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Originally published In Press as doi:10.1074/jbc.M312054200 on November 17, 2003

J. Biol. Chem., Vol. 279, Issue 6, 5008-5016, February 6, 2004
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c-Jun-NH2 Kinase (JNK) Contributes to the Regulation of c-Myc Protein Stability*

Dania Alarcon-Vargas and Ze'ev Ronai{ddagger}

From the Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, November 3, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In accord with the central role c-Myc plays in control of cell growth and death, the stability of this protein is tightly regulated. Although the NH2-terminal domain of c-Myc has been implicated in the regulation of its stability, c-Myc-S, which lacks this domain, is equally unstable, pointing to the role of additional domains in the regulation of c-Myc stability. Our former studies revealed that amino acids (aa) 127-189 of c-Myc are responsible for stress-induced stability of the c-Myc protein. This region of c-Myc shares homology with the {delta} domain of c-Jun, which is required for JNK association and subsequent targeting of c-Jun for ubiquitination under non-stressed growth conditions. Here we demonstrate that JNK associates with, and mediates, c-Myc ubiquitination and degradation. Addition of JNK increased the degree of c-Myc ubiquitination in in vitro ubiquitination reactions. Increased c-Myc stability following MEKK1/JNK stimuli is abolished upon mutation within the {delta}-like domain of c-Myc (aa 166-181), as well as deletion of aa 127-189. Significantly, inhibition of JNK expression via small interfering RNA increased c-Myc protein expression. Similarly, squelching JNK association with c-Myc by overexpression of a peptide corresponding to aa 127-189 of c-Myc increased endogenous c-Myc stability and elevated the fraction of cells within the G2/M phase of the cell cycle. In all, these findings point to the contribution of JNK to the regulation of c-Myc protein stability under normal growth conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The c-myc protooncogene is a basic helix-loop-helix leucine zipper transcription factor involved in the regulation of cell proliferation, differentiation, and apoptosis (reviewed in Refs. 1 and 2). As a critical regulator of these distinct cellular functions, expression of c-Myc is tightly regulated at multiple levels, including transcription, as well as mRNA and protein stability (1). c-Myc protein has a relatively short half-life of about 20-30 min (2-4), similar to that reported for other regulatory transcription factors, such as p53 (3, 5). Ubiquitin-dependent c-Myc protein degradation has been demonstrated (4) and can be blocked by treatment of cells with proteasome inhibitors (4, 6-9), which was also shown for n-Myc (10, 11). The amino-terminal domain of c-myc, and specifically, two highly conserved regions, box I and box II, have been implicated in regulation of c-Myc protein stability (4, 7). Intriguingly, a naturally occurring form of c-Myc, c-Myc S, which lacks box I and most of the transactivation domain, exhibits a similar half-life as full-length c-Myc, pointing to the role of other c-Myc protein regions in regulation of c-Myc protein stability (12). Furthermore, c-Myc S mediates a number of c-Myc-specific functions, including repression of transcription from several promoters, regulation of proliferation and apoptosis, as well as rescue of phenotypes in myc null fibroblasts (13-15). These results suggest that amino acids (aa)1 that lie downstream of those missing in c-Myc S (aa 1-100) are responsible for regulation of c-Myc protein stability. Recently, the F box protein Skp2, along with its associated E3 ligase complex, was shown to mediate the destabilization of c-Myc via box II and helix-loop-helix leucine zipper domains of c-Myc (16, 17).

The expression of c-myc is regulated differently in normal growth conditions than under conditions of cellular stress or in human cancers, albeit with different outcomes of c-myc function. For example, human Papillomavirus E6-16 high risk oncoprotein, but not the low risk E6-11 oncoprotein, was shown to induce the ubiquitin-dependent, proteasomal degradation of Myc (11), presumably acting to prevent apoptotic induction by c-Myc. However, significant increases in c-Myc protein half-life have been noted in human glioma and in Burkitt's lymphoma cell lines (4, 8, 18). The opposing level of c-Myc proteins in different tumor types suggests that regulation of c-Myc stability is adjusted depending on its cell type-specific function.

Point mutations frequently found to occur around Thr-58 and Ser-62 within the transactivation domain of c-Myc have been associated with increased c-Myc protein stability (4, 8, 18, 19). These mutational hotspot residues are phosphorylated by GSK3 and MAPK family members (20-24). Additionally, JNK has been shown to phosphorylate c-Myc Ser-62 and Thr-71 (25). Regulation of c-Myc protein stability as well as subsequent gene activation and S phase induction have been associated with Ras signaling (6, 19, 26). However, mutation of c-myc is not essential for its stabilization in certain Burkitt's lymphoma cells, suggesting that other mechanisms may contribute to the regulation of c-Myc protein stability (8).

The stability of several transcription factors, including c-Jun, ATF2, JunB, and p53, was shown to be affected by JNK (5, 27-30). JNK-targeted ubiquitination and degradation of these transcription factors takes place under normal, non-stressed conditions, when JNK is not active as a kinase (reviewed in Ref. 31). In all cases, JNK serves as an adaptor to an unknown ligase, different from those implicated in the regulation of the stability of these proteins (i.e. independent of Mdm2, in the case of p53; (5, 29)).

We previously identified a novel region of c-Myc from aa 127-189 involved in mediating an MEKK1-dependent, stress-induced increase in c-Myc protein stability (32). In this study, we further characterize the role of aa 127-189 in mediating altered c-Myc stability under normal growth conditions. Here we identify JNK as a targeting factor that increases the ubiquitin-dependent degradation of c-Myc via a {delta}-like domain on c-Myc. In addition, we developed a peptide of aa 127-189 of c-Myc capable of blocking endogenous c-Myc degradation by competing for JNK binding, resulting in altered cell cycle distribution.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—TIG-3, normal human lung fibroblasts, and NIH3T3, mouse embryo fibroblasts, were grown in Dulbecco's modified Eagle's medium (Mediatech, Inc.) supplemented with 10% calf serum (Invitrogen) and 100 units and 100 µg/ml penicillin/streptomycin (Mediatech, Inc.). MCF-7, breast carcinoma cells, 293T, human adenovirus-transformed kidney cells expressing SV40 large T antigen cells, and HeLa, cervical carcinoma cells, were grown in Dulbecco's modified Eagle's medium (Mediatech, Inc.) supplemented with 10% fetal bovine serum (Sigma) and 100 units and 100 µg/ml penicillin/streptomycin (Mediatech, Inc.). Cell cultures were maintained at 37 °C in 5% CO2.

