Glutathione S-transferase P1–1 (GSTP1–1) Inhibits c-Jun N-terminal Kinase (JNK1) Signaling through Interaction with the C Terminus

Abstract

c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulated endogenously in part by protein-protein interactions with glutathione S-transferase P1–1 (GSTP1–1) (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar). Using purified recombinant proteins, combined with fluorescence resonance energy transfer technology, we have found that the C terminus of JNK is critical to the interaction with GSTP1–1. The apparent K d for full-length JNK was 188 nm and for a C-terminal fragment (residues 200–424) 217 nm. An N-terminal fragment (residues 1–206) did not bind to GSTP1–1. Increased expression of the C-terminal JNK fragment in a tetracycline-inducible transfected NIH3T3 cell line produced a concentration-dependent increase in the kinase activity of JNK under normal, unstressed growth conditions indicating a dominant-negative effect. This suggests that the fragment can compete with endogenous full-length functional JNK resulting in dissociation of the GSTP1–1-JNK interaction and concomitant JNK enzyme activation. By using an antibody to hemagglutinin-tagged C-JNK, a concentration-dependent co-immunoprecipitation of GSTP1–1 was achieved. These data provide evidence for direct interactions between the C-terminal of JNK and GSTP1–1 and a rationale for considering GSTP1–1 as a critical ligand-binding protein with a role in regulating kinase pathways. c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulated endogenously in part by protein-protein interactions with glutathione S-transferase P1–1 (GSTP1–1) (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar). Using purified recombinant proteins, combined with fluorescence resonance energy transfer technology, we have found that the C terminus of JNK is critical to the interaction with GSTP1–1. The apparent K d for full-length JNK was 188 nm and for a C-terminal fragment (residues 200–424) 217 nm. An N-terminal fragment (residues 1–206) did not bind to GSTP1–1. Increased expression of the C-terminal JNK fragment in a tetracycline-inducible transfected NIH3T3 cell line produced a concentration-dependent increase in the kinase activity of JNK under normal, unstressed growth conditions indicating a dominant-negative effect. This suggests that the fragment can compete with endogenous full-length functional JNK resulting in dissociation of the GSTP1–1-JNK interaction and concomitant JNK enzyme activation. By using an antibody to hemagglutinin-tagged C-JNK, a concentration-dependent co-immunoprecipitation of GSTP1–1 was achieved. These data provide evidence for direct interactions between the C-terminal of JNK and GSTP1–1 and a rationale for considering GSTP1–1 as a critical ligand-binding protein with a role in regulating kinase pathways. c-jun N-terminal kinase mitogen-activated protein kinase glutathione S-transferases apoptosis-signaling kinase whole cell extract fluorescence resonance energy transfer hemagglutinin enhanced chemiluminescence c-Jun N-Terminal kinase (JNK),1 or stress activated kinase, is a member of the mitogen activated stress kinase family (MAPK), which also includes extracellular signal regulated kinase and p38-MAPK. JNK activation was initially identified as a cellular response to environmental stresses, proinflammatory cytokines, and interleukins (2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1369) Google Scholar). In addition, the JNK pathway participates in activating transcription factor 2 (3Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar), ELK-1 (4Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (873) Google Scholar), and the Sap-1a transcription factor (5Janknecht R. Hunter T. J. Biol. Chem. 1997; 272: 4219-4224Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and JNK may influence p53 (6Fuchs S.Y. Adler V. Pincus M.R. Ronai Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10541-10546Crossref PubMed Scopus (449) Google Scholar, 7Buschmann T. Yin Z. Bhoumik A. Ronai Z. J. Biol. Chem. 2000; 275: 16590-16596Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and NF-κB pathways (8Meyer C.F. Wang X. Chang C. Templeton