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Examining signaling specificity to transcription factors
Methods 26 (2002) 217–225 www.academicpress.com
Examining signaling specificity to transcription factors Alan J. Whitmarsh* School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK Accepted 1 February 2002
Abstract The activities of many transcription factors are controlled by intracellular signal transduction pathways that respond to changes in the extracellular environment. The resulting changes in gene expression lead to appropriate physiological responses. Elucidating the signaling pathways that target transcription factors is an important goal as they represent potential targets for therapeutic intervention in many diseases. A number of tools and techniques have been developed that can be used for studying signaling specificity toward transcription factors. This article describes how (1) mutant alleles of signaling enzymes and (2) protein/peptide inhibitors can be used in conjunction with phosphorylation-specific antibodies and reporter gene assays to examine the targeting of transcription factors by signaling pathways in vivo. 2002 Elsevier Science (USA). All rights reserved. Keywords: Transcription factor; Signal transduction; Protein kinase; Phosphorylation; Mitogen-activated protein kinase; Reporter gene; Phosphorylation-specific antibody
1. Introduction Alterations in the patterns of gene expression in response to extracellular signaling cues are mediated by the integration of multiple intracellular signal transduction pathways that target transcription factors, transcriptional coregulators, and chromatin-modifying factors [1–3]. Protein phosphorylation and dephosphorylation constitute the most common regulatory mechanism, particularly for the rapid modulation of transcription factor activity [1–3]. However, other forms of posttranslational modification of proteins including acetylation, methylation, ubiquitination, and SUMOconjugation are also important for regulating transcription factor function [4–7]. A number of in vitro assays can be performed (e.g., protein kinase assays and acetylation assays) to address whether particular transcription factors are likely targets of protein modification enzymes. However, the physiological relevance of transcription factor modifications observed in vitro needs to be confirmed by in vivo experiments involving the specific activation or inhibition
*
Fax: +44-(0)161-275-5082. E-mail address: [email protected] (A.J. Whitmarsh).
of signaling pathways in cells. Two methods of blocking signaling pathways are (1) employing small molecule inhibitors and (2) generating cells with targeted deletions of genes encoding signaling proteins. Only a few genuinely specific small molecule inhibitors of signaling enzymes are commercially available; therefore their general use for determining signaling specificity toward transcription factors has been limited. Gene-targeted cells from mice have proved to be an increasingly important tool for delineating signaling pathways that target transcription factors. However, in a number of cases cells cannot always be derived if the targeted gene is essential for early embryonic development of the mice. In this article two additional methods for specifically modulating signaling pathways that target transcription factors are described. These are the use of activated mutant alleles or dominant-negative mutant alleles of signaling enzymes either to activate or to block specific pathways, and the use of small inhibitory proteins or peptides that inhibit specific protein–protein interactions between signaling proteins and transcription factors. These tools can be employed in conjunction with assays that measure the effect of signaling pathways on the modification and function of transcription factors. These include the generation of antibodies that specifically recognize posttranslationally modified forms of
1046-2023/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 0 2 5 - 7
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transcription factors, and the use of reporter gene assays to assess transcriptional activity. The examples discussed below focus on examining the targeting of transcription factors by the stress-activated group of the mitogen-activated protein (MAP) kinase family of signaling pathways. However, in most cases the techniques described are generally applicable to the study of many of the signaling pathways that target transcription factors. There are three major groups of MAP kinases (ERK, JNK, and p38) that are activated by sequential phosphorylation by signaling modules consisting of a MAP kinase kinase (MKK) and a MAP kinase kinase kinase (MKKK) [8,9] (Fig. 1). Members of the Ras and Rho families of GTPases lie upstream of these signaling modules and control their activity [8,9]. The ERK pathway is stimulated mainly by mitogenic stimuli (e.g.,
growth factors, hormones) and regulates the activity of the transcription factors Elk-1 and c-Myc [8,9]. The JNK and p38 pathways are stimulated mainly by stress stimuli (e.g., osmotic stress, oxidative stress, UV radiation, proinflammatory cytokines) and are collectively referred to as the stress-activated protein kinases (SAPKs). JNK regulates the activity of c-Jun, ATF-2, NFAT4, and Elk-1, while p38 regulates the activity of ATF-2, CHOP, MEF2C, and Elk-1 [8–11]. The specific protocols that are described are (1) the use of an activated allele of the MAP kinase kinase MKK6 to determine the effect of the p38 MAP kinase signaling pathway on the activity of transcription factors (measured by a luciferase reporter gene assay), and (2) the use of the JNK-binding domain (JBD) of the scaffold protein JNK-interacting protein 1 (JIP-1) to inhibit JNK MAP kinase signaling to transcription factors (measured by antibodies that recognize the JNK phosphorylation sites on the transcription factors).
2. Expression of an activated allele of MKK6 to selectively stimulate p38 MAP kinase-mediated transcriptional activity: Use of luciferase reporter gene assays 2.1. Activated allele of MKK6
Fig. 1. Tools to activate and inhibit MAP kinase signaling pathways that target transcription factors. The MAP kinase signaling module consists of a MAP kinase (MAPK) that is activated by sequential phosphorylation by a MAP kinase kinase (MKK) and a MAP kinase kinase kinase (MKKK). MKKKs may be activated by members of the Ras and Rho families of GTPases. To specifically activate MAP kinase pathways activated alleles of MKKKs (MKKK*) or MKKs (MKK*) can be used. Constitutively activated MAP kinases can be generated by fusing the MAPK to the MKK. Inducible activation systems are also in use. Some MKKKs are active as dimers. These MKKKs can be fused to the B domain of DNA gyrase (GyrB) which forms dimers in the presence of coumermycin, which can be added to cells expressing the fusion proteins. A second inducible system involves the fusion of activated alleles of MAP kinase pathway components to a mutated hormone binding domain of the estrogen receptor, for example ERMKK*. To inhibit signaling through MAP kinase pathways dominant-negative (DN) mutants of MKKKs, MKKs, or MAPKs have proved useful. In addition, small protein or peptide inhibitors of interactions between MAPKs and their transcription factor targets can be employed.
The overexpression of signaling enzymes in cells is sometimes sufficient to trigger the activation of downstream signaling events. However, often more potent activation of signaling pathways occurs if constitutively activated mutant enzymes (i.e., active in the absence of stimuli) are created. This can be achieved in a number of ways depending on the characteristics of the particular enzyme. The most common is the mutation of the activating phosphorylation sites in the enzyme to acidic residues to mimic the charge effect of phosphorylation. These mutations in MKK6 (Ser-207 and Thr-211 to Glu) result in a mutant enzyme that, when expressed in cells, potently and selectively activates the p38 MAP kinase signaling pathway [12]. 2.2. Reporter gene assay Reporter gene assays can be used to address which stimuli and signaling pathways regulate the transcriptional activity of a transcription factor. The principle of the assay is that the transcriptional activation domain of a transcription factor is fused to a heterologous DNA binding domain (usually the DNA binding domain of the transcription factor GAL4 or the repressor LexA) and cotransfected with a reporter plasmid containing sites for the DNA binding domain upstream of a minimal promoter (e.g., E1b promoter) and the firefly luciferase gene. The modification of the transcription factor fusion protein (e.g., by protein kinases) leads to
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increased or decreased expression of the luciferase gene, which can be measured by enzymatic assay. As a control for transfection efficiency, a plasmid featuring a b-galactosidase reporter gene is also transfected and b-galactosidase activity measured (alternatively a second type of luciferase reporter can be used featuring the Renilla luciferase gene). The reporter gene system used in the protocol below features the transcriptional activation domain of the p38 substrate ATF-2 fused to the DNA binding domain of GAL4 [13] and a luciferase reporter gene containing five GAL4 binding sites [14]. The protocol works well in a number of cell lines (e.g., NIH 3T3, HeLa, COS-7, CHO) and can be adapted for use with other activated signaling enzymes and GAL4 transcription factor fusion proteins. As an alternative to the described protocol, commercial kits are available (e.g., from Promega, Madison, WI) that feature dual luciferase reporter systems. 2.3. Appropriate control experiments To test whether a particular transcription factor is a novel target for regulation by the p38 MAP kinase signaling pathway, it is important to perform control experiments to demonstrate that the p38 pathway has been activated by the expression of the MKK6 mutant. This can be achieved by carrying out a parallel experiment using a known p38 target (e.g., GAL4-ATF-2) or immunoprecipitating p38 MAP kinase from the cells and measuring its activity in vitro [15]. Other important control experiments are: (1) expressing the GAL4 DNA binding domain alone to ensure that the MKK6 mutant is not inducing luciferase gene transcription in the absence of the transcription factor, and (2) including a mutant version of the GAL4 transcription factor fusion with the phosphorylation sites mutated to nonphosphorylatable residues to demonstrate that any effects on transcriptional activity are directly due to the phosphorylation of the transcription factor. 2.4. Protocol 2.4.1. Materials and reagents 2.4.1.1. Buffers. • Phosphate-buffered saline (PBS): 20 mM sodium phosphate, pH 7.4, 150 mM NaCl. • Potassium phosphate buffer, pH 7.8. • Luciferase assay buffer: 15 mM potassium phosphate, pH 7.8, 25 mM glycylglycine, pH 7.8, 15 mM Mg2 SO4 , 4 mM EGTA, 2 mM ATP (made as 100 mM stock and adjusted to pH 7.8), 1 mM dithiothreitol. • Buffer Z: 100 mM sodium phosphate, pH 7.0, 10 mM KCl, 1 mM Mg2 SO4 , 1 M Na2 CO3 .