Plasmid Constructs—The wild type Myc HA (MycWT) and Myc{Delta}C (deleted of amino acids 127-189) HA cloned in pCGN under the control of a cytomegalovirus-driven promoter were kindly provided by Dr. William P. Tansey (Cold Spring Harbor Laboratory). Myc deleted of aa 166-181 (Myc{Delta}166-181), Myc point mutation of Leu-176 and Leu-178 to Ala (Myc2PM), and 4-point mutant of Leu-176, Leu-178, Ser-186, and Glu-187 to Ala (Myc4PM) were created on the MycWT HA-tagged backbone using the QuikChange site-directed mutagenesis kit (Stratagene). MycWT tagged with the V5 epitope was kindly provided by Dr. Elizabeth Flinn (7). {Delta}MEKK1, constitutively active MEKK1 lacking amino acids 1-351, and TR-MEKK1, catalytically inactive MEKK1 with a mutation at Lys-432 to Met, were kindly provided by Dr. Audrey Minden (33). JNKK2-CAA, constitutively active JNKK2, and JNKK2-AA, catalytically inactive JNKK2, were kindly provided by Dr. Michael Karin (34). His MycWT and His Myc{Delta}C were subcloned from the pCGN vectors into BamHI and EcoRI sites of pTrcHis A bacterial expression plasmid (Invitrogen). MycWT was cloned into BamHI and EcoRI sites of pcDNA vector (Invitrogen) with two FLAG tags to create Flag-MycWT. pRETRO-SUPER (pRS-C) vector was kindly provided by Dr. Reuven Agami (35). JNK sequences were cloned into the BglII and HindIII sites of pRETRO-SUPER to create pRETRO-SUPER JNK (pRS-JNK). pBabe puro vector was kindly provided by Dr. Greg P. Nolan. GFP was cloned into the pBabe puro vector to produce pBabe GFP. pEF vector, driven by the elongation factor 1 promoter, was kindly provided by Dr. Patricia Cortes. HA tag, penetratin sequence, and GFP tag were cloned into the pEF vector to create vector GFP (V-GFP). Myc aa 127-189 was PCR-amplified from WT Myc sequence with BamHI and SalI restriction sites for subcloning into the GFP vector backbone to create HA Myc C peptide GFP (Myc-pepWT), and Myc aa 127-189 with point mutations at Leu-176 and Leu-178 to Ala were similarly cloned from Myc Leu-176,178 Ala into the pEF vector to create HA Myc LL-C peptide GFP (Myc-pep2PM) and from Myc aa 127-189 with point mutations at Leu-176, Leu-178, Ser-186, and Glu-187 to Ala from 4PM Myc to create HA Myc 4PM C peptide GFP (Myc-pep4PM). pMD.OGP encoding gag-pol and pMD.G encoding vesicular stomatitis virus G protein (VSV-G) plasmids were kindly provided by Dr. Adrian Ting (36). All constructs were verified for integrity by sequencing.

Bacterial Protein Expression—pTrcHis A MycWT and Myc{Delta}C were transformed into Escherichia coli strain BL21. Cultures were grown to OD 0.6 and induced with 1 mM isopropyl-{beta}-D-thiogalactopyranoside for 4 h at 37 °C. Cells were harvested by centrifugation at 6000 rpm for 10 min at 4 °C. The bacterial pellet was lysed in buffer with 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and 10 mM imidazole. After a brief sonication, the lysate was centrifuged at 15,000 rpm for 30 min at 4 °C. Cleared supernatant was then incubated with 50% Ni-NTA resin (Qiagen) on a rotator for 1 h at 4 °C. Myc proteins bound to the beads were washed and eluted against a buffer with 250 mM imidazole and dialyzed against a solution containing 50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM benzamidine, 0.1% {beta}-mercaptoethanol, 0.2% Nonidet P-40, and 5% glycerol. Proteins were again bound to 50% Ni-NTA resin (Qiagen), and then washed and analyzed for purity and concentration by SDS-PAGE and Coomassie Brilliant Blue staining using bovine serum albumin (Pierce) as a standard.

In Vitro Ubiquitination—Empty nickel NTA beads (Qiagen) or nickel beads bound to bacterially expressed MycWT or Myc{Delta}C (deleted of aa 127-189) cloned in pTrcA his vector (Invitrogen) were preincubated for targeting with control buffer (composed of 20 mM HEPES, pH 7.6, 50 mM KCl, 1 mM dithiothreitol, 0.25% Nonidet P-40, 5 mM NaF, 1 mM EGTA, 5 mM MgCl2, 0.25 mM phenylmethylsulfonyl fluoride); or with 100 µg of TIG-3 cell extract lysed with lysis buffer composed of control buffer with 350 mM NaCl and 20% glycerol; or with 2 µg of purified JNK (isolated from extracts by c-Jun protein coupled to GST beads) as indicated for 30 min at 37 °C, and then washed four times in control buffer and one time in 50 mM Tris, pH 7.4, 1 mM dithiothreitol. In vitro ubiquitination in the presence of ubiquitination buffer (50 mM Tris HCl, pH 8.0, 5 mM MgCl2, 0.5 mM dithiothreitol, 2 mM NaF, 3 µM okadaic acid), 2 µg of ubiquitin HA, 2 mM ATP, 1.5 mM ATP{gamma}S, and rabbit reticulocyte lysate depleted of JNK by preincubation with GST-c-Jun beads was performed for 1 h at 37 °C. The reaction was stopped using STOP buffer (8 M urea, pH 6.5, 0.1% Nonidet P-40), beads were washed three times in STOP buffer and one time with phosphate-buffered saline, 0.5% Triton X-100, and protein complexes were eluted in 3x SDS sample buffer. The proteins were separated on 8% SDS-PAGE gels, and the upper portion of the blot was probed with HA antibody (12CA5; Berkeley Antibody) to detect polyubiquitin HA complexes formed on c-Myc, whereas the lower portion of the blot was probed with c-Myc polyclonal antibody (Sigma) to confirm the loading of equal amounts of c-Myc in the reactions compared.

Transfection—TIG-3, normal human lung fibroblasts, were transfected with 4 µg of the Myc forms indicated under "Results" and 400 ng of the kinases indicated using LipofectAMINE Plus reagent, according to manufacturer's protocol (Invitrogen). MCF-7, breast carcinoma cells, and HeLa, cervical carcinoma cells, were transiently transfected with the indicated constructs (see Fig. 3) using LipofectAMINE Plus (Invitrogen). 293T, human adenovirus-transformed kidney cells expressing SV40 large T antigen, were transfected using calcium phosphate with gag-pol, VSV-G, and pRS plasmids as described (36). MCF-7, breast carcinoma cells, were transfected with 4 µg of the HA-GFP (V-GFP), or Myc-pepWT, Myc-pep2PM, or Myc-pep4PM, with and without 0.8 µg of pBabe puro using LipofectAMINE Plus (Invitrogen).