D. Tan T.H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 9Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). c-Jun, a component of the transcription factor AP-1 complex is the most characterized downstream target of JNK, where regulation is achieved by phosphorylation at Ser-63 or Ser-73 (3Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar). Extracellular signals such as growth factors, transforming oncoproteins, and UV irradiation stimulate phosphorylation of c-Jun at ser-63/73 and activate c-Jun-dependent transcription. The binding of JNK to the N-terminal region of c-Jun permits substrate phosphorylation. The JNK pathway has also been shown to be important in the control of cell survival and death pathways, and interference with the JNK pathway suppresses the induction of apoptosis by a variety of agents (10Yin Z. Ivanov V. Habelhah H. Tew K.D. Ronai Z. Cancer Res. 2000; 60: 4053-4057PubMed Google Scholar). The glutathione S-transferases (GST) are a multigene family of isozymes that catalyze the nucleophilic attack of the sulfur atom of glutathione on electrophilic groups of substrate molecules. Although catalytic efficiencies for many of the substrates are low, their biological relevance as enzymes has frequently been linked to pleiotropic substrate specificity. Among this family of isozymes, GSTP1–1 is generally the most prevalent in mammalian cells. The link between GST overexpression, especially with respect to GSTP1–1, and anticancer drug resistance has been extensively studied (11Tew K.D. Cancer Res. 1994; 54: 4313-4320PubMed Google Scholar). Because of the defined role of GST in drug metabolism, elevated expression of GSTP1–1 in solid tumors or in drug-resistant cells has been associated frequently with detoxification reactions even in instances where there is no evidence that the selecting drug is a substrate for GSTP1–1. More recently, however, the link between the redox active components of GSTP1–1 and stress-activated kinases, such as JNK, has been redefined as a non-catalytic, ligand binding activity that mediates both stress and apoptotic responses (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar, 10Yin Z. Ivanov V. Habelhah H. Tew K.D. Ronai Z. Cancer Res. 2000; 60: 4053-4057PubMed Google Scholar). In a parallel series of studies, either GSTμ or thioredoxin has also been identified as a ligand-binding partner for apoptosis-signaling kinase (ASK1) (12Saitoh M. Nishitoh H. Fuji M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2045) Google Scholar, 13Cho, S.-G., Lee, Y. H., Park, H.-S., Ryoo, K., Kang, K. W., Park, J., Eom, S.-J., Kim, M. J., Chang, T.-W., Choi, S.-Y., Shim, J., Kim, Y., Dong, M.-S., Lee, M.-J., Kim, S. G., Ichijo, H., and Choi, E.-J. (2001) J. Biol. Chem., in press.Google Scholar), extending the role that “redox proteins” may have in kinase regulation. To elucidate further the mechanism by which GSTP1–1 mediates JNK signal transduction, we have used biophysical methods in reconstituted protein systems to provide direct evidence for protein-protein interactions and to measure binding affinities. Our results demonstrate that GSTP1–1 has significant affinity for the C terminus of JNK and confirm the ligand-binding regulatory role for this ubiquitously expressed protein. Recombinant JNK1 protein was expressed in Escherichia coli. The polymerase chain reaction product was constructed with a leader His-tag, digested with NdeI and BamHI, purified, and ligated intoNdeI-digested, BamHI-digested, and phosphatase-treated pET-15b to create JNK.pET-15b. The JNK1 protein was expressed in E. coli and purified using a standard Ni2+ column for His-tagged proteins. The purified protein was homogeneous as judged by the single polypeptide band of the predicted M r on silver-stained SDS-polyacrylamide gels (data not shown). Both the C-terminal and N-terminal truncated proteins expressed inE. coli were found mainly in the insoluble fraction of the bacterial extracts. Thus, inclusion bodies were solubilized in 50 mm CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), pH 11.0, with 0.3% N-lauroylsarcosine supplemented with 1 mm dithiothreitol and slowly diluted to 20 mmTris-HCl, pH 8.5, before subjection to Ni2+ column chromatography. Renaturation was accomplished by dialyzing for 12 h at 4 °C with a change of buffer with dithiothreitol and continued dialysis for an additional 6 h. To promote refolding, a further period of dialysis in the above buffer (20 mm Tris, pH 8.5) without dithiothreitol for the same length of time was enacted. The GSTP1–1 construct was made by direct digestion of GSTP1–1 from PCR2.1(+) with HindIII and XhoI followed by cloning into pET21 vector previously treated with HindIII and XhoI. This was expressed in E. coli, and protein was purified using GSH affinity chromatography. Circular dichroic spectra of the C-terminal and N-terminal JNK1 in 20 mm PO43+ were obtained using an Aviv 62A DS spectrometer at the Fox Chase Cancer Center core facility. The corresponding structural elements were calculated using an algorithm for the estimation of the percentages of protein secondary structure from UV circular dichroism spectra using a Kohonen neural network with a two-dimensional output layer using K2D software (14Andrade M.A. Chacon P. Merelo J.J. Morán F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (945) Google Scholar,15Merelo J.J. Andrade M.A. Prieto A. Morán F. Neurocomputing. 1994; 6: 443-454Crossref Scopus (123) Google Scholar). The program uses a self-organizing neural network to extract the secondary structure features present in the data from a set of circular dichroism spectra ranging from 200–241 nm. The result provides an estimate of percentage helix, β-sheet, and random structure for the protein. K2D also estimates the probable error in the estimation based on the training procedure results. GSTP1–1 was labeled at a 1:1 molar ratio (probe:protein) with the fluorescence energy transfer donor 7-methoxycoumarin succinyl ester using previously described procedures (16Wang T. Dowal L. El-Maghrabi M.R. Rebecchi M. Scarlata S. J. Biol. Chem. 2000; 275: 7466-7469Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The JNK1 partner proteins (full-length and partial) were similarly labeled with the non-fluorescence energy transfer acceptor, 4(dimethylamino) phenylazophenyl-4-sulfonyl chloride succinyl ester (Molecular Probes Inc., Eugene, OR). The protein:probe labeling ratios were determined by absorption spectroscopy (17Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar) using the calculated extinction coefficients for the protein and the coefficients provided by the probe manufacturer. NIH3T3 mouse fibroblast cells that stably express the pSV40-Hyg plasmid (CLONTECH, Palo Alto, CA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Life Technologies Inc.). Cells were grown at 37 °C with 5% CO2. The pTet-C-JNK was assembled by subcloning the cDNA of the C-terminal 224 amino acids of wild type JNK1 into the tet-regulated promoter of the pUHD-10−3 vector. Cell clones that stably express both constructs were selected in 600 μg/ml geneticin in the presence of hygromycin (100 μg/ml) 24 h afterN,N,N-trimethyl 1–2-3-bis (1-oxo-9-octa-decenyl)oxy-(Z,Z)-1-propanaminium methyl sulfate transfection with the pTet-C-JNK-UHD-10−3 construct. Cells expressing C-terminal JNK were maintained in Dulbecco's modified Eagle's medium containing fetal bovine serum (10%), hygromycin (100 μg/ml), and geneticin (400 μg/ml). To maintain suppression of C-terminal JNK expression, tetracycline was added to the medium every 3 days (to a final concentration of 1 μg/ml). Analysis of JNK1 activity was achieved by measurement of phosphorylation of c-Jun as a substrate (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar). Antibodies to C-JNK and phospho-c-Jun were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and New England BioLabs (Beverly, MA). Immunoblot analysis was performed using 200 μg of whole cell extract (WCE) separated on SDS-polyacrylamide gel electrophoresis followed by electrotransfer to a polyvinylidene difluoride membrane in 20 mm CHAPS (3-(3-cholamidopropyl) dimethylammonio-1-propane-sulfonate)) buffer, pH 11, for 30 min membrane blocking (5% non-fat milk) and incubating with the respective antibodies. Reactions were visualized using chemiluminescence (ECL) reagents. Immunoprecipitations were carried out following the protocol from