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2.4.1.2. Reagents. • D -Luciferin (Pharmingen): 1 mM stock solution made by dissolving 10 mg D -L uciferin in 31.5 ml water and adjusting to pH 6.0–6.3 by adding 100 mM NaOH solution dropwise. Stored frozen. • O-Nitrophenyl-b-D -galactopyranoside (ONPG) (U.S. Biochemical): 4 mg/ml in 100 mM potassium phosphate, pH 7.0, filter sterilized and stored frozen. • pCDNA3-Flag-MKK6(Glu): encodes a mutant form of MKK6 featuring mutation of Ser-207 and Thr211 to Glu [12]. • pSG424-ATF-2: encodes a fusion of ATF-2 amino acids 1–109 with the GAL4 DNA binding domain [13]. • pG5E1bLuc: firefly luciferase gene reporter plasmid featuring five GAL4 binding sites and the minimal E1b promoter [14]. • pCH110: b-galactosidase reporter plasmid (Amersham–Pharmacia). • Lipofectamine (Life Technologies). • Chinese hamster ovary (CHO) cells (American Type Culture Collection, Manassas, VA). 2.4.1.3. Equipment. • 1.5-ml microfuge tubes. • Luminometer cuvettes. • Luminometer. • 37 C heating block • Spectrophotometer • Six-well tissue culture dishes (35-mm diameter) (Corning). 2.4.2. Luciferase reporter gene assay 1. Transfect CHO cells that are grown to 50–70% confluence in six-well dishes with 0.1 lg pSG424-ATF-2, 0.25 lg pG5E1bLuc, and 0.25 lg pCH110 using Lipofectamine (as described by the manufacturer). Cotransfect pCDNA3-Flag-MKK6(Glu). The amount of plasmid transfected for a particular experiment needs to be determined empirically. High expression levels can result in toxicity to the cells. The expression level of Flag-MKK6(Glu) can be monitored by immunoblotting cell lysates using the Flag-M2 antibody (Sigma). 2. Forty-eight hours posttransfection wash the cells in ice-cold PBS (3 3 ml) and scrape them into 200 ll of ice-cold 15 mM potassium phosphate buffer, pH 7.8. Transfer the cells to a 1.5-ml microfuge tube. 3. Lyse the cells by freeze–thawing them three times (i.e., incubate on dry ice for 5 min and then thaw at 37 C for 5 min). Remove the insoluble material by centrifugation at 15,000 rpm for 15 min at 4 C. 4. Aliquot the luciferase assay buffer into luminometer cuvettes (300 ll per cuvette) and adjust the luminometer to inject 100 ll of the D -luciferin stock solution per sample.
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5. Add the clarified cell lysate from step 2 (usually between 1 and 20 ll depending on the expected signal) to the luciferase assay buffer in the cuvettes and read the luminescence values 10 s following injection of the D -luciferin stock solution. 6. b-Galactosidase assay: Add 15 ll of the cell lysate from step 2 to 200 ll Buffer Z and 100 ll ONPG. Incubate the reaction mixture at 37 C until a yellow color appears. Terminate the reaction by adding 500 ll of 1 M Na2 CO3 . Clarify the samples by centrifugation at 15,000 rpm for 5 min at room temperature and measure the optical density of the supernatants by spectrophotometry (wavelength, 420 nm).
3. Expressing the JBD of JIP-1 to inhibit signaling by the JNK pathway to transcription factors: Use of phosphorylation-specific antibodies 3.1. Inhibition of JNK signaling by the JIP-1 JBD Specific protein–protein interactions are critical for the fidelity of cell signaling and transcriptional responses [16]. The identification of discrete protein binding motifs in many signaling molecules and transcription factors has been exploited to generate specific protein/peptide inhibitors of signaling pathways in vivo. The protein sequences corresponding to the binding motifs can be expressed in cells, or they can be synthesized as cellpermeable peptides by fusing them with sequences that facilitate the uptake of proteins by cells. The expressed proteins and cell-permeable peptides competitively inhibit the interaction of the endogenous proteins and can be used to selectively block signaling to transcription factor targets. Many signaling pathways and transcription factor complexes are coordinated by scaffold or regulatory proteins [16–18]. Small proteins or peptides based on the sequences of the interaction motifs in these proteins or the proteins they bind have proved useful as inhibitors of signaling pathways that target transcription factors. One example is the MAP kinase scaffold protein JNKinteracting protein (JIP) 1 which coordinates components of the stress-activated JNK MAP kinase pathway [19,20]. The efficient phosphorylation of JNK substrates requires that JNK binds to docking sites on the substrates [21]. JIP-1 binds to JNK via a similar docking site but with greatly enhanced affinity compared with the affinity of JNK for its transcription factor substrates [19]. Therefore, the overexpression of the JNK-binding domain (JBD) of JIP-1 blocks the association of JNK with its transcription factor substrates, resulting in their inefficient phosphorylation and the inhibition of JNKmediated transcriptional events [19]. This strategy has been used to block JNK signaling in a number of systems. It is reported that the expression of JIP-1 blocks
JNK-mediated apoptosis of neuronal cells [19,22] and b cells [23], and inhibits JNK-mediated transformation of pre-B cells by the Bcr-Abl oncogene [19]. The protocol below describes the inhibition of JNK MAP kinase signaling to the transcription factor c-Jun by the expression of the JBD of JIP-1. The experiment is performed in COS-7 cells which transfect to high efficiency using the Lipofectamine reagent. If the cell line that is used transfects poorly, then this transfection protocol is not suitable as most of the cells will not express the JBD and therefore little effect on the phosphorylation status of endogenous transcription factors will be observed. As an alternative to transfection, both retroviral and adenoviral constructs that express the JBD have been successfully used to infect difficult-totransfect cell lines and primary cells [19,22]. 3.2. Phosphorylation-specific antibodies Antibodies that recognize the phosphorylated forms of proteins are becoming extremely powerful tools to address the phosphorylation status of proteins in vivo [24]. These antibodies are generated by immunizing rabbits or mice with a peptide sequence that spans the phosphorylation site in the protein and is phosphorylated at the appropriate residue. The antibodies can be used to detect a phosphorylated protein in a cell lysate by immunoblotting, or to examine the phosphorylation of a protein in whole cells or tissues by immunocytochemistry. The protocol below describes how an antibody that specifically recognizes one of the JNK phosphorylation sites (Ser-63) on c-Jun can be used to monitor the effect of JIP-1 JBD expression on JNK signaling in cells. The protocol can be adapted to test whether known regulatory phosphorylation sites on other transcription factors are targets of the JNK signaling pathway in vivo. The immunoblotting and immunocytochemistry protocols that are described work well with a number of phosphorylation-specific antibodies including those directed against the MAP kinase phosphorylation sites in other transcription factors (e.g., ATF2 and Elk-1). Alternatively the antibody manufacturer’s protocols can be followed. 3.3. Appropriate control experiments When testing whether a regulatory phosphorylation site on a transcription factor is a novel target for the JNK MAP kinase signaling pathway in response to stress stimuli, it is important to perform control experiments to demonstrate that JNK signaling in the cells has been blocked by the expression of the JBD. A parallel experiment employing the phosphorylation-specific c-Jun antibody would be a suitable control experiment as c-Jun is phosphorylated by JNK in response to stress stimuli and this phosphorylation is blocked by the
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expression of the JBD. It should be noted that the immunoprecipitation of JNK MAP kinase and the measurement of its activity by in vitro protein kinase assay do not constitute a suitable control experiment as the JBD does not block JNK activation, rather it inhibits signaling downstream of JNK. 3.4. Protocol 3.4.1. Materials and reagents 3.4.1.1. Buffers. • Triton lysis buffer (TLB): 20 mM Tris–HCl, pH7.4, 137 mM NaCl, 25 mM sodium b-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 5 lg/ml leupeptin, 5 lg/ml aprotinin, 0.5 mM dithiothreitol, 10% glycerol, 1% Triton X-100. • Western transfer buffer (WTB): 25 mM Tris, 200 mM glycine, 20% methanol, 0.1% SDS. • Western blotting buffer (WBB): 15 mM Tris–HCl, pH 7.4, 150 mM NaCl. • Phosphate-buffered saline (PBS): 20 mM sodium phosphate, pH 7.4, 150 mM NaCl. • 3% paraformaldehyde/PBS: per 10 ml, dissolve 0.3 g paraformaldehyde in 8.5 ml water and 20 ll of 10 M NaOH. Once dissolved add 1 ml 10 PBS, 50 ll of 1 M MgCl2 , and 210 ll of 1 M HCl. Check that pH is between 7.2 and 7.4. • Tris-buffered saline (TBS): 50 mM Tris–HCl, pH 7.4, 150 mM NaCl. • Tris-buffered saline plus Triton X-100 (TBST): TBS/ 0.1% Triton X- 100. • Blocking buffer: 3% w/v bovine serum albumin/TBS. 3.4.1.2. Reagents. • Lipofectamine (Life Technologies). • COS-7 cells (American Type Culture Collection). • pCDNA3-Flag-JBD: encodes the JBD of JIP-1 fused to the Flag epitope tag [19]. • Methanol • Horse serum (GG free, Life Technologies). • Tween 20 (Sigma). • 10% SDS-polyacrylamide gels. • Enhanced chemiluminescence (ECL) reagents (New England Nuclear). • Paraformaldehyde (Sigma). • Bovine serum albumin (Sigma). • Vectashield (Vector Labs Inc.). • Phosphorylation-specific (Ser-63) c-Jun antibody (New England Biolabs). • Anti-rabbit Ig secondary antibody (Amersham–Pharmacia). • Texas red-conjugated anti-rabbit Ig secondary antibody (Jackson Immunoresearch). • 4,6-Diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR).