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  		FIG. 3.
A peptide corresponding to aa 127-189 of c-Myc binds to JNK and increases expression of endogenous Myc. As shown in A, TIG-3 cells were transfected with vector GFP or the peptides, as indicated in the figure. Whole cell extract was used in direct Western analysis (WCE lanes) and for immunoprecipitation with HA antibody (IP HA), as indicated in the figure. Proteins were separated on a 10% SDS-PAGE gel and immunoblotted (IB) with the antibodies indicated. As shown in B, HeLa cells were transfected with pcDNA FLAG-tagged MycWT (0.5 µg) and GFP peptides (1 µg), as indicated in the figure, and 24 h later, cells were fixed, permeabilized, and subjected to immunocytochemistry using polyclonal FLAG antibodies to detect FLAG-tagged MycWT. Normal rabbit Ig was used as a negative control.

 
Stable Cell Line Production—MCF-7 stable mass cultures were established by co-transfection of the peptides described above and V-GFP vector as control along with pBabe puro for selection using lipofectAMINE Plus (Invitrogen). Cells were selected using 1 µg/ml puromycin (Sigma) treatment for 2 weeks, and selected cells were sterile-sorted based on GFP expression to enrich for the GFP-positive population by MoFlo high speed cell sorter (Flow Cytometry Shared Research Facility, Mount Sinai). Whole cell extract was used in direct Western analysis (see Fig. 3, WCE lanes) and for immunoprecipitation using HA antibody (12CA5; Berkeley Antibody).

Cycloheximide Chase—Equal numbers of transfected MCF-7 cells (as outlined above) were plated. 24 h later, cells were treated with 20 µg/ml cycloheximide (Sigma) for 40 or 80 min or untreated (0 min). Cells were harvested, and proteins were prepared and separated on 10% SDS-PAGE gels. Immunoblot analysis was performed using c-Myc polyclonal antibody (Sigma). The blots were stripped and reprobed using HA antibody (12CA5; Berkeley Antibody), as well as monoclonal {beta}-actin antibody (Sigma).

Chemical Treatment—TIG-3 cells were treated 24 h after transfection with Me2SO (Sigma) or 40 µM MG132 (Peptides International) for 4 h before harvesting for co-immunoprecipitation.

Co-immunoprecipitation—TIG-3 cells were transiently transfected with V5-tagged c-Myc and were treated 24 h after transfection with Me2SO (Sigma) or 40 µM MG132 (Peptides International) for 4 h before harvesting. Immunoprecipitation (IP) of V5 Myc was performed using V5 antibody (Invitrogen), IP of JNK was performed using monoclonal JNK antibody (Pharmingen), and IP of HA was performed using HA antibody (Berkeley Antibody) on 1 mg of total protein at 4 °C for 16 h. Protein G agarose beads (Invitrogen) were incubated with reaction mixtures at 4 °C for 2 h. Immune complexes were washed in Dulbecco's phosphate-buffered salt solution (Mediatech, Inc.) two times followed by three times without 0.5 M lithium chloride (Sigma). Bound proteins were eluted by boiling in SDS-sample buffer and separated on 10% SDS-PAGE gels, blotted onto nitrocellulose membranes, and probed with Myc polyclonal antibody (RDI). The blot was then stripped and incubated with JNK monoclonal antibody (666; Pharmingen) or JNK polyclonal antibodies (Pocono Rabbit Farm, Poconos, PA).

Small Interfering RNA Infection—3T3 cells were infected for 16 h with 4 µg/ml polybrene and retroviral supernatant collected from 293T cells co-transfected with gag-pol and VSV-G along either with empty pRS vector (pRS-C) or with pRS vector containing small interfering RNA against JNK (pRS-JNK) prepared as described (36). Following change to fresh media, cells were harvested after 48 h. 100 µg of cell lysate was run on a 10% SDS-PAGE gel and immunoblotted using Myc polyclonal antibody (Sigma). The blot was stripped and reprobed with antibodies to JNK (666; Pharmingen) to confirm reduction of JNK, as well as with {beta}-actin monoclonal antibody (Sigma) to confirm equal protein loading. Results shown are representative of three experiments.

Immunoblotting—Proteins were prepared 48 h after transfection using lysis buffer (control buffer with 350 mM NaCl and 20% glycerol) with protease inhibitor mixture (Sigma), and protein concentrations were determined using Coomassie protein assay reagent (Pierce) and bovine serum albumin (Pierce) as standard. Proteins were boiled in SDS-sample buffer and separated on SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane. The blots were blocked with 5% non-fat dry milk in TBST (50 mM Tris-Cl, pH 7.6, 100 mM NaCl, and 0.1% Tween 20) at room temperature for 1 h and incubated either for 1 h at room temperature or for 16 h at 4 °C with the indicated antibodies followed by washes in TBST and incubation with horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody at a 1:10,000 dilution (Amersham Biosciences) for 1 h at room temperature. Following washes in TBST, bands were visualized using enhanced chemiluminescence (ECL) according to manufacturer's instructions (Amersham Biosciences).

Antibodies—Immunoblot for HA-tagged proteins was performed using monoclonal HA antibody (12CA5; Berkeley Antibody). Immunoblot for bacterially expressed Myc and endogenous c-Myc was performed using polyclonal Myc antibody (Sigma), unless otherwise indicated. Immunoblot of JNK was performed using monoclonal antibodies to JNK (666; Pharmingen) or JNK polyclonal antibody (Pocono Rabbit Farm). Immunoblot for {beta}-actin was performed using monoclonal antibodies to {beta}-actin (Sigma). Immunoprecipitation of V5-Myc complex was performed using monoclonal V5 antibody (Invitrogen).

Immunocytochemistry—HeLa cells plated on coverslips in 6-well dishes were transfected with 0.5 µg of pcDNA FLAG-MycWT and 1 µg of GFP peptides using LipofectAMINE (Invitrogen). 24 h later, cells were fixed in 3% paraformaldehyde, 2% sucrose in phosphate-buffered saline for 10 min at room temperature, permeabilized in 0.5% Triton X-100, 3 mM MgCl2, and 6.8% sucrose for 5 min on ice, and blocked for 1 h in 5% bovine serum albumin in phosphate-buffered saline at room temperature. Cells were incubated with FLAG polyclonal primary antibody diluted 1:500 (Sigma) or normal rabbit Ig (Molecular Probes) as a negative control for 30 min at room temperature, and then cells were washed and incubated with Alexa red 568 secondary antibody (Molecular Probes) diluted 1:1,000 for 30 min at room temperature. Cells were washed and stained with 4',6-diamidino-2-phenylindole (Sigma) for 1 min at room temperature to stain DNA and visualize the nucleus. Staining was visualized using a four-channel UV confocal laser scanning microscope (Microscopy Shared Research Facility, Mount Sinai).