the manual provided by Roche Diagnostics Corporation using 1 mg of WCE and 2 μg of the respective antibodies for 16 h at 4 °C. Protein G beads (Roche Molecular Biochemicals) were added (15 μl) for 4 h at 4 °C. Immunoprecipitated material was subjected to immunoblot analysis. Protein kinase assays were carried out using JNK kinase assay kits from New England BioLabs according the to manufacturer's instructions. The kinase reaction was carried out in the presence of excess unlabeled ATP. c-Jun phosphorylation was selectively measured using a phospho-c-Jun antibody. This antibody specifically measures JNK-induced phosphorylation of c-Jun at Ser-63, an important site for c-Jun-dependent transcriptional activity. Associations between GSTP1–1 and JNK were measured by fluorescence resonance energy transfer (FRET) as described previously (16Wang T. Dowal L. El-Maghrabi M.R. Rebecchi M. Scarlata S. J. Biol. Chem. 2000; 275: 7466-7469Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Protein concentrations were measured using UV absorption (A 280) and colorimetric assays. Extinction coefficients for each protein were assessed as described previously (18Wang T. Pentyala S. Elliott J.T. Dowal L. Gupta E. Rebecchi M.J. Scarlata S. Proc. Natl. Acad. Sci. U. S. A. 1999; 6: 7843-7846Crossref Scopus (53) Google Scholar). FRET was performed on a PC1 (photon counting) spectrofluorimeter (ISS, Inc., Champagne, IL) using a 3-mm cuvette with a magnetic stirrer. Coumarin-labeled proteins were excited at 350 nm and scanned from 380–480 nm. To determine the binding affinities for protein-protein interactions, the energy transfer data were analyzed using Sigma Plot. All immunoblot and SDS-polyacrylamide gel electrophoresis gels were scanned using a UMAX Powerbook III flatbed with Adobe Photoshop software. Scanned images were quantified by NIH image program. Prism software was used for data analysis. Full-length JNK1 was soluble and purified directly by Ni2+chromatography. Both truncated forms were insoluble in aqueous buffer and were initially solubilized in detergent followed by slow dialysis against renaturing The proteins were similarly purified by Ni2+ chromatography. The of proteins was by single band on SDS-polyacrylamide gel electrophoresis (data not shown). Circular dichroism of purified proteins was used to the 1 the secondary structure of the truncated from the circular dichroism These data were analyzed further using a Kohonen neural network with two-dimensional output layer using K2D software (14Andrade M.A. Chacon P. Merelo J.J. Morán F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (945) Google Scholar, J.J. Andrade M.A. Prieto A. Morán F. Neurocomputing. 1994; 6: 443-454Crossref Scopus (123) Google Scholar). JNK1 a to a protein which has been X. Y. Fox T. J.T. M.A. 1998; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). The C-terminal of is of the N-terminal is of are in with the data shown in that in has software analysis of circular dichroism in a In between the purified JNK proteins and GSTP1–1 was analyzed using (FRET) and is shown in GSTP1–1 was labeled with the succinyl ester of Full-length and truncated JNK1 was labeled with the non-fluorescence energy transfer acceptor, phenylazophenyl-4-sulfonyl chloride succinyl binding were used to measure the binding at at concentrations of the protein to determine 2 that for the full-length JNK1 a in 7-methoxycoumarin fluorescence was as the protein with GSTP1–1. result was found for the C-terminal fragment the N-terminal fragment buffer conditions produced change in The d for GSTP1–1 with full-length JNK1 was 188 nm and for the C-terminal 217 nm. These data provide evidence for the direct protein-protein interaction between GSTP1–1 and These results were to a cellular by using a construct of To measure of C-JNK and GSTP1–1, of WCE from cells expressing C-JNK was of C-JNK expression following of tetracycline in an enhanced of GSTP1–1 co-immunoprecipitation an antibody was used These data are with the that C-JNK and GSTP1–1 in a cellular the expression of the C-terminal truncated of JNK1 to an increase in JNK1 kinase activity as a of for the GSTP1–1 binding site and active JNK1 that the C-terminal of JNK1 is critical to the a tetracycline-inducible expression was used to the expression of the C-terminal truncated JNK1 have a dominant-negative GSTP1–1 of JNK1 activity. 