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3.4.1.3. Equipment. • Six-well tissue culture dishes, 35-mm diameter (Corning). • SDS–PAGE apparatus and semi-dry Western transfer apparatus (Hoefer Scientific). • Parafilm (Sigma). • Coverslips (Corning). • Glass slides (VWR). • Immobilon-P transfer membrane (Millipore). • Gel blot paper (Whatman). • Zeiss axiophot microscope. • X-ray film or chemiluminescent imaging equipment. 3.4.2. Immunoblotting 1. Transfect COS-7 cells seeded in a six-well dish and grown to 50–70% confluency with the plasmid pCDNA3-Flag-JBD using Lipofectamine reagent (according to manufacturer’s instructions). FlagJBD expression can be checked by immunoblotting cell lysates or immunostaining the cells using the Flag-M2 antibody (Sigma). 2. Forty-eight hours posttransfection subject the cells to stress stimuli that induce activation of the JNK signaling pathway. These could include treatment with UV radiation (80 J/m2 for 1 min, then leave to recover for 60 min), anisomycin (5 lg=ml for 30 min), or interleukin-1 (10 nM for 20 min). 3. Aspirate the culture medium, wash the cells with icecold PBS (2 3 ml per well), and then scrape into 0.5 ml PBS and transfer to a 1.5-ml microfuge tube. Pellet the cells by centrifugation at 3000 rpm for 5 min at 4 C. 4. Resuspend the pellet in 20–50 ll of TLB and leave on ice for 10 min for cells to lyse. Remove the insoluble material by centrifugation at 15,000 rpm for 10 min at 4 C. 5. Perform SDS–PAGE using 50–100 lg of cell lysate per sample. 6. Soak the gel in WTB for 5 min. Immerse the Immobilon-P transfer membrane in methanol for a few seconds to wet it and then soak it in WTB for 5 min. Sandwich the gel and membrane with two sheets of gel blot paper on either side, and perform Western transfer (2–3 h at 15 V using the semidry transfer apparatus). 7. Block the membrane by incubating with 200 ml 20% horse serum/WBB for 2 h at room temperature with gentle shaking. 8. Primary antibody incubation: Incubate the membrane overnight at 4 C with gentle shaking in 1:1000 dilution of phosphorylation-specific c-Jun antibody in 20% horse serum/WBB. 9. The following day, wash the membrane twice for 10 min in 100 ml 0.5% Tween 20/WBB at room temperature with shaking.
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10. Secondary antibody incubation: Incubate the membrane with anti-rabbit Ig secondary antibody diluted 1:10,000 in 20% horse serum/WBB for 30 min at room temperature with gentle shaking. 11. Wash the membrane four times for 15 min with 0.5% Tween 20/WBB at room temperature with shaking, and then for 5 min with WBB. 12. Develop the immunoblot using ECL reagents and detect the signal by exposing to X-ray film.
for 5 min in 2 ml TBS. To prevent exposure to light, wrap the six-well dishes in aluminum foil during the washes. 11. Lay the coverslips on top of a drop of Vectashield that is spotted onto glass slides and visualize the staining pattern by fluorescence microscopy. Store the slides in the dark to preserve the fluorescent staining.
3.4.3. Immunocytochemistry 1. Transfect COS-7 cells grown on glass coverslips [for some cell types the coverslips need to be treated with appropriate attachment factors, e.g., poly(L -lysine), collagen] in six-well dishes as described under Immunoblotting. 2. Forty-eight hours posttransfection aspirate the cell culture medium and wash the cells with ice-cold PBS (2 3 ml per well). 3. Fix the cells with 1 ml 3% paraformaldehyde/PBS for 10 min at 4 C. Wash the cells with TBST (2 ml per well) twice for 5 min at room temperature with shaking, and then rinse the cells with TBS. 4. Permeabilize the cells by incubating with 1 ml methanol (prechilled at )20 C) for 10 min at )20 C, and then wash twice for 10 min in 2 ml TBST at room temperature with shaking. 5. Blocking: Incubate the cells with 2 ml blocking buffer for 60 min at room temperature with gentle shaking. 6. Primary antibody incubation: Spot 35 ll of phosphorylation-specific c-Jun antibody (diluted 1:200 with blocking buffer) onto Parafilm and carefully lay the coverslips on top. Place the Parafilm with the coverslips in a damp environment (to prevent drying out, a 150-mm-diameter culture dish covered in aluminum foil and with damp paper towel attached to the inside of the lid can be used) overnight at 4 C. 7. The next day place the coverslips back into the sixwell tissue culture dish and wash three times for 5 min with 2 ml TBST with shaking, and once for 5 min with TBS. 8. Secondary antibody incubation (in the dark): Spot 35 ll of diluted Texas red-conjugated anti-rabbit Ig secondary antibody (diluted 1:100 in blocking buffer) onto Parafilm and carefully lay the coverslips on top. Incubate in the dark at room temperature for 45 min. All the subsequent steps should be performed in the dark due to the light sensitivity of the secondary antibody. 9. DNA staining: Place the coverslips back in the sixwell dish and rinse with TBS. Incubate the coverslips with DAPI DNA stain (diluted 1:10,000 in TBS) in the dark for 2 min at room temperature. 10. Wash the coverslips three times for 10 min in 2 ml TBST at room temperature with shaking and then
4. Discussion 4.1. Mutated enzymes that activate or inhibit signaling pathways The mutation of the activating phosphorylation sites on MKKs from serine and threonine residues to acidic residues to mimic the charge effect of phosphorylation often results in mutant proteins that, when expressed in cells, potently activate endogenous MAP kinases and downstream signaling events (Fig. 1). The first protocol utilizes such a mutant of MKK6 to selectively activate the p38 MAP kinase pathway. This approach has also been successfully used with MKK3, a second MKK that selectively activates p38 [12], and MEK1, an activator of the ERK pathway [25]. However, similar mutations in the JNK activators MKK4 and MKK7 have proved less useful due to their relatively weak effect in activating endogenous JNK in most cell lines. In addition, the mutation of the threonine and tyrosine phosphorylation sites to acidic residues on the MAP kinases themselves does not result in constitutively active mutants. A number of other approaches may be taken to generate activated signaling enzymes apart from mutating the activating phosphorylation sites. These include the deletion of regulatory domains or fusing the enzyme to other proteins that confer particular properties, for example, inducible oligomerization. An alternative approach to generate constitutively activated forms of ERK and JNK MAP kinases is to fuse them with their respective MKKs [26,27] (Fig. 1). The MEK1–ERK2 fusion protein behaves as a constitutively activated form of ERK2 in cells [26], while a MKK7– JNK1 fusion protein behaves as a constitutively activated form of JNK1 [27]. Mutated forms of MKKKs have also proved useful for activating MAP kinase pathways in cells. For example, deletion of the aminoterminal regulatory domains of Raf-1 [28] and MEKK1 [29,30] results in the constitutive activity of their catalytic domains. The Raf-1 mutant preferentially activates the ERK pathway [28] and the MEKK1 mutant preferentially activates the JNK pathway [29,30]. In some cases oligomerization of MKKKs is required for their activity. This has been exploited to generate systems for inducing MKKK oligomerization resulting in activated forms of the enzymes. The fusion of MKKKs to either a
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domain of the B subunit of bacterial DNA gyrase (GyrB) [31] (Fig. 1) or to the FKBP12 protein [32] allows the inducible oligomerization of the MKKKs in cells by the addition of coumermycin (which acts as a natural dimerizer of GyrB) or FK1012A (a synthetic dimerizer that binds to FKBP12), respectively. These oligomerization-inducible systems have been used to activate Raf-1 [31,32] and the MKKK ASK1 [33], a component of JNK and p38 signaling pathways. To specifically block signaling pathways, dominant negative mutant alleles can be created by mutating the activating phosphorylation sites on the enzymes to nonphosphorylatable residues. Dominant-negative mutants exert their effect by binding and sequestering their substrates or upstream signaling components. Mutation of the threonine and tyrosine phosphorylation sites on MAP kinases to alanine and phenylalanine residues, respectively, creates dominant negative mutants that have been used to specifically block signaling in a number of systems (e.g., Refs. [34–37]) (Fig. 1). Similarly, the overexpression of mutant forms of MKKs that have their serine and threonine phosphorylation sites mutated to alanine leads to the inhibition of signaling through their respective pathways (e.g., Refs. [12,38–40]) (Fig. 1). Catalytically inactivated forms of components of MAP kinase pathways are a second type of dominant negative mutant that can be used to block signaling (e.g., Refs. [41,42]). These usually feature the mutation of a conserved lysine residue in the catalytic site of the enzymes. The prolonged overexpression of constitutively activated or dominant negative mutants of signaling enzymes is sometimes toxic to cells, resulting in cell death. This makes their effect on transcription factor function difficult to ascertain. To try and circumvent these problems, inducible expression systems can be used. This involves generating stable transfected cell lines that express the mutant enzyme under the control of an inducible promoter. A number of inducible expression systems are commercially available including tetracycline-inducible systems (e.g., the Tet-On and Tet-Off systems from Clontech) and the ecdysone-inducible system (available from Invitrogen) [43]. In contrast to controlling the level of protein expression, other inducible systems regulate the activity of the protein. The GyrB- and FKBP12-inducible oligomerization systems mentioned previously are one example. A second example involves the fusion of mutant signaling enzymes to a mutated form of the hormone binding domain (HBD) of the estrogen receptor [44] (Fig. 1). The mutated HBD selectively binds to synthetic ligands such as 4-hydroxytamoxifen (4HT) rather than the natural ligands [44]. The fused enzyme is inactive in the absence of 4HT, but becomes rapidly activated on addition of 4HT to cells. The exact mechanism by which this occurs is not fully understood. In the absence of ligand the