FACS Analysis—Duplicate sets of TIG-3 cells were transfected with the indicated constructs using LipofectAMINE Plus (Invitrogen). 24 h later, one set of cells was trypsinized, fixed with ice-cold ethanol, and stained with propidium iodide (PI). Flow cytometry analysis was performed using a FACScalibur (BD Biosciences) and analyzed with CellQuest software. The control panel (see Fig. 6, No GFP) represents a profile obtained with cells that were transfected with empty vector. The other profiles shown reflect GFP-positive, PI-positive cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A {delta} Domain-like Region Is Required for MEKK1-mediated Increase in c-Myc Expression—Using a panel of c-myc deletion mutants in which successive regions of 60 amino acids were deleted, we have previously identified aa 127-189 as the region of c-Myc that mediates an increase in c-Myc protein stability following MEKK1 stimuli (32). Using the Block Maker homology search program (37), we identified an area within aa 166-181 of c-Myc that shared significant homology (50% similarity) with the {delta} domain of Jun family members (aa 31-59; Fig. 1A). This finding raised the possibility that JNK may associate with c-Myc through the {delta} domain-like sequence, resulting in targeting of c-Myc for ubiquitination and degradation, similar to its effects on c-Jun, JunB, ATF2, and p53 (5, 27-30). To further address this possibility, we sought to identify the specific residues involved in mediating the effect of MEKK1 on c-Myc protein levels. Point mutation (PM) of Leu-40 and Leu-42 and Leu-49 and Lys-50 (Leu-50 and Lys-51 in JunD) within the {delta} domain of the Jun family proteins to alanine abrogates binding of JNK (38). Based on homology with the c-Myc sequence, we deleted aa 166-181 of c-Myc, thereby creating a more defined deletion of the region that resembles the {delta} domain of c-Jun. We also mutated either two leucines (Leu-176 and Leu-178) of c-Myc to create Myc2PM or 4 aa (Leu-176, Leu-178, Ser-186, and Glu-187) to create Myc4PM, sites that are homologous to the residues shown previously to be critical for JNK binding to c-Jun (Fig. 1A). To assess the MEKK1 responsiveness of the more defined mutant Myc forms, we co-transfected TR-MEKK1, a kinase-dead form of MEKK1, or {Delta}MEKK, a constitutive active MEKK1, with either wild type or mutant Myc forms, as indicated in Fig. 1B. Deletion of Myc aa 127-189 (Myc{Delta}C) resulted in loss of MEKK1-dependent increase in Myc expression (Fig. 1B), consistent with our previous studies (32). In addition, Myc mutants, Myc{Delta}166-181, Myc2PM, and Myc4PM, also failed to increase expression upon co-transfection of {Delta}MEKK1 (Fig. 1B). These data suggest that the {delta} domain-like region of c-Myc is required for mediating an MEKK1-dependent increase in c-Myc expression.



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  		FIG. 1.
Stress kinase-mediated increases in c-Myc protein levels require the {delta} domain-like region of c-Myc. In A, a schematic outline of the human c-Myc protein highlights the regions involved in stress kinase-mediated changes in c-Myc protein levels and the area of homology with the Jun family proteins revealed by Block Maker homology search. The C peptide of c-Myc consists of aa 127-189. The {delta} domain-like region corresponds to aa 166-181 ({Delta}). Arrowheads point to the four residues (Leu-176, Leu-178, Ser-186, and Glu-187 of c-Myc and Leu-40, Leu-42, Leu-49, Lys-50 of Jun proteins, except JunD Leu-50 and Lys-51) that are required for JNK docking. As shown in B, TIG-3, normal human lung fibroblast cells, were transfected with 4 µg of pcDNA (mock), HA-tagged c-Myc wild type (MycWT), or the mutant forms of Myc as indicated (Myc{Delta}C, deleted of aa 127-189; Myc{Delta}166-181, lacking aa 166-181; Myc2PM, Myc mutated at L176,178A; or Myc4PM, mutated at Leu-176, Leu-178, Ser-186, Glu-187 to Ala), along with 400 ng of catalytically inactive MEKK1, TR-MEKK1, or constitutive active MEKK1, {Delta}MEKK1, as indicated. Proteins were prepared 24 h after transfection, and 200 µg of total cell lysate was separated on a 10% SDS-PAGE gel followed by immunoblotting (IB) using antibodies to the HA tag. As shown in C, TIG-3 cells were transfected with 4 µg of HA-tagged MycWT along with either 400 ng of JNKK2-AA, a catalytically inactive JNKK2, or 400 ng of JNKK2-CAA, a constitutive active JNKK2. Proteins were prepared and analyzed, as indicated in panel B. Blots were stripped and reprobed with antibodies to {beta}-actin. As shown in D, TIG-3 cells were transfected with 4 µg of V5-tagged MycWT, and 24 h after transfection, cells were treated with Me2SO as control (- lanes) or 40 µM MG132 (+ lanes) for 4 h before proteins were prepared. 10% input represents 100 µg of total protein cell lysate, of which 1 mg was used in the IP. IP of V5 Myc was performed using V5 antibody, and IP of JNK was performed using monoclonal JNK antibody. Immunoblot of Myc was performed using c-Myc polyclonal antibody (RDI). The blot was subsequently stripped and incubated with monoclonal antibodies to JNK. Results shown are representative of three experiments.

 
Since MEKK1 effects are mediated via JNK, the ability of a constitutive active JNKK2 (JNKK2-CAA) to increase Myc levels was assessed. Indeed, overexpression of constitutive active JNKK2-CAA was able to increase c-Myc expression levels unlike its kinase-dead counterpart, JNKK2-AA (Fig. 1C). JNK-dependent targeting of its transcription factor substrates for ubiquitination occurs under non-stressed normal growth conditions where JNK is inactive as a kinase (27, 28, 38). Accordingly, mutant forms of Myc exhibit higher basal expression levels as compared with MycWT (Fig. 1B). This finding provided the first indication for the possible role of JNK in regulation of basal c-Myc stability. To further assess this possibility, we sought to determine whether JNK and c-Myc associate, which is a prerequisite for JNK to affect the stability of its associated proteins (27). IP of c-Myc from cells that overexpress this protein allowed the identification of JNK as a Myc-associated protein. Similarly, IP of JNK identified Myc as a JNK-bound protein (Fig. 1D). Importantly, the identification of this complex required the presence of proteasome inhibitor, further pointing to the unstable nature of this complex. These results allude to the involvement of JNK in the regulation of c-Myc stability.