4 the increase in expression of the C-terminal truncated JNK1 fragment following of of the C-terminal fragment was by 1 h and for at h. In same full-length JNK1 expression was and this period JNK1 activity was measured by the phosphorylation of the downstream c-Jun substrate The increase in c-Jun was with the enhanced expression of the truncated C-terminal tetracycline was not from the there was no enhanced of c-Jun product results the that the C-JNK fragment binding with GSTP1–1 can in a dominant-negative and increase the catalytic activity of S-transferases are a multigene family of isozymes that have been linked with or In GSTP1–1 is frequently in solid tumors and in cells that have resistance to anticancer even the selected drug is not a GST substrate (11Tew K.D. Cancer Res. 1994; 54: 4313-4320PubMed Google Scholar). The for the of expression in cells has not been we have carried out a series of to that GSTP1–1 can as a ligand-binding protein and an endogenous for the control of the catalytic activity of JNK1 (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar, 10Yin Z. Ivanov V. Habelhah H. Tew K.D. Ronai Z. Cancer Res. 2000; 60: 4053-4057PubMed Google Scholar). This has to that GSTP1–1 is in control of apoptosis S. F. C. S. C. L. R. J. Cell. Biochem. 2000; PubMed Scopus Google Scholar). of cellular that influence cell growth and apoptosis be In such as may influence signaling kinase pathways in stress response and cell survival Ronai Z. Tew K.D. J. Google Scholar). to that GSTP1–1 can against apoptosis by by the of kinase activity by JNK1 cellular such as extracellular signal regulated kinase, kinase, and (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Z. Ivanov V. Habelhah H. Tew K.D. Ronai Z. Cancer Res. 2000; 60: 4053-4057PubMed Google Scholar). Our interactions between GSTP1–1 and JNK1 on In this we a of to provide evidence for direct protein-protein interaction between GSTP1–1 and These data also binding for proteins with K d in the The of the C terminus of JNK1 was by a of that the N-terminal and unlabeled proteins, of which to The affinity of the truncated C-JNK fragment for GSTP1–1 was to the full-length protein. JNK1 a of amino with By we were to that the of the C-terminal truncated JNK1 was to the structure of by data X. Y. Fox T. J.T. M.A. 1998; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). Both the in and cellular data that the truncated C-terminal JNK1 amino that can be in the binding to GSTP1–1. R. M.J. Chen S. Adler V. Ronai Z. Pincus M.R. J. Protein Chem. 1999; 18: PubMed Scopus Google Scholar) has used of to that of GSTP1–1 may be in the interaction with This that are or groups in their analysis of the C terminus of JNK1 both a region and an in and These are either in the structure or the of the protein and can a binding of with GSTP1–1. in is on the of JNK1 and in a to protein-protein interaction with GSTP1–1. 6 the for the C-terminal components of JNK family X. Y. Fox T. J.T. M.A. 1998; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). The interaction site in the analysis R. M.J. Chen S. Adler V. Ronai Z. Pincus M.R. J. Protein Chem. 1999; 18: PubMed Scopus Google Scholar) is to the GST and A. M. J. Biol. 1994; PubMed Scopus Google Scholar) that JNK may in with GSTP1–1. In addition, the catalytic kinase is in the N-terminal of results that the of GSTP1–1 to JNK enzyme activity be an This may be in the of of the ligand-binding of proteins. this on JNK1 may kinase expression by influence apoptosis (10Yin Z. Ivanov V. Habelhah H. Tew K.D. Ronai Z. Cancer Res. 2000; 60: 4053-4057PubMed Google Scholar). have previously shown that a of GSTP1–1 Tew K.D. Cancer 1996; PubMed Scopus Google Scholar) a dissociation of the complex resulting in JNK catalytic activity (1Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar). This drug has significant on pathways in cells. J. L. C. C. J., and K. D. (2001) J. in is that with such agents control of the regulation of stress with a significant in the of Yin for the JNK for the NIH3T3 cell and Scarlata for of