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HBD binds to heat shock protein 90 (Hsp90). It is thought that Hsp90 may sterically block the fused enzyme from interacting with its substrates, thereby inhibiting its activity. The addition of ligand relieves Hsp90 binding to the HBD and allows the fused enzyme to become active [44]. This strategy has been used to successfully regulate the activity of components of MAP kinase signaling pathways as well as other signaling pathways [44]. It should be emphasized that the strategies that have been discussed will not work in every case and the best approach for a particular experiment needs to be determined empirically. A second caveat to these types of studies is that overexpression of the mutated enzymes in cells may trigger the activation of, or inhibit, nonphysiologically relevant signaling pathways. For example a number of kinase-inactive versions of protein kinases will block all signaling from upstream activators rather than being specific for their particular pathway. Therefore, it is critical to perform appropriate control experiments to establish the specificity of the mutant enzymes. 4.2. Protein/peptide inhibitors of signaling pathways. A few pharmacological agents that specifically block intracellular signaling pathways are commercially available. For example, the ERK MAP kinase signaling pathway can be blocked by inhibitors of MEK activity such as PD98059 (available from Calbiochem) and U0126 (available from Promega) [45], while a family of pyridinylimadazole-based drugs (the most widely used of which is SB203580, available from Calbiochem) can be used to inhibit the protein kinase activity of p38 MAP kinase isoforms a and b2 [46]. These drugs have been very useful for determining the roles of the ERK and p38 pathways in cells. However, they are not completely specific for their desired targets [15,45,46] so some care has to be taken in interpreting the data obtained. In the absence of specific pharmacological inhibitors for many signaling pathways, the use of protein/peptide inhibitors is proving to be a viable alternative for disrupting signaling in cells. There are no commercially available pharmacological inhibitors of JNK MAP kinase; therefore, the JBD of the scaffold protein JIP-1 has become an important tool for disrupting JNK signaling in cells. The success of this approach relies on the fact that the JIP-1 JBD has a much higher binding affinity for JNK than the transcription factor substrates of JNK, and thereby prevents JNK from associating with and phosphorylating its substrates [19]. It is likely that in the future the protein interaction motifs of other MAP kinase scaffold proteins [47–49] may also become useful tools for specifically blocking these signaling pathways.
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Protein or peptide sequences spanning the protein interaction motifs on protein kinases can also be generated to block the interaction of protein kinases with key regulatory proteins. For example, cell-permeable peptides have been used to inhibit the transcription factor NFjB by blocking the interaction of the regulatory protein NEMO (also named IKKc) with the IjBkinases (IKKs) IKKa and IKKb [50]. IjB binds to NF-jB and inhibits its ability to activate transcription. The phosphorylation of IjB by the IKK complex (IKKa, IKKb, and NEMO) targets it for degradation by the proteosome, leading to the release of NFjB, which can then activate gene transcription [51]. The cellpermeable peptides span the NEMO binding sites on IKKa and IKKb and block the interaction of NEMO with these protein kinases, leading to inhibition of IjB phosphorylation and NF-jB activation [50]. In addition to exploiting the high-affinity interactions between scaffold/regulatory proteins and signaling enzymes or transcription factors, cell-permeable peptides have been used to inhibit specific enzyme–transcription factor interactions. For example, the transcription factor NFAT is activated by dephosphorylation by the protein phosphatase calcineurin (also named protein phosphatase 2B), which results in nuclear translocation of NFAT and NFAT-mediated gene expression [52]. Calcineurin binding to NFAT is required for efficient dephosphorylation [52]. The introduction into cells of peptides spanning the calcineurin binding site on NFAT inhibits the ability of calcineurin to bind to and dephosphorylate NFAT, but does not affect the activity of calcineurin toward other substrates [53]. Subsequently, mutant NFAT peptides with increased affinity for calcineurin have been isolated by screening peptide libraries [54]. These behave as highly specific and potent inhibitors of NFAT-mediated gene transcription, and demonstrate the therapeutic potential of this approach [54]. A second advantage of the peptide approach is that it can be employed with cells that are difficult to transfect. For a more detailed discussion of protocols for the generation and use of cell-permeable peptides (see Refs. [55] and [56]). 4.3. Assays that measure transcription factor phosphorylation and activation The use of phosphorylation-specific antibodies and reporter gene experiments are two important assays for determining the phosphorylation status and the transcriptional activity of transcription factors, respectively. These assays can be combined with the tools discussed in the previous sections to determine the specificity of signaling pathways toward transcription factors in vivo. The reporter gene system described in the protocol features the transcriptional activation domain of ATF-2 fused to the GAL4 DNA binding domain. Repression
domains of transcription factors can also be fused to GAL4 to determine if signaling pathways regulate repression activity. When investigating repression domains a more active promoter (e.g., SV40 promoter) upstream of the luciferase gene may be required to be able to observe significant repression of luciferase expression. More complex reporter gene systems can be constructed to assay transrepression activities of transcription factors. For example a LexA-GAL4-luciferase reporter gene (which features tandem LexA and GAL4 DNA binding sites upstream of a minimal promoter and the firefly luciferase gene) allows a GAL4-repressor fusion to be assayed in the presence of a LexA-activator fusion [57]. Phosphorylation-specific antibodies are becoming the most widely used method to determine the phosphorylation status of proteins in cells. Many antibodies that recognize specific phosphorylation sites on transcription factors are commercially available. One major advantage of this approach over other methods in detecting phosphorylation in vivo is that it does not require the labeling of cells with radioactive phosphate and subsequent phosphopeptide mapping. Caveats associated with the approach are that it relies on the antibodies being highly specific for the modified sites, and second, the modification of nearby sites in vivo may prevent antibody recognition. In addition to phosphorylation, many transcription factors are subjected to other types of posttranslational modifications that regulate their activity, including acetylation and methylation. Specific antibodies that recognize these modifications have also become useful tools for understanding the regulation of transcription factors by signaling pathways.
5. Concluding remarks A number of approaches can be taken to elucidate the signaling pathways that target transcription factors in vivo. These include the use of gene-targeting strategies, small molecule inhibitors, and the two approaches described in this article: activated and dominant negative signaling enzymes, and protein/peptide inhibitors. This article has focused on applying these approaches to study the regulation of transcription factors by MAP kinase pathways; however, they can be applied equally well to many of the signaling pathways that target transcription factors (e.g., protein kinase B (PKB)/Akt pathways, the IjB-kinase complex, calcium/calmodulindependent protein kinase pathways, calcineurin/NFAT). Two methods for assaying the targeting of transcription factors by signaling pathways, modificationspecific antibodies and reporter gene assays, are also described. These assays can be employed in conjunction with activated/dominant negative signaling enzymes and protein/peptide inhibitors to provide a greater
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understanding of the signaling pathways that regulate transcription factor function. In the future, the development of novel highly specific peptide inhibitors and the increased use of modification-specific antibodies are likely to lead to further rapid progress in dissecting the intracellular signaling pathways that regulate gene expression. Acknowledgments Work in the author’s laboratory is sponsored by the Wellcome Trust, the Cancer Research Campaign (CRC), and the Royal Society. The author is a Lister Institute-Jenner Research Fellow. References [1] C.S. Hill, R. Treisman, Cell 80 (1995) 199–211. [2] P. Shore, A.D. Sharrocks, in: J. Locker (Ed.), Transcription Factors, BIOS Scientific, Oxford, 2001, pp. 113–135. [3] A.J. Whitmarsh, R.J. Davis, Cell. Mol. Life Sci. 57 (2000) 1172– 1183. [4] A.J. Bannister, E.A. Miska, Cell. Mol. Life Sci. 57 (2000) 1184– 1192. [5] J.C. Rice, C.D. Allis, Curr. Opin. Cell Biol. 13 (2001) 263–273. [6] K.A. Mowen, J. Tang, W. Zhu, B.T. Schurter, K. Shuai, H.R. Herschman, M. David, Cell 104 (2001) 731–741. [7] J.M. Desterro, M.S. Rodriguez, R.T. Hay, Cell. Mol. Life Sci. 57 (2000) 1207–1219. [8] A.J. Whitmarsh, R.J. Davis, J. Mol. Med. 74 (1996) 589–607. [9] T.S. Lewis, P.S. Shapiro, N.G. Ahn, Adv. Cancer Res. 74 (1998) 49–139. [10] R.J. Davis, Cell 103 (2000) 239–252. [11] K. Ono, J. Han, Cell Signal. 12 (2000) 1–13. [12] J. Raingeaud, A.J. Whitmarsh, T. Barrett, B. Derijard, R.J. Davis, Mol. Cell. Biol. 16 (1996) 1247–1255. [13] S. Gupta, D. Campbell, B. Derijard, R.J. Davis, Science 267 (1995) 389–393. [14] S. Gupta, A. Seth, R.J. Davis, Proc. Natl. Acad. Sci. USA 90 (1993) 3216–3220. [15] A.J. Whitmarsh, R.J. Davis, Methods Enzymol. 332 (2001) 319– 336. [16] T. Pawson, P. Nash, Genes Dev. 14 (2000) 1027–1047. [17] T. Pawson, J.D. Scott, Science 278 (1997) 2075–2080. [18] A.J. Whitmarsh, R.J. Davis, Trends Biochem. Sci. 23 (1998) 481– 485. [19] M. Dickens, J.S. Rogers, J. Cavanagh, A. Raitano, Z. Xia, J.R. Halpern, M.E. Greenberg, C.L. Sawyers, R.J. Davis, Science 277 (1997) 693. [20] A.J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R.J. Davis, Science 281 (1998) 1671–1674. [21] A.D. Sharrocks, S.H. Yang, A. Galanis, Trends Biochem. Sci. 26 (2000) 448–453. [22] T.C. Harding, L. Xue, A. Bienemann, D. Haywood, M. Dickens, A.M. Tolkovsky, J.B. Uney, J. Biol. Chem. 276 (2001) 4531–4534. [23] C. Bonny, A. Oberson, M. Steinmann, D.F. Schorderet, P. Nicod, G. Waeber, J. Biol. Chem. 275 (2000) 16466–16472. [24] J.P. Blaydes, B. Vojtesek, G.B. Bloomberg, T.R. Hupp, Methods Mol. Biol. 99 (2000) 177–189. [25] S.J. Mansour, W.T. Matten, A.S. Hermann, J.M. Candia, S. Rong, K. Fukasawa, G.F. Vande Woude, N.G. Ahn, Science 265 (1994) 966–970.