JNK Increases the in Vitro Ubiquitination of c-Myc via aa 127-189—To further assess the possible role of JNK in the regulation of c-Myc stability, we have carried out in vitro ubiquitination reactions. Bacterially expressed MycWT or Myc{Delta}C were incubated either with extracts from TIG-3 cells or with purified JNK protein prior to the in vitro ubiquitination reaction. Preincubation with cell extract resulted in substantial ubiquitination of c-Myc, which was lower when c-Myc lacking aa 127-189 (Myc{Delta}C) was used as a substrate (Fig. 2A, compare lanes 3 and 6). This finding suggests that aa 127-189 contribute to the ubiquitination of the c-Myc protein; however, such ubiquitination also relies on other domains of c-Myc. Notably, addition of purified JNK to the in vitro ubiquitination reaction resulted in the ubiquitination of MycWT, but not Myc{Delta}C (Fig. 2A, compare lanes 4 and 7). These results provide direct evidence for the role of JNK in targeting the ubiquitination of c-Myc and the requirement of aa 127-189 of c-Myc for JNK targeting. The degree of c-Myc ubiquitination by JNK is lower, when compared with cell extracts, suggesting that JNK is one of the components involved in the ubiquitination of c-Myc and that cooperating factors, missing from the purified JNK fraction, limit the degree of Myc ubiquitination. Since JNK used in these reactions was immunoprecipitated from JNK overexpressing cells, we assume that either that the JNK-associated ligase was co-immunoprecipitated or that such a ligase is present in the reticulocyte lysates (depleted of JNK), thereby enabling Myc ubiquitination.



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  		FIG. 2.
JNK targeted ubiquitination of c-Myc requires aa 127-189. As shown in A, empty nickel beads or those bound to bacterially expressed MycWT or Myc{Delta}C were preincubated with control buffer, with TIG-3 cell extract (100 µg), or with affinity-purified JNK (2 µg) for 30 min at 37 °C followed by extensive washes. In vitro ubiquitination was carried out in the presence of ubiquitin HA (UB-HA) and rabbit reticulocyte lysate, which was immunodepleted of JNK for 1 h at 37 °C. The reaction was stopped, beads were washed extensively, and protein complexes were eluted and separated on an 8% SDS-PAGE gel. The upper portion of the blot was probed with HA antibody to detect polyubiquitin HA complexes formed on c-Myc, whereas the lower portion of the blot was probed with c-Myc polyclonal antibody. IB, immunoblot. As shown in B, 3T3 cells were infected with supernatant expressing either empty pRS control vector (pRS-C) or pRS vector containing small interfering RNA against JNK (pRS-JNK). Proteins were prepared 48 h after infection and subjected to immunoblot analysis using Myc polyclonal antibody. The blot was stripped and reprobed with antibodies to JNK, as well as with {beta}-actin monoclonal antibody. Results shown are representative of three experiments.

 
RNA Interference of JNK Increases c-Myc Expression—To confirm the role of JNK in the regulation of c-Myc stability, we employed retroviruses that express small interfering RNA oligonucleotides that are designed to selectively inhibit JNK1 and JNK2 (pRS-JNK) or control pRS-C (35). Upon infection of 3T3 cells with supernatant expressing pRS-JNK, but not pRS-C, we were able to reduce the expression of endogenous JNK protein (Fig. 2B). Cells that exhibited reduced JNK expression were also found to possess elevated expression of endogenous c-Myc as compared with pRS-C-infected cells (Fig. 2B). This increase in endogenous c-Myc levels provides an important confirmation for the role of JNK in regulation of c-Myc stability. Similarly, inhibition of JNK expression by antisense oligonucleotides (39) also resulted in increased c-Myc expression (data not shown).

A Peptide of c-Myc aa 127-189 Binds to JNK and Increases Endogenous c-Myc Expression—Although the use of JNK RNA interference, which increases expression of endogenous c-Myc, provides compelling evidence for the role of JNK in the regulation of c-Myc stability, we cannot exclude the possibility that the effect of JNK may be indirect, given the large number of target molecules and signaling cascades regulated by JNK. Therefore, we have conducted additional experiments, which relied on the JNK binding domain of c-Myc to selectively address possible changes in c-Myc stability upon abrogating association with JNK. To achieve this, we cloned WT or mutant (2PM and 4PM) forms of c-Myc peptide (aa 127-189), which contains the {delta} domain-like region of c-Myc implicated in JNK association, into a vector containing an amino-terminal HA tag followed by a nuclear localization sequence to ensure expression in the nucleus and GFP, which is driven by the strong elongation factor 1 (EF1) promoter.

Possible changes in the expression of endogenous c-Myc protein were monitored upon expression of WT or mutant peptides in normal human fibroblasts (TIG-3), which have a relatively low basal level of endogenous c-Myc. Expression of endogenous c-Myc increased in cells that express Myc-pepWT, when compared with control vector (Fig. 3A). Notably, mutant Myc-pep4PM failed to increase c-Myc expression (Fig. 3A). However, Myc-pep2PM was still capable of increasing endogenous c-Myc expression (Fig. 3A). We next tested whether the peptide, which contains a {delta} domain-like sequence expected to mediate an interaction with JNK, would block the association of JNK with endogenous c-Myc by squelching. To this end, WT or mutant forms of the Myc peptide were transfected into TIG-3 cells, and lysates were subject to co-immunoprecipitation with HA antibody against the tagged peptides followed by immunoblot with antibodies to JNK. Although immunoprecipitation of the Myc-pepWT identified JNK2 as a Myc peptide-associated protein, neither vector GFP nor mutant Myc peptides were able to associate with JNK (Fig. 3A). The lack of association between either Myc-pep2PM or Myc-pep4PM with JNK is consistent with previous observations that point mutations of the corresponding residues of c-Jun to alanine abrogates JNK association (38). The specific association of the Myc-pepWT with JNK2 suggests that the increase in endogenous c-Myc expression is achieved via competition for JNK binding. The lack of association of Myc-pep4PM with JNK is consistent with the inability of this peptide to increase endogenous c-Myc protein levels (Fig. 3A). However, Myc-pep2PM resulted in an increase in endogenous c-Myc, despite the lack of any significant binding with JNK, suggesting that other cellular factors (i.e. the actual ligase) may be out-competed by this peptide.

To determine whether the cellular localization of Myc is altered in response to peptide overexpression, we transiently co-transfected FLAG-tagged WT Myc and HA peptides with a GFP tag. The GFP-Myc peptides were localized in the nucleus, as expected upon expression as a fusion protein with a nuclear localization signal (Fig. 3B). The FLAG-tagged WT Myc was found in the nucleus along with Myc-pepWT as well as Myc-pep2PM and Myc-pep4PM mutants (Fig. 3B), similar to what was observed upon expression of the control vector GFP (data not shown). Additionally, FLAG-Myc{Delta}C, Myc2PM, and Myc4PM were also found in the nucleus when co-transfected with Myc-pepWT GFP (data not shown). These data demonstrate that the Myc peptides are expressed in the nucleus where c-Myc is also found and that the effect of these peptides on c-Myc is not due to altered subcellular distribution.

