mutant K-ras alleles using a new concept of tail-primer design and is likely to assist in identifying patients at risk to develop pancreatic, colon, or lung cancer, which harbor high incidence of mutant ras alleles.

Base Sequence↗

PubMed1997

Selective inhibition of oncogenic ras-p21 in vivo by agents that block its interaction with jun-N-kinase (JNK) and jun proteins. Implications for the design of selective chemotherapeutic agents.

We have obtained evidence that oncogenic and activated normal ras-p21 proteins utilize overlapping but distinct signal transduction pathways. Recently, we found that ras-p21 binds to both jun and its kinase, jun kinase (JNK). We now present evidence that suggests that oncogenic but not normal activated p21 depends strongly on early activation of JNK/jun. This early activation most likely involves direct interaction between oncogenic p21 and JNK/jun because p21 peptides that blocked the binding of p21 to JNK and jun strongly inhibited oncogenic p21-induced oocyte maturation while they did not inhibit insulin-activated normal cellular p21-induced maturation. Very similar results were also obtained for a newly characterized specific inhibitor of JNK which blocked oncogenic but not normal activated p21-induced oocyte maturation. We also found that both jun and JNK strongly enhanced oncogenic p21-induced oocyte maturation while they inhibited insulin-activated normal p21-induced oocyte maturation. These results suggest that the peptides and JNK inhibitor may be useful agents in selectively blocking the effects of oncogenic but not normal p21 in cells.

Animals↗

PubMed1997

Combined analysis of microsatellite instability and K-ras mutation increases detection incidence of normal samples from colorectal cancer patients.

Microsatellite instability (MI) and K-ras oncogene mutation have been widely used as biomarkers of genetic changes in colorectal cancer (CRC). Each of these biomarkers was independently found in normal-appearing colonic mucosa at stages preceding the development of CRC, albeit at a relatively low incidence. To assess the potential value of combined MI and K-ras mutation analysis in the detection of normal-appearing colonic mucosa samples taken from patients with CRC, we have chosen to analyze multiple (3-7) normal colonic mucosa samples and the respective colorectal tumor tissues from 20 patients with CRC. As a control, we have used 54 normal mucosa samples obtained from 9 autopsies of patients without CRC. In at least 1 of 5 loci analyzed, MI was found in 8 of 20 patients via analysis of multiple normal-appearing colonic mucosa samples from each patient. Combined analysis of MI and mutant ras alleles in normal-appearing colonic mucosa samples enabled the identification of 11 of 20 patients with CRC. None of the 54 normal colonic mucosa samples obtained from 9 patients without CRC were found to carry mutant ras or MI. The ability to detect 55% of patients with CRC via the analysis of normal mucosa samples provides an important advance in our approach toward early detection of individuals who may be at risk to develop this tumor type.

Aged↗

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↗