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Examining signaling specificity to transcription factors Alan J. Whitmarsh* School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK Accepted 1 February 2002
Abstract The activities of many transcription factors are controlled by intracellular signal transduction pathways that respond to changes in the extracellular environment. The resulting changes in gene expression lead to appropriate physiological responses. Elucidating the signaling pathways that target transcription factors is an important goal as they represent potential targets for therapeutic intervention in many diseases. A number of tools and techniques have been developed that can be used for studying signaling specificity toward transcription factors. This article describes how (1) mutant alleles of signaling enzymes and (2) protein/peptide inhibitors can be used in conjunction with phosphorylation-specific antibodies and reporter gene assays to examine the targeting of transcription factors by signaling pathways in vivo. 2002 Elsevier Science (USA). All rights reserved. Keywords: Transcription factor; Signal transduction; Protein kinase; Phosphorylation; Mitogen-activated protein kinase; Reporter gene; Phosphorylation-specific antibody
1. Introduction Alterations in the patterns of gene expression in response to extracellular signaling cues are mediated by the integration of multiple intracellular signal transduction pathways that target transcription factors, transcriptional coregulators, and chromatin-modifying factors [1–3]. Protein phosphorylation and dephosphorylation constitute the most common regulatory mechanism, particularly for the rapid modulation of transcription factor activity [1–3]. However, other forms of posttranslational modification of proteins including acetylation, methylation, ubiquitination, and SUMOconjugation are also important for regulating transcription factor function [4–7]. A number of in vitro assays can be performed (e.g., protein kinase assays and acetylation assays) to address whether particular transcription factors are likely targets of protein modification enzymes. However, the physiological relevance of transcription factor modifications observed in vitro needs to be confirmed by in vivo experiments involving the specific activation or inhibition
*
Fax: +44-(0)161-275-5082. E-mail address: [email protected] (A.J. Whitmarsh).
of signaling pathways in cells. Two methods of blocking signaling pathways are (1) employing small molecule inhibitors and (2) generating cells with targeted deletions of genes encoding signaling proteins. Only a few genuinely specific small molecule inhibitors of signaling enzymes are commercially available; therefore their general use for determining signaling specificity toward transcription factors has been limited. Gene-targeted cells from mice have proved to be an increasingly important tool for delineating signaling pathways that target transcription factors. However, in a number of cases cells cannot always be derived if the targeted gene is essential for early embryonic development of the mice. In this article two additional methods for specifically modulating signaling pathways that target transcription factors are described. These are the use of activated mutant alleles or dominant-negative mutant alleles of signaling enzymes either to activate or to block specific pathways, and the use of small inhibitory proteins or peptides that inhibit specific protein–protein interactions between signaling proteins and transcription factors. These tools can be employed in conjunction with assays that measure the effect of signaling pathways on the modification and function of transcription factors. These include the generation of antibodies that specifically recognize posttranslationally modified forms of
1046-2023/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 0 2 5 - 7
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transcription factors, and the use of reporter gene assays to assess transcriptional activity. The examples discussed below focus on examining the targeting of transcription factors by the stress-activated group of the mitogen-activated protein (MAP) kinase family of signaling pathways. However, in most cases the techniques described are generally applicable to the study of many of the signaling pathways that target transcription factors. There are three major groups of MAP kinases (ERK, JNK, and p38) that are activated by sequential phosphorylation by signaling modules consisting of a MAP kinase kinase (MKK) and a MAP kinase kinase kinase (MKKK) [8,9] (Fig. 1). Members of the Ras and Rho families of GTPases lie upstream of these signaling modules and control their activity [8,9]. The ERK pathway is stimulated mainly by mitogenic stimuli (e.g.,
growth factors, hormones) and regulates the activity of the transcription factors Elk-1 and c-Myc [8,9]. The JNK and p38 pathways are stimulated mainly by stress stimuli (e.g., osmotic stress, oxidative stress, UV radiation, proinflammatory cytokines) and are collectively referred to as the stress-activated protein kinases (SAPKs). JNK regulates the activity of c-Jun, ATF-2, NFAT4, and Elk-1, while p38 regulates the activity of ATF-2, CHOP, MEF2C, and Elk-1 [8–11]. The specific protocols that are described are (1) the use of an activated allele of the MAP kinase kinase MKK6 to determine the effect of the p38 MAP kinase signaling pathway on the activity of transcription factors (measured by a luciferase reporter gene assay), and (2) the use of the JNK-binding domain (JBD) of the scaffold protein JNK-interacting protein 1 (JIP-1) to inhibit JNK MAP kinase signaling to transcription factors (measured by antibodies that recognize the JNK phosphorylation sites on the transcription factors).
2. Expression of an activated allele of MKK6 to selectively stimulate p38 MAP kinase-mediated transcriptional activity: Use of luciferase reporter gene assays 2.1. Activated allele of MKK6
Fig. 1. Tools to activate and inhibit MAP kinase signaling pathways that target transcription factors. The MAP kinase signaling module consists of a MAP kinase (MAPK) that is activated by sequential phosphorylation by a MAP kinase kinase (MKK) and a MAP kinase kinase kinase (MKKK). MKKKs may be activated by members of the Ras and Rho families of GTPases. To specifically activate MAP kinase pathways activated alleles of MKKKs (MKKK*) or MKKs (MKK*) can be used. Constitutively activated MAP kinases can be generated by fusing the MAPK to the MKK. Inducible activation systems are also in use. Some MKKKs are active as dimers. These MKKKs can be fused to the B domain of DNA gyrase (GyrB) which forms dimers in the presence of coumermycin, which can be added to cells expressing the fusion proteins. A second inducible system involves the fusion of activated alleles of MAP kinase pathway components to a mutated hormone binding domain of the estrogen receptor, for example ERMKK*. To inhibit signaling through MAP kinase pathways dominant-negative (DN) mutants of MKKKs, MKKs, or MAPKs have proved useful. In addition, small protein or peptide inhibitors of interactions between MAPKs and their transcription factor targets can be employed.