Expression of Myc aa 127-189 Increases Endogenous c-Myc Stability—We next determined whether increased steady state levels of c-Myc, seen upon expression of Myc peptides, is due to increased c-Myc stability. Cycloheximide chase experiments revealed the half-life of endogenous c-Myc to be about 30 min in GFP-expressing cells (Fig. 4), similar to what has been reported previously (2-4), indicating that expression of vector GFP does not have an effect on endogenous c-Myc protein stability. Expression of Myc pepWT results in a prolonged half-life of endogenous c-Myc over 80 min (Fig. 4). Expression of Myc-pep2PM also increased Myc stability, albeit to a lesser degree (Fig. 4). Notably, expression of Myc-pep4PM did not increase the half-life of c-Myc. These data indicate that the Myc-pepWT and to a lesser degree Myc-pep2PM increase c-Myc protein stability. The mutant Myc-pep4PM, which is defective in JNK binding, also fails to increase c-Myc steady state levels and is unable to affect the half-life of the endogenous c-Myc protein. Along these lines, each of the peptides that failed to associate with JNK (Fig. 3A), also exhibited prolonged half-lives of up to 80 min (Fig. 4). Significantly, the Myc-pepWT protein decays with a half-life of about 30 min, similar to that reported for full-length Myc protein (Fig. 4). This suggests that aa 127-189 of c-Myc are sufficient for targeting by JNK for degradation, as seen for the full-length protein. Altogether, these results identify the contribution of JNK to the stability of the c-Myc protein, which can be abrogated by introduction of a peptide of Myc aa 127-189 that is able to associate with JNK, resulting in degradation of the peptide while blocking the targeting of endogenous c-Myc for degradation.



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  		FIG. 4.
Expression of c-Myc aa 127-189 peptide increases endogenous c-Myc stability. MCF-7 cells were transfected with vector GFP or the peptides as indicated. 48 h later, cells were treated with 20 µg/ml cycloheximide (CHX) for the time periods indicated. Proteins prepared at the indicated time points (100 µg) were subjected to immunoblot (IB) analysis using antibodies indicated in the figure. *ns indicates the position of a non-specific band detected in the immunoblot with HA antibody.

 
Increased c-Myc Expression Affects Cell Cycle Distribution—To determine whether the increased expression of c-Myc resulting upon peptide overexpression retained Myc functionality, we monitored possible changes in cell cycle distribution, which were reported to be altered in cells overexpressing Myc (38). To this end, we carried out FACS analysis of normal human fibroblasts (TIG-3), which were transfected with control or Myc peptide constructs. Notably, expression of Myc-pepWT and Myc-pep2PM resulted in a significant decrease (Fig. 5, *1 = p < 0.01) in the fraction of cells found in the G0/G1 phases (51% of vector GFP-expressing cells as compared with 34% of both Myc-pepWT and Myc-pep2PM GFP-expressing cells). Myc-pep4PM GFP-expressing cells exhibit distribution that resembled that seen in controls (48%: Fig. 5A). Similarly, a concomitant significant increase (*2 = p < 0.01) in the fraction of cells within the G2/M phases of the cell cycle was found with Myc-pepWT and Myc-pep2PM GFP-expressing cells as compared with controls (30% of vector GFP-expressing cells as compared with 52% of Myc-pepWT and 53% of Myc-pep2PM GFP-expressing cells), whereas Myc-pep4PM GFP-expressing cells exhibited distribution similar to control (32%; Fig. 5A). The S phase fraction remained relatively unchanged (Fig. 5A). The profiles of the GFP-negative, PI-positive cells closely resemble that of the negative control (Fig. 5A), demonstrating that expression of GFP alone did not alter the cell cycle distribution in any significant manner (data not shown). Additionally, the expression of exogenous full-length c-Myc caused changes in cell cycle distribution similar to the Myc-pepWT GFP-expressing cells (data not shown). A duplicate set of transfected TIG cells was subject to Western immunoblot analysis to confirm the changes in endogenous c-Myc levels upon expression of the corresponding peptides (Fig. 5B). These findings suggest that increased c-Myc expression that results upon squelching of JNK targeting retains the ability of c-Myc to mediate cellular changes seen upon its elevated expression.



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  		FIG. 5.
Elevated Myc expression increases fraction of cells within the G22/M phase of the cell cycle. As shown in A, a duplicate set of TIG-3 cells was transfected with the indicated constructs, and 24 h later, one set of cells was harvested, fixed, and stained with PI followed by FACS analysis using a FACScalibur (BD Biosciences) and CellQuest software. The No GFP control panel represents a profile obtained with cells that were transfected with empty vector. The other profiles shown reflect GFP-positive, PI-positive cells. *1 and *2 indicate significant p values > 0.01 as compared with the control No GFP panel, according to two-tailed Welch's t test (InStat Program). As shown in B, the second set of cells was harvested 24 h after transfection, and total proteins (200 µg) were subjected to immunoblot analysis using the antibodies indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The current study identifies the role of JNK in the regulation of c-Myc protein stability. The role of JNK as an adaptor protein, which mediates c-Myc ubiquitination and degradation, is supported by findings that demonstrate that: (a) inhibition of JNK expression by RNA interference results in an increase of endogenous c-Myc expression; (b) addition of JNK to in vitro ubiquitination reactions induces c-Myc ubiquitination in a manner that depends on aa 127-189; (c) a peptide corresponding to the JNK docking site, which contains a {delta}-like domain, binds to JNK and exhibits stability similar to that of full-length c-Myc; (d) expression of Myc peptide increases endogenous c-Myc protein stability by competing for JNK targeting; (e) JNK and MEKK1-signaling increase Myc expression in a manner dependent on the presence of aa 127-189 of c-Myc. Altogether, our data establish the role of JNK in the regulation of c-Myc protein stability.

JNK-targeted ubiquitination and degradation of its associated proteins has been demonstrated previously by us and others for c-Jun, ATF2, JunB, and p53 (5, 27-31). In all cases, this targeting occurs under non-stressed, normal growth conditions, when JNK is inactive as a kinase, as such targeting can be efficiently mediated by a kinase-dead form of JNK (28). Importantly, JNK targeting depends on its ability to associate with the protein, even if it is not capable of phosphorylating the substrate, as shown for JunB (27). Although former studies have demonstrated that JNK phosphorylates c-Myc on Ser-71 and on Ser-62 (25), these phosphoacceptor sites can be also phosphorylated by other MAPKs (20-24), suggesting that stabilization of Myc upon stress or cytokine stimuli may occur via alternate pathways, all of which would inactivate JNK-dependent targeting of c-Myc ubiquitination and degradation.

