Victor Adler

Regulation of JNK signaling by GSTp.

Studies of low basal Jun N‐terminal kinase (JNK) activity in non‐stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S‐transferase Pi (GSTp) and was characterized as a JNK‐associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp–JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun–JNK complex caused a dose‐dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp‐derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c‐Jun ubiquitination and decreased c‐Jun‐mediated transcription. Co‐transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1–MKK4 module. Mouse embryo fibroblasts from GSTp‐null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases.

Role of redox potential and reactive oxygen species in stress signaling.

Stress-activated signaling cascades are affected by altered redox potential. Key contributors to altered redox potential are reactive oxygen species (ROS) which are formed, in most cases, by exogenous genotoxic agents including irradiation, inflammatory cytokines and chemical carcinogens. ROS and altered redox potential can be considered as the primary intracellular changes which regulate protein kinases, thereby serving as an important cellular component linking external stimuli with signal transduction in stress response. The mechanisms, which underlie the ROS-mediated response, involve direct alteration of kinases and transcription factors, and indirect modulation of cysteine-rich redox-sensitive proteins exemplified by thioredoxin and glutathione S-transferase. This review summarizes the current understanding of the mechanisms contributing to ROS-related changes in key stress activated signaling cascades.

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.

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 growingversus stressed cells are discussed.

Small, highly structured RNAs participate in the conversion of human recombinant PrPSen to PrPRes in vitro

We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP). We also demonstrate the ability of this shsRNA to form highly stable nucleoprotein complexes with hrPrP and cellular PrP (PrP(C)) from various cell extracts and mammalian brain homogenates. The apparent mass of the nucleoprotein complex is dependent on the molar ratio of hrPrP to RNA during complex formation. The hrPrP in these complexes acquires resistance to degradation by Proteinase K (PK). Other shsRNAs, however, under identical conditions, neither form stable complexes with hrPrP nor do they induce resistance to PK digestion. We also demonstrate that the RNAs in these nucleoprotein complexes become resistant to ribonuclease A hydrolysis. These interactions between shsRNAs and hrPrP suggest possible roles of RNAs in the modulation of PrP structure and perhaps disease development. ShsRNAs that bind to hrPrP with high affinity and induce resistance to PK digestion can be used to develop molecular biology assays for the screening of compounds associated with PrP structure transformation or for drugs that inhibit this process.

ATF2 confers radiation resistance to human melanoma cells.

We have previously identifed 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 (ATF2Δ1 – 195). MeWo cells that stably express ATF2Δ1 – 195 showed weaker transcriptional activities and an altered pattern of homo/hetero dimers. ATF2Δ1-195 clones exhibited up to tenfold lower resistance to irradiation by either U.V. or X-rays. The degree of resistance to radiation in the ATF2Δ1 – 195-expressing clones could be increased upon transient transfection with ATF2wt, but not with phosphorylation-defective mutant ATF269,71. Similarly, transfection of ATF2wt to WM3211, an early stage human melanoma cells line, increased resistance to radiation. Finally, changes elicited through ATF2Δ1 – 195 also led to reduced drug resistance, as shown for MMC, araC and cis-platinum. 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.

Contribution of phosphatidylinositol 3‐kinase to radiation resistance in human melanoma cells

The activity of phosphatidylinositol 3‐kinase (PI3K), a key component of multiple signal transduction pathways, was investigated in early‐ and late‐stage melanoma cells that have varying degrees of radiation resistance. Analysis of PI3K biproducts (PI‐3,4‐P2 and PI‐3,4,5‐triphosphate) revealed a direct correlation between radiation resistance and levels of PI3K activity. Treating melanoma cells with wortmanin or LY294002, two different PI3K inhibitors, decreased PI3K activity and caused a dose‐dependent decrease in resistance to ultraviolet radiation. Lower resistance to radiation elicited by LY294002 coincided with increased apoptosis. To further establish the role of PI3K in radiation resistance, we transfected early‐stage melanoma cells with the cDNA of p85, the regulatory subunit of PI3K. Clones that constitutively overexpressed p85 exhibited a higher degree of PI‐3,4‐P2 synthesis and a corresponding increase in their resistance to ultraviolet radiation. The results of this study point to the role of PI3K and its biproducts in radiation resistance of human melanoma cells. Mol. Carcinog. 24:64–69, 1999.

Concentration and removal of prion proteins from biological solutions

The pathogenesis of prion diseases is characterized by the accumulation of amyloid‐like rods or scrapie‐associated fibrils. The major protein component of scrapie‐associated fibrils is an abnormally folded isoform of the normal cellular prion protein (PrPC) that is resistant to digestion by proteinase K and is referred to as PrPSc. Purified human recombinant (hr PrP) was used to characterize the binding of a set of RNAs with affinity to PrP proteins. We report here that hr PrP has two RNA‐binding activities at physiological pH. One activity is capable of binding all of the screened RNAs with high affinity, whereas the other activity can bind only to a subset of the RNAs with high affinity in the presence of non‐specific competitor RNAs. A novel RNA belonging to the latter class, RQ11+12, bound to hr PrP with high affinity in the presence of vast molar excesses of competing RNAs. Beads impregnated with the RQ11+12 RNA were used to construct a filtration column. The column efficiently bound hr PrP and native PrPC from serum and urine. Importantly, the filtration device was also capable of binding proteinase K‐treated PrPSc from serum and urine. The level of sensitivity of detection of PrP by standard Western blotting was increased at least 1000‐fold by first concentrating PrP from solution with the filtration column.

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 π-isozyme (GST-π), 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-π 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-π 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-π to block the in vitro phosphorylation of jun by JNK. These results suggest that this region of GST-π is critical to its functioning as a newly discovered regulator of signal transduction.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK-MAP kinase signal transduction pathway

Purpose: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. Methods: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MAP kinase-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. Results: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. Conclusion: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MAP kinase inhibitors. The results imply that blockade of both MAP kinase and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras- induced tumors.