The overexpression of signaling enzymes in cells is sometimes sufficient to trigger the activation of downstream signaling events. However, often more potent activation of signaling pathways occurs if constitutively activated mutant enzymes (i.e., active in the absence of stimuli) are created. This can be achieved in a number of ways depending on the characteristics of the particular enzyme. The most common is the mutation of the activating phosphorylation sites in the enzyme to acidic residues to mimic the charge effect of phosphorylation. These mutations in MKK6 (Ser-207 and Thr-211 to Glu) result in a mutant enzyme that, when expressed in cells, potently and selectively activates the p38 MAP kinase signaling pathway [12]. 2.2. Reporter gene assay Reporter gene assays can be used to address which stimuli and signaling pathways regulate the transcriptional activity of a transcription factor. The principle of the assay is that the transcriptional activation domain of a transcription factor is fused to a heterologous DNA binding domain (usually the DNA binding domain of the transcription factor GAL4 or the repressor LexA) and cotransfected with a reporter plasmid containing sites for the DNA binding domain upstream of a minimal promoter (e.g., E1b promoter) and the firefly luciferase gene. The modification of the transcription factor fusion protein (e.g., by protein kinases) leads to
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increased or decreased expression of the luciferase gene, which can be measured by enzymatic assay. As a control for transfection efficiency, a plasmid featuring a b-galactosidase reporter gene is also transfected and b-galactosidase activity measured (alternatively a second type of luciferase reporter can be used featuring the Renilla luciferase gene). The reporter gene system used in the protocol below features the transcriptional activation domain of the p38 substrate ATF-2 fused to the DNA binding domain of GAL4 [13] and a luciferase reporter gene containing five GAL4 binding sites [14]. The protocol works well in a number of cell lines (e.g., NIH 3T3, HeLa, COS-7, CHO) and can be adapted for use with other activated signaling enzymes and GAL4 transcription factor fusion proteins. As an alternative to the described protocol, commercial kits are available (e.g., from Promega, Madison, WI) that feature dual luciferase reporter systems. 2.3. Appropriate control experiments To test whether a particular transcription factor is a novel target for regulation by the p38 MAP kinase signaling pathway, it is important to perform control experiments to demonstrate that the p38 pathway has been activated by the expression of the MKK6 mutant. This can be achieved by carrying out a parallel experiment using a known p38 target (e.g., GAL4-ATF-2) or immunoprecipitating p38 MAP kinase from the cells and measuring its activity in vitro [15]. Other important control experiments are: (1) expressing the GAL4 DNA binding domain alone to ensure that the MKK6 mutant is not inducing luciferase gene transcription in the absence of the transcription factor, and (2) including a mutant version of the GAL4 transcription factor fusion with the phosphorylation sites mutated to nonphosphorylatable residues to demonstrate that any effects on transcriptional activity are directly due to the phosphorylation of the transcription factor. 2.4. Protocol 2.4.1. Materials and reagents 2.4.1.1. Buffers. • Phosphate-buffered saline (PBS): 20 mM sodium phosphate, pH 7.4, 150 mM NaCl. • Potassium phosphate buffer, pH 7.8. • Luciferase assay buffer: 15 mM potassium phosphate, pH 7.8, 25 mM glycylglycine, pH 7.8, 15 mM Mg2 SO4 , 4 mM EGTA, 2 mM ATP (made as 100 mM stock and adjusted to pH 7.8), 1 mM dithiothreitol. • Buffer Z: 100 mM sodium phosphate, pH 7.0, 10 mM KCl, 1 mM Mg2 SO4 , 1 M Na2 CO3 .
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2.4.1.2. Reagents. • D -Luciferin (Pharmingen): 1 mM stock solution made by dissolving 10 mg D -L uciferin in 31.5 ml water and adjusting to pH 6.0–6.3 by adding 100 mM NaOH solution dropwise. Stored frozen. • O-Nitrophenyl-b-D -galactopyranoside (ONPG) (U.S. Biochemical): 4 mg/ml in 100 mM potassium phosphate, pH 7.0, filter sterilized and stored frozen. • pCDNA3-Flag-MKK6(Glu): encodes a mutant form of MKK6 featuring mutation of Ser-207 and Thr211 to Glu [12]. • pSG424-ATF-2: encodes a fusion of ATF-2 amino acids 1–109 with the GAL4 DNA binding domain [13]. • pG5E1bLuc: firefly luciferase gene reporter plasmid featuring five GAL4 binding sites and the minimal E1b promoter [14]. • pCH110: b-galactosidase reporter plasmid (Amersham–Pharmacia). • Lipofectamine (Life Technologies). • Chinese hamster ovary (CHO) cells (American Type Culture Collection, Manassas, VA). 2.4.1.3. Equipment. • 1.5-ml microfuge tubes. • Luminometer cuvettes. • Luminometer. • 37 C heating block • Spectrophotometer • Six-well tissue culture dishes (35-mm diameter) (Corning). 2.4.2. Luciferase reporter gene assay 1. Transfect CHO cells that are grown to 50–70% confluence in six-well dishes with 0.1 lg pSG424-ATF-2, 0.25 lg pG5E1bLuc, and 0.25 lg pCH110 using Lipofectamine (as described by the manufacturer). Cotransfect pCDNA3-Flag-MKK6(Glu). The amount of plasmid transfected for a particular experiment needs to be determined empirically. High expression levels can result in toxicity to the cells. The expression level of Flag-MKK6(Glu) can be monitored by immunoblotting cell lysates using the Flag-M2 antibody (Sigma). 2. Forty-eight hours posttransfection wash the cells in ice-cold PBS (3 3 ml) and scrape them into 200 ll of ice-cold 15 mM potassium phosphate buffer, pH 7.8. Transfer the cells to a 1.5-ml microfuge tube. 3. Lyse the cells by freeze–thawing them three times (i.e., incubate on dry ice for 5 min and then thaw at 37 C for 5 min). Remove the insoluble material by centrifugation at 15,000 rpm for 15 min at 4 C. 4. Aliquot the luciferase assay buffer into luminometer cuvettes (300 ll per cuvette) and adjust the luminometer to inject 100 ll of the D -luciferin stock solution per sample.
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5. Add the clarified cell lysate from step 2 (usually between 1 and 20 ll depending on the expected signal) to the luciferase assay buffer in the cuvettes and read the luminescence values 10 s following injection of the D -luciferin stock solution. 6. b-Galactosidase assay: Add 15 ll of the cell lysate from step 2 to 200 ll Buffer Z and 100 ll ONPG. Incubate the reaction mixture at 37 C until a yellow color appears. Terminate the reaction by adding 500 ll of 1 M Na2 CO3 . Clarify the samples by centrifugation at 15,000 rpm for 5 min at room temperature and measure the optical density of the supernatants by spectrophotometry (wavelength, 420 nm).
3. Expressing the JBD of JIP-1 to inhibit signaling by the JNK pathway to transcription factors: Use of phosphorylation-specific antibodies 3.1. Inhibition of JNK signaling by the JIP-1 JBD Specific protein–protein interactions are critical for the fidelity of cell signaling and transcriptional responses [16]. The identification of discrete protein binding motifs in many signaling molecules and transcription factors has been exploited to generate specific protein/peptide inhibitors of signaling pathways in vivo. The protein sequences corresponding to the binding motifs can be expressed in cells, or they can be synthesized as cellpermeable peptides by fusing them with sequences that facilitate the uptake of proteins by cells. The expressed proteins and cell-permeable peptides competitively inhibit the interaction of the endogenous proteins and can be used to selectively block signaling to transcription factor targets. Many signaling pathways and transcription factor complexes are coordinated by scaffold or regulatory proteins [16–18]. Small proteins or peptides based on the sequences of the interaction motifs in these proteins or the proteins they bind have proved useful as inhibitors of signaling pathways that target transcription factors. One example is the MAP kinase scaffold protein JNKinteracting protein (JIP) 1 which coordinates components of the stress-activated JNK MAP kinase pathway [19,20]. The efficient phosphorylation of JNK substrates requires that JNK binds to docking sites on the substrates [21]. JIP-1 binds to JNK via a similar docking site but with greatly enhanced affinity compared with the affinity of JNK for its transcription factor substrates [19]. Therefore, the overexpression of the JNK-binding domain (JBD) of JIP-1 blocks the association of JNK with its transcription factor substrates, resulting in their inefficient phosphorylation and the inhibition of JNKmediated transcriptional events [19]. This strategy has been used to block JNK signaling in a number of systems. It is reported that the expression of JIP-1 blocks
JNK-mediated apoptosis of neuronal cells [19,22] and b cells [23], and inhibits JNK-mediated transformation of pre-B cells by the Bcr-Abl oncogene [19]. The protocol below describes the inhibition of JNK MAP kinase signaling to the transcription factor c-Jun by the expression of the JBD of JIP-1. The experiment is performed in COS-7 cells which transfect to high efficiency using the Lipofectamine reagent. If the cell line that is used transfects poorly, then this transfection protocol is not suitable as most of the cells will not express the JBD and therefore little effect on the phosphorylation status of endogenous transcription factors will be observed. As an alternative to transfection, both retroviral and adenoviral constructs that express the JBD have been successfully used to infect difficult-totransfect cell lines and primary cells [19,22]. 3.2. Phosphorylation-specific antibodies Antibodies that recognize the phosphorylated forms of proteins are becoming extremely powerful tools to address the phosphorylation status of proteins in vivo [24]. These antibodies are generated by immunizing rabbits or mice with a peptide sequence that spans the phosphorylation site in the protein and is phosphorylated at the appropriate residue. The antibodies can be used to detect a phosphorylated protein in a cell lysate by immunoblotting, or to examine the phosphorylation of a protein in whole cells or tissues by immunocytochemistry. The protocol below describes how an antibody that specifically recognizes one of the JNK phosphorylation sites (Ser-63) on c-Jun can be used to monitor the effect of JIP-1 JBD expression on JNK signaling in cells. The protocol can be adapted to test whether known regulatory phosphorylation sites on other transcription factors are targets of the JNK signaling pathway in vivo. The immunoblotting and immunocytochemistry protocols that are described work well with a number of phosphorylation-specific antibodies including those directed against the MAP kinase phosphorylation sites in other transcription factors (e.g., ATF2 and Elk-1). Alternatively the antibody manufacturer’s protocols can be followed. 3.3. Appropriate control experiments When testing whether a regulatory phosphorylation site on a transcription factor is a novel target for the JNK MAP kinase signaling pathway in response to stress stimuli, it is important to perform control experiments to demonstrate that JNK signaling in the cells has been blocked by the expression of the JBD. A parallel experiment employing the phosphorylation-specific c-Jun antibody would be a suitable control experiment as c-Jun is phosphorylated by JNK in response to stress stimuli and this phosphorylation is blocked by the
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expression of the JBD. It should be noted that the immunoprecipitation of JNK MAP kinase and the measurement of its activity by in vitro protein kinase assay do not constitute a suitable control experiment as the JBD does not block JNK activation, rather it inhibits signaling downstream of JNK. 3.4. Protocol 3.4.1. Materials and reagents 3.4.1.1. Buffers. • Triton lysis buffer (TLB): 20 mM Tris–HCl, pH7.4, 137 mM NaCl, 25 mM sodium b-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 5 lg/ml leupeptin, 5 lg/ml aprotinin, 0.5 mM dithiothreitol, 10% glycerol, 1% Triton X-100. • Western transfer buffer (WTB): 25 mM Tris, 200 mM glycine, 20% methanol, 0.1% SDS. • Western blotting buffer (WBB): 15 mM Tris–HCl, pH 7.4, 150 mM NaCl. • Phosphate-buffered saline (PBS): 20 mM sodium phosphate, pH 7.4, 150 mM NaCl. • 3% paraformaldehyde/PBS: per 10 ml, dissolve 0.3 g paraformaldehyde in 8.5 ml water and 20 ll of 10 M NaOH. Once dissolved add 1 ml 10 PBS, 50 ll of 1 M MgCl2 , and 210 ll of 1 M HCl. Check that pH is between 7.2 and 7.4. • Tris-buffered saline (TBS): 50 mM Tris–HCl, pH 7.4, 150 mM NaCl. • Tris-buffered saline plus Triton X-100 (TBST): TBS/ 0.1% Triton X- 100. • Blocking buffer: 3% w/v bovine serum albumin/TBS. 3.4.1.2. Reagents. • Lipofectamine (Life Technologies). • COS-7 cells (American Type Culture Collection). • pCDNA3-Flag-JBD: encodes the JBD of JIP-1 fused to the Flag epitope tag [19]. • Methanol • Horse serum (GG free, Life Technologies). • Tween 20 (Sigma). • 10% SDS-polyacrylamide gels. • Enhanced chemiluminescence (ECL) reagents (New England Nuclear). • Paraformaldehyde (Sigma). • Bovine serum albumin (Sigma). • Vectashield (Vector Labs Inc.). • Phosphorylation-specific (Ser-63) c-Jun antibody (New England Biolabs). • Anti-rabbit Ig secondary antibody (Amersham–Pharmacia). • Texas red-conjugated anti-rabbit Ig secondary antibody (Jackson Immunoresearch). • 4,6-Diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR).