Clearly, JNK is only one of the adaptor molecules important for regulation of c-Myc stability. This notion is supported by the finding that c-Myc that no longer binds JNK (Myc{Delta}C) is still targeted for ubiquitination in vitro, albeit to a substantially lower degree. Furthermore, the ability of the Myc peptide mutated on 2 leucines (Myc-pep2PM), which are critical for the {delta} domain-like conformation and for JNK binding, to also increase c-Myc stability, albeit to a lower degree as compared with Myc-pepWT, suggests that other cellular proteins may compete with JNK in the regulation of c-Myc stability.

The domain of c-Myc studied here is different from those currently implicated in the regulation of c-Myc stability, which have focused on the amino-terminal box I region of c-Myc that harbors the well characterized phosphoacceptor sites Thr-58 and Ser-62. It is plausible that the C peptide studied here is responsible for proper conformation of the amino-terminal region, and as such, plays a central role in the regulation of c-Myc stability. This possibility is supported by the finding that c-myc S, a form of myc initiated from downstream methionines as a result of a leaky translational scanning mechanism, which lacks the amino-terminal 100 amino acids, is as unstable as full-length c-Myc (12).

Mutation of Leu-176, albeit in the context of other mutations, has been reported in a Burkitt's lymphoma-derived allele, B17, with altered c-Myc activity (41, 42). Significantly, a mutation of Leu-178 was reported, also as one of a number of other mutations, in a CA46 Burkitt's lymphoma-derived cell line to result in the extension of the half-life of mutated c-Myc protein (8, 18). These observations indirectly support the model proposed in our study concerning the domain of c-Myc involved in mediating the role of JNK in the regulation of c-Myc stability.

As JNK is not expected to have E3 ligase activity, but rather serves as a targeting (adaptor) molecule, one question that remains to be addressed is the identity of the E3 ligase for c-Myc. Gross-Mesilaty et al. (11) reported increased ubiquitination of c-Myc by human papillomavirus E6 complex with E6-AP E3 ligase, which resembles the targeting of p53 for ubiquitination and degradation by this complex (43). Interestingly, the docking site of E6 to human p53 (aa 92-112) overlaps with the residues reported to mediate regulation of human p53 stability by JNK (aa 97-116; 5). These findings point to a possible interplay between E6 and JNK in regulation of the ubiquitination and degradation of their associated proteins, including p53 and c-Myc. It is expected that the cellular E3 ligase will be found and shown to regulate stability of c-Myc. Skp2 is among proteins implicated in the regulation of c-Myc stability (16, 17), which also require domains important for JNK association. The relationship between Skp2 and JNK as well as additional F box proteins is expected to shed further light on the regulation of c-Myc stability.

Expression of the C peptide of c-Myc increased endogenous c-Myc expression, resulting in a greater proportion of cells in the G2/M phases of the cell cycle. A number of c-Myc target genes have been reported to affect cells in G2, including p53 and Cdc25A (40). Induction of apoptosis by etoposide treatment has been reported to occur at times of maximum G2/M content of c-Myc-positive cultures as opposed to c-Myc null cells (44), pointing to the possible use of the Myc peptide studied here for sensitization of selective cell types to treatment. Altogether, the characterization of JNK in regulation of c-Myc stability adds an additional level of complexity to our understanding of mechanisms underlying c-Myc stability and activities.


   FOOTNOTES
 
* This work was supported by National Research Service Award Grant CA85197 (to D. A.-V.) and National Cancer Institute Grant CA78419 (to Z. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Fax: 212-849-2446; E-mail: zeev.ronai{at}mssm.edu.

1 The abbreviations used are: aa, amino acids; JNK, c-Jun-NH2 kinase; MAPK, mitogen-activated protein kinase; MEKK, MAPK/extracellular signal-regulated kinase kinase; HA, hemagglutinin; WT, wild type; PM, Point mutation; GFP, green fluorescent protein; V-GFP, vector GFP; Ni-NTA, nickel-nitrilotriacetic acid; IP, immunoprecipitation; PI, propidium iodide; FACS, fluorescence-activated cell sorter; E3, ubiquitin-protein isopeptide ligase; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate); VSV-G, vesicular stomatitis virus G protein. Back