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3.4.1.3. Equipment. • Six-well tissue culture dishes, 35-mm diameter (Corning). • SDS–PAGE apparatus and semi-dry Western transfer apparatus (Hoefer Scientific). • Parafilm (Sigma). • Coverslips (Corning). • Glass slides (VWR). • Immobilon-P transfer membrane (Millipore). • Gel blot paper (Whatman). • Zeiss axiophot microscope. • X-ray film or chemiluminescent imaging equipment. 3.4.2. Immunoblotting 1. Transfect COS-7 cells seeded in a six-well dish and grown to 50–70% confluency with the plasmid pCDNA3-Flag-JBD using Lipofectamine reagent (according to manufacturer’s instructions). FlagJBD expression can be checked by immunoblotting cell lysates or immunostaining the cells using the Flag-M2 antibody (Sigma). 2. Forty-eight hours posttransfection subject the cells to stress stimuli that induce activation of the JNK signaling pathway. These could include treatment with UV radiation (80 J/m2 for 1 min, then leave to recover for 60 min), anisomycin (5 lg=ml for 30 min), or interleukin-1 (10 nM for 20 min). 3. Aspirate the culture medium, wash the cells with icecold PBS (2 3 ml per well), and then scrape into 0.5 ml PBS and transfer to a 1.5-ml microfuge tube. Pellet the cells by centrifugation at 3000 rpm for 5 min at 4 C. 4. Resuspend the pellet in 20–50 ll of TLB and leave on ice for 10 min for cells to lyse. Remove the insoluble material by centrifugation at 15,000 rpm for 10 min at 4 C. 5. Perform SDS–PAGE using 50–100 lg of cell lysate per sample. 6. Soak the gel in WTB for 5 min. Immerse the Immobilon-P transfer membrane in methanol for a few seconds to wet it and then soak it in WTB for 5 min. Sandwich the gel and membrane with two sheets of gel blot paper on either side, and perform Western transfer (2–3 h at 15 V using the semidry transfer apparatus). 7. Block the membrane by incubating with 200 ml 20% horse serum/WBB for 2 h at room temperature with gentle shaking. 8. Primary antibody incubation: Incubate the membrane overnight at 4 C with gentle shaking in 1:1000 dilution of phosphorylation-specific c-Jun antibody in 20% horse serum/WBB. 9. The following day, wash the membrane twice for 10 min in 100 ml 0.5% Tween 20/WBB at room temperature with shaking.
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10. Secondary antibody incubation: Incubate the membrane with anti-rabbit Ig secondary antibody diluted 1:10,000 in 20% horse serum/WBB for 30 min at room temperature with gentle shaking. 11. Wash the membrane four times for 15 min with 0.5% Tween 20/WBB at room temperature with shaking, and then for 5 min with WBB. 12. Develop the immunoblot using ECL reagents and detect the signal by exposing to X-ray film.
for 5 min in 2 ml TBS. To prevent exposure to light, wrap the six-well dishes in aluminum foil during the washes. 11. Lay the coverslips on top of a drop of Vectashield that is spotted onto glass slides and visualize the staining pattern by fluorescence microscopy. Store the slides in the dark to preserve the fluorescent staining.
3.4.3. Immunocytochemistry 1. Transfect COS-7 cells grown on glass coverslips [for some cell types the coverslips need to be treated with appropriate attachment factors, e.g., poly(L -lysine), collagen] in six-well dishes as described under Immunoblotting. 2. Forty-eight hours posttransfection aspirate the cell culture medium and wash the cells with ice-cold PBS (2 3 ml per well). 3. Fix the cells with 1 ml 3% paraformaldehyde/PBS for 10 min at 4 C. Wash the cells with TBST (2 ml per well) twice for 5 min at room temperature with shaking, and then rinse the cells with TBS. 4. Permeabilize the cells by incubating with 1 ml methanol (prechilled at )20 C) for 10 min at )20 C, and then wash twice for 10 min in 2 ml TBST at room temperature with shaking. 5. Blocking: Incubate the cells with 2 ml blocking buffer for 60 min at room temperature with gentle shaking. 6. Primary antibody incubation: Spot 35 ll of phosphorylation-specific c-Jun antibody (diluted 1:200 with blocking buffer) onto Parafilm and carefully lay the coverslips on top. Place the Parafilm with the coverslips in a damp environment (to prevent drying out, a 150-mm-diameter culture dish covered in aluminum foil and with damp paper towel attached to the inside of the lid can be used) overnight at 4 C. 7. The next day place the coverslips back into the sixwell tissue culture dish and wash three times for 5 min with 2 ml TBST with shaking, and once for 5 min with TBS. 8. Secondary antibody incubation (in the dark): Spot 35 ll of diluted Texas red-conjugated anti-rabbit Ig secondary antibody (diluted 1:100 in blocking buffer) onto Parafilm and carefully lay the coverslips on top. Incubate in the dark at room temperature for 45 min. All the subsequent steps should be performed in the dark due to the light sensitivity of the secondary antibody. 9. DNA staining: Place the coverslips back in the sixwell dish and rinse with TBS. Incubate the coverslips with DAPI DNA stain (diluted 1:10,000 in TBS) in the dark for 2 min at room temperature. 10. Wash the coverslips three times for 10 min in 2 ml TBST at room temperature with shaking and then
4. Discussion 4.1. Mutated enzymes that activate or inhibit signaling pathways The mutation of the activating phosphorylation sites on MKKs from serine and threonine residues to acidic residues to mimic the charge effect of phosphorylation often results in mutant proteins that, when expressed in cells, potently activate endogenous MAP kinases and downstream signaling events (Fig. 1). The first protocol utilizes such a mutant of MKK6 to selectively activate the p38 MAP kinase pathway. This approach has also been successfully used with MKK3, a second MKK that selectively activates p38 [12], and MEK1, an activator of the ERK pathway [25]. However, similar mutations in the JNK activators MKK4 and MKK7 have proved less useful due to their relatively weak effect in activating endogenous JNK in most cell lines. In addition, the mutation of the threonine and tyrosine phosphorylation sites to acidic residues on the MAP kinases themselves does not result in constitutively active mutants. A number of other approaches may be taken to generate activated signaling enzymes apart from mutating the activating phosphorylation sites. These include the deletion of regulatory domains or fusing the enzyme to other proteins that confer particular properties, for example, inducible oligomerization. An alternative approach to generate constitutively activated forms of ERK and JNK MAP kinases is to fuse them with their respective MKKs [26,27] (Fig. 1). The MEK1–ERK2 fusion protein behaves as a constitutively activated form of ERK2 in cells [26], while a MKK7– JNK1 fusion protein behaves as a constitutively activated form of JNK1 [27]. Mutated forms of MKKKs have also proved useful for activating MAP kinase pathways in cells. For example, deletion of the aminoterminal regulatory domains of Raf-1 [28] and MEKK1 [29,30] results in the constitutive activity of their catalytic domains. The Raf-1 mutant preferentially activates the ERK pathway [28] and the MEKK1 mutant preferentially activates the JNK pathway [29,30]. In some cases oligomerization of MKKKs is required for their activity. This has been exploited to generate systems for inducing MKKK oligomerization resulting in activated forms of the enzymes. The fusion of MKKKs to either a
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domain of the B subunit of bacterial DNA gyrase (GyrB) [31] (Fig. 1) or to the FKBP12 protein [32] allows the inducible oligomerization of the MKKKs in cells by the addition of coumermycin (which acts as a natural dimerizer of GyrB) or FK1012A (a synthetic dimerizer that binds to FKBP12), respectively. These oligomerization-inducible systems have been used to activate Raf-1 [31,32] and the MKKK ASK1 [33], a component of JNK and p38 signaling pathways. To specifically block signaling pathways, dominant negative mutant alleles can be created by mutating the activating phosphorylation sites on the enzymes to nonphosphorylatable residues. Dominant-negative mutants exert their effect by binding and sequestering their substrates or upstream signaling components. Mutation of the threonine and tyrosine phosphorylation sites on MAP kinases to alanine and phenylalanine residues, respectively, creates dominant negative mutants that have been used to specifically block signaling in a number of systems (e.g., Refs. [34–37]) (Fig. 1). Similarly, the overexpression of mutant forms of MKKs that have their serine and threonine phosphorylation sites mutated to alanine leads to the inhibition of signaling through their respective pathways (e.g., Refs. [12,38–40]) (Fig. 1). Catalytically inactivated forms of components of MAP kinase pathways are a second type of dominant negative mutant that can be used to block signaling (e.g., Refs. [41,42]). These usually feature the mutation of a conserved lysine residue in the catalytic site of the enzymes. The prolonged overexpression of constitutively activated or dominant negative mutants of signaling enzymes is sometimes toxic to cells, resulting in cell death. This makes their effect on transcription factor function difficult to ascertain. To try and circumvent these problems, inducible expression systems can be used. This involves generating stable transfected cell lines that express the mutant enzyme under the control of an inducible promoter. A number of inducible expression systems are commercially available including tetracycline-inducible systems (e.g., the Tet-On and Tet-Off systems from Clontech) and the ecdysone-inducible system (available from Invitrogen) [43]. In contrast to controlling the level of protein expression, other inducible systems regulate the activity of the protein. The GyrB- and FKBP12-inducible oligomerization systems mentioned previously are one example. A second example involves the fusion of mutant signaling enzymes to a mutated form of the hormone binding domain (HBD) of the estrogen receptor [44] (Fig. 1). The mutated HBD selectively binds to synthetic ligands such as 4-hydroxytamoxifen (4HT) rather than the natural ligands [44]. The fused enzyme is inactive in the absence of 4HT, but becomes rapidly activated on addition of 4HT to cells. The exact mechanism by which this occurs is not fully understood. In the absence of ligand the
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HBD binds to heat shock protein 90 (Hsp90). It is thought that Hsp90 may sterically block the fused enzyme from interacting with its substrates, thereby inhibiting its activity. The addition of ligand relieves Hsp90 binding to the HBD and allows the fused enzyme to become active [44]. This strategy has been used to successfully regulate the activity of components of MAP kinase signaling pathways as well as other signaling pathways [44]. It should be emphasized that the strategies that have been discussed will not work in every case and the best approach for a particular experiment needs to be determined empirically. A second caveat to these types of studies is that overexpression of the mutated enzymes in cells may trigger the activation of, or inhibit, nonphysiologically relevant signaling pathways. For example a number of kinase-inactive versions of protein kinases will block all signaling from upstream activators rather than being specific for their particular pathway. Therefore, it is critical to perform appropriate control experiments to establish the specificity of the mutant enzymes. 4.2. Protein/peptide inhibitors of signaling pathways. A few pharmacological agents that specifically block intracellular signaling pathways are commercially available. For example, the ERK MAP kinase signaling pathway can be blocked by inhibitors of MEK activity such as PD98059 (available from Calbiochem) and U0126 (available from Promega) [45], while a family of pyridinylimadazole-based drugs (the most widely used of which is SB203580, available from Calbiochem) can be used to inhibit the protein kinase activity of p38 MAP kinase isoforms a and b2 [46]. These drugs have been very useful for determining the roles of the ERK and p38 pathways in cells. However, they are not completely specific for their desired targets [15,45,46] so some care has to be taken in interpreting the data obtained. In the absence of specific pharmacological inhibitors for many signaling pathways, the use of protein/peptide inhibitors is proving to be a viable alternative for disrupting signaling in cells. There are no commercially available pharmacological inhibitors of JNK MAP kinase; therefore, the JBD of the scaffold protein JIP-1 has become an important tool for disrupting JNK signaling in cells. The success of this approach relies on the fact that the JIP-1 JBD has a much higher binding affinity for JNK than the transcription factor substrates of JNK, and thereby prevents JNK from associating with and phosphorylating its substrates [19]. It is likely that in the future the protein interaction motifs of other MAP kinase scaffold proteins [47–49] may also become useful tools for specifically blocking these signaling pathways.
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Protein or peptide sequences spanning the protein interaction motifs on protein kinases can also be generated to block the interaction of protein kinases with key regulatory proteins. For example, cell-permeable peptides have been used to inhibit the transcription factor NFjB by blocking the interaction of the regulatory protein NEMO (also named IKKc) with the IjBkinases (IKKs) IKKa and IKKb [50]. IjB binds to NF-jB and inhibits its ability to activate transcription. The phosphorylation of IjB by the IKK complex (IKKa, IKKb, and NEMO) targets it for degradation by the proteosome, leading to the release of NFjB, which can then activate gene transcription [51]. The cellpermeable peptides span the NEMO binding sites on IKKa and IKKb and block the interaction of NEMO with these protein kinases, leading to inhibition of IjB phosphorylation and NF-jB activation [50]. In addition to exploiting the high-affinity interactions between scaffold/regulatory proteins and signaling enzymes or transcription factors, cell-permeable peptides have been used to inhibit specific enzyme–transcription factor interactions. For example, the transcription factor NFAT is activated by dephosphorylation by the protein phosphatase calcineurin (also named protein phosphatase 2B), which results in nuclear translocation of NFAT and NFAT-mediated gene expression [52]. Calcineurin binding to NFAT is required for efficient dephosphorylation [52]. The introduction into cells of peptides spanning the calcineurin binding site on NFAT inhibits the ability of calcineurin to bind to and dephosphorylate NFAT, but does not affect the activity of calcineurin toward other substrates [53]. Subsequently, mutant NFAT peptides with increased affinity for calcineurin have been isolated by screening peptide libraries [54]. These behave as highly specific and potent inhibitors of NFAT-mediated gene transcription, and demonstrate the therapeutic potential of this approach [54]. A second advantage of the peptide approach is that it can be employed with cells that are difficult to transfect. For a more detailed discussion of protocols for the generation and use of cell-permeable peptides (see Refs. [55] and [56]). 4.3. Assays that measure transcription factor phosphorylation and activation The use of phosphorylation-specific antibodies and reporter gene experiments are two important assays for determining the phosphorylation status and the transcriptional activity of transcription factors, respectively. These assays can be combined with the tools discussed in the previous sections to determine the specificity of signaling pathways toward transcription factors in vivo. The reporter gene system described in the protocol features the transcriptional activation domain of ATF-2 fused to the GAL4 DNA binding domain. Repression
domains of transcription factors can also be fused to GAL4 to determine if signaling pathways regulate repression activity. When investigating repression domains a more active promoter (e.g., SV40 promoter) upstream of the luciferase gene may be required to be able to observe significant repression of luciferase expression. More complex reporter gene systems can be constructed to assay transrepression activities of transcription factors. For example a LexA-GAL4-luciferase reporter gene (which features tandem LexA and GAL4 DNA binding sites upstream of a minimal promoter and the firefly luciferase gene) allows a GAL4-repressor fusion to be assayed in the presence of a LexA-activator fusion [57]. Phosphorylation-specific antibodies are becoming the most widely used method to determine the phosphorylation status of proteins in cells. Many antibodies that recognize specific phosphorylation sites on transcription factors are commercially available. One major advantage of this approach over other methods in detecting phosphorylation in vivo is that it does not require the labeling of cells with radioactive phosphate and subsequent phosphopeptide mapping. Caveats associated with the approach are that it relies on the antibodies being highly specific for the modified sites, and second, the modification of nearby sites in vivo may prevent antibody recognition. In addition to phosphorylation, many transcription factors are subjected to other types of posttranslational modifications that regulate their activity, including acetylation and methylation. Specific antibodies that recognize these modifications have also become useful tools for understanding the regulation of transcription factors by signaling pathways.
5. Concluding remarks A number of approaches can be taken to elucidate the signaling pathways that target transcription factors in vivo. These include the use of gene-targeting strategies, small molecule inhibitors, and the two approaches described in this article: activated and dominant negative signaling enzymes, and protein/peptide inhibitors. This article has focused on applying these approaches to study the regulation of transcription factors by MAP kinase pathways; however, they can be applied equally well to many of the signaling pathways that target transcription factors (e.g., protein kinase B (PKB)/Akt pathways, the IjB-kinase complex, calcium/calmodulindependent protein kinase pathways, calcineurin/NFAT). Two methods for assaying the targeting of transcription factors by signaling pathways, modificationspecific antibodies and reporter gene assays, are also described. These assays can be employed in conjunction with activated/dominant negative signaling enzymes and protein/peptide inhibitors to provide a greater
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