   ACKNOWLEDGMENTS
 
We thank Serge Fuchs for advice during the early phase of these studies and Lu-Hai Wang and Mitchell Goldfarb as well as members of the Ronai Laboratory for extensive discussions. We also thank Anindita Bhoumik for technical assistance, advice, and support. We are grateful to William P. Tansey, Michael Karin, and Roger Davis for providing us with the constructs used in the course of these studies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Potter, M., and Marcu, K. B. (1997) Curr. Top. Microbiol. Immunol. 224, 1-17[Medline] [Order article via Infotrieve]
  2. Luscher, B., and Eisenman, R. N. (1988) Mol. Cell Biol. 8, 2504-2512[Abstract/Free Full Text]
  3. Ciechanover, A., DiGiuseppe, J. A., Bercovich, B., Orian, A., Richter, J. D., Schwartz, A. L., and Brodeur, G. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 139-143[Abstract/Free Full Text]
  4. Salghetti, S. E., Kim, S. Y., and Tansey, W. P. (1999) EMBO J. 18, 717-726[CrossRef][Medline] [Order article via Infotrieve]
  5. Fuchs, S. Y., Adler, V., Buschmann, T., Yin, Z., Wu, X., Jones, S. N., and Ronai, Z. (1998) Genes Dev. 12, 2658-2663[Abstract/Free Full Text]
  6. Sears, R., Leone, G., DeGregori, J., and Nevins, J. R. (1999) Mol. Cell 3, 169-179[CrossRef][Medline] [Order article via Infotrieve]
  7. Flinn, E. M., Busch, C. M., and Wright, A. P. (1998) Mol. Cell Biol. 18, 5961-5969[Abstract/Free Full Text]
  8. Gregory, M. A., and Hann, S. R. (2000) Mol. Cell Biol. 20, 2423-2435[Abstract/Free Full Text]
  9. Gavine, P. R., Neil, J. C., and Crouch, D. H. (1999) Oncogene 18, 7552-7558[CrossRef][Medline] [Order article via Infotrieve]
  10. Bonvini, P., Nguyen, P., Trepel, J., and Neckers, L. M. (1998) Oncogene 16, 1131-1139[CrossRef][Medline] [Order article via Infotrieve]
  11. Gross-Mesilaty, S., Reinstein, E., Bercovich, B., Tobias, K. E., Schwartz, A. L., Kahana, C., and Ciechanover, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8058-8063[Abstract/Free Full Text]
  12. Chen, L., Smith, L., Accavitti-Loper, M. A., Omura, S., and Bingham Smith, J. (2000) Arch. Biochem. Biophys. 374, 306-312[CrossRef][Medline] [Order article via Infotrieve]
  13. Mateyak, M. K., Obaya, A. J., Adachi, S., and Sedivy, J. M. (1997) Cell Growth & Differ. 8, 1039-1048[Abstract]
  14. Xiao, Q., Claassen, G., Shi, J., Adachi, S., Sedivy, J., and Hann, S. R. (1998) Genes Dev. 12, 3803-3808[Abstract/Free Full Text]
  15. Spotts, G. D., Patel, S. V., Xiao, Q., and Hann, S. R. (1997) Mol. Cell Biol. 17, 1459-1468[Abstract]
  16. Kim, S. Y., Herbst, A., Tworkowski, K. A., Salghetti, S. E., and Tansey, W. P. (2003) Mol. Cell 11, 1177-1188[CrossRef][Medline] [Order article via Infotrieve]
  17. von der Lehr, N., Johansson, S., Wu, S., Bahram, F., Castell, A., Cetinkaya, C., Hydbring, P., Weidung, I., Nakayama, K., Nakayama, K. I., Soderberg, O., Kerppola, T. K., and Larsson, L. G. (2003) Mol. Cell 11, 1189-1200[CrossRef][Medline] [Order article via Infotrieve]
  18. Bahram, F., von der Lehr, N., Cetinkaya, C., and Larsson, L. G. (2000) Blood 95, 2104-2110[Abstract/Free Full Text]
  19. Sears, R., Nuckolls, F., Haura, E., Taya, Y., Tamai, K., and Nevins, J. R. (2000) Genes Dev. 14, 2501-2514[Abstract/Free Full Text]
  20. Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285[Abstract/Free Full Text]
  21. Seth, A., Alvarez, E., Gupta, S., and Davis, R. J. (1991) J. Biol. Chem. 266, 23521-23524[Abstract/Free Full Text]
  22. Henriksson, M., Bakardjiev, A., Klein, G., and Luscher, B. (1993) Oncogene 8, 3199-3209[Medline] [Order article via Infotrieve]
  23. Lutterbach, B., and Hann, S. R. (1994) Mol. Cell Biol. 14, 5510-5522[Abstract/Free Full Text]
  24. Pulverer, B. J., Fisher, C., Vousden, K., Littlewood, T., Evan, G., and Woodgett, J. R. (1994) Oncogene 9, 59-70[Medline] [Order article via Infotrieve]
  25. Noguchi, K., Kitanaka, C., Yamana, H., Kokubu, A., Mochizuki, T., and Kuchino, Y. (1999) J. Biol. Chem. 274, 32580-32587[Abstract/Free Full Text]
  26. Cheng, M., Wang, D., and Roussel, M. F. (1999) J. Biol. Chem. 274, 6553-6558[Abstract/Free Full Text]
  27. Fuchs, S. Y., Xie, B., Adler, V., Fried, V. A., Davis, R. J., and Ronai, Z. (1997) J. Biol. Chem. 272, 32163-32168[Abstract/Free Full Text]
  28. Fuchs, S. Y., Dolan, L., Davis, R. J., and Ronai, Z. (1996) Oncogene 13, 1531-1535[Medline] [Order article via Infotrieve]
  29. Fuchs, S. Y., Adler, V., Pincus, M. R., and Ronai, Z. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10541-10546[Abstract/Free Full Text]
  30. Musti, A. M., Treier, M., and Bohmann, D. (1997) Science 275, 400-402[Abstract/Free Full Text]
  31. Fuchs, S. Y., Fried, V. A., and Ronai, Z. (1998) Oncogene. 17, 1483-1490[CrossRef][Medline] [Order article via Infotrieve]
  32. Alarcon-Vargas, D., Tansey, W. P., and Ronai, Z. (2002) Oncogene 21, 4384-4391[CrossRef][Medline] [Order article via Infotrieve]
  33. Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., Johnson, G. L., and Karin, M. (1994) Science 266, 1719-1723[Abstract/Free Full Text]
  34. Chen, C. Y., Del Gatto-Konczak, F., Wu, Z., and Karin, M. (1998) Science 280, 1945-1949[Abstract/Free Full Text]
  35. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) Cancer Cell 2, 243-247[CrossRef][Medline] [Order article via Infotrieve]
  36. He, K. L., and Ting, A. T. (2002) Mol. Cell Biol. 22, 6034-6045[Abstract/Free Full Text]
  37. Henikoff, S., Pietrokovski, S., and Henikoff, J. G. (1998) Nucleic Acids Res. 26, 309-312[Abstract/Free Full Text]
  38. Kallunki, T., Deng, T., Hibi, M., and Karin, M. (1996) Cell 87, 929-939[CrossRef][Medline] [Order article via Infotrieve]
  39. Buschmann, T., Potapova, O., Bar-Shira, A., Ivanov, V. N., Fuchs, S. Y., Henderson, S., Fried, V. A., Minamoto, T., Alarcon-Vargas, D., Pincus, M. R., Gaarde, W. A., Holbrook, N. J., Shiloh, Y., and Ronai, Z. (2001) Mol. Cell Biol. 21, 2743-2754[Abstract/Free Full Text]
  40. Felsher, D. W., Zetterberg, A., Zhu, J., Tlsty, T., and Bishop, J. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10544-10548[Abstract/Free Full Text]
  41. Hoang, A. T., Lutterbach, B., Lewis, B. C., Yano, T., Chou, T. Y., Barrett, J. F., Raffeld, M., Hann, S. R., and Dang, C. V. (1995) Mol. Cell Biol. 15, 4031-4042[Abstract]
  42. Kuttler, F., Ame, P., Clark, H., Haughey, C., Mougin, C., Cahn, J. Y., Dang, C. V., Raffeld, M., and Fest, T. (2001) Oncogene 20, 6084-6094[CrossRef][Medline] [Order article via Infotrieve]
  43. Scheffner, M., Huibregtse, J. M., Vierstra, R. D., and Howley, P. M. (1993) Cell 75, 495-505[CrossRef][Medline] [Order article via Infotrieve]
  44. Adachi, S., Obaya, A. J., Han, Z., Ramos-Desimone, N., Wyche, J. H., and Sedivy, J. M. (2001) Mol. Cell Biol. 21, 4929-4937[Abstract/Free Full Text]

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