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Molecular and Cellular Biology, June 2005, p. 4956-4968, Vol. 25, No. 12
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.12.4956-4968.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

RelA/p65 Regulation of IBß

Erin Hertlein,^1,2 Jingxin Wang,¹ Katherine J. Ladner,¹ Nadine Bakkar,^1,3 and Denis C. Guttridge^1,2,3,4,5*

Human Cancer Genetics Program,¹ Department of Molecular Virology, Immunology and Medical Genetics,⁴ Integrated Biomedical Graduate Program,² Molecular, Cellular, and Developmental Biology Graduate Program,³ The Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio⁵

Received 2 December 2004/ Returned for modification 19 January 2005/ Accepted 17 March 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
IB inhibitor proteins are the primary regulators of NF-B. In contrast to the defined regulatory interplay between NF-B and IB, much less is known regarding the regulation of IBß by NF-B. Here, we describe in detail the regulation of IBß by RelA/p65. Using p65^–/– fibroblasts, we show that IBß is profoundly reduced in these cells, but not in other NF-B subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65^–/– cells and tissues, IB is also reduced, but not nearly to the same extent as IBß, thus highlighting the degree to which IBß is dependent on p65. This dependence is based on the ability of p65 to stabilize IBß protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IBß was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65^–/– fibroblasts, expression of a proteolysis-resistant form of IBß, but not IB, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IBß protein by p65 is necessary for the maintenance of cellular homeostasis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The NF-B transcription factor is a vital regulator of cellular processes involved in immune response, cellular proliferation, differentiation, and apoptosis (6, 38, 47, 53). Constitutive activation of NF-B is also thought to contribute to multiple pathophysiological conditions such as rheumatoid arthritis (55), inflammatory bowel disease (60), and AIDS (22) and, with ever increasing evidence, cancer (1, 5, 33, 48). In mammals, the NF-B family consists of RelA (from here on referred to as p65), c-Rel, and RelB, as well as p105 and p100 and their processed forms, p50 and p52, respectively (31). Each subunit contains a Rel homology domain (RHD) specifying DNA binding, protein dimerization, and nuclear localization. In addition, p65, c-Rel, and RelB contain transactivation domains (TAD) located at the carboxy terminus. Although in vitro most NF-B subunits possess the ability to homo- or heterodimerize, in vivo, NF-B primarily exists as a p50/p65 heterodimer.

Unlike the majority of transcription factors that reside in the nucleus, NF-B is sequestered predominantly in the cytoplasm bound to IB inhibitor proteins. IB is the prototypical IB protein within a family that includes IBß, IB, p100, p105, Bcl-3, and newly described IB (6, 7, 32, 71). With the exceptions of Bcl-3 and IB, these proteins function as inhibitors through ankyrin repeats that interact with the RHD of NF-B to mask the nuclear localization signal (NLS) and inhibit nuclear translocation. Classical activation of NF-B proceeds by the degradation of IB proteins, which is mediated by the activity of the IB kinase complex (IKK) (31, 47). At the core of this large 700- to 900-kDa subunit complex are two catalytic subunits, IKK and IKKß, and a regulatory subunit, IKK or NEMO/IKKAP. In response to a multitude of factors that include inflammatory cytokines, bacterial products, viruses, double-stranded RNA, reactive oxygen species, and irradiation, IKK is activated, leading to the IKKß-dependent phosphorylation of IB on two N-terminal serine residues (74). This triggers polyubiquitination on neighboring lysines and subsequent IB proteolysis via the 26S proteasome (11, 47). Upon IB degradation, NF-B translocates to the nucleus, where it binds to its cognate DNA sequence and interacts with basal transcription and chromatin remodeling factors to activate gene expression (4, 15, 61, 63, 75).

Over the past decade, there has been a concerted effort to understand the function by which IB proteins regulate NF-B activity. Earlier studies demonstrated that shortly following NF-B activation, IB is resynthesized in an NF-B-dependent manner (24, 44, 51). Following this resynthesis, IB enters the nucleus by a yet to be confirmed mechanism, where it then binds and removes NF-B from the DNA and exports the complex back to the cytoplasm (2, 13, 20, 65). By this autoregulatory mechanism, NF-B transcriptional activity remains transient, lasting between 1 and 4 h in most cells, with the exception in mature splenic B cells, where the p50/c-Rel complex is constitutively active (34, 54, 59). Disruption of this regulatory loop via the deletion of IB expression leads to persistent p65 activity and postnatal lethality (8, 49). More recently, structural analysis of IB in complex with the p50/p65 heterodimer revealed that IB ankyrin repeats 3 to 6 contribute the majority of p50/p65 binding while repeats 1 and 2 are more loosely associated with the NLS site of NF-B (42, 45). The p50 NLS remains exposed when bound to IB (42, 45), which is thought to allow cytoplasmic to nuclear shuttling of the complex (56), while reverse shuttling is regulated by nuclear export signals located in the carboxyl- and amino-terminal halves of IB (3, 41, 46, 67), as well as in the TAD of p65 (37).

In contrast to this well-described regulatory interplay between NF-B and IB, the regulation of NF-B by IBß and a potential feedback mechanism has not been described. Unlike IB, the beta isoform is not rapidly degraded by classical NF-B-inducing signals, and following NF-B activation, IBß is also not resynthesized in an NF-B-dependent manner. Depending on the cell type or stimulus, IBß may instead undergo persistent degradation, leading to constitutive NF-B activity (12, 50, 69). Constitutive NF-B activity is also regulated by a hypophosphorylated form of IBß that is capable of competing with IB for NF-B binding but is incapable of dislodging NF-B from the DNA (23, 66). Basal phosphorylation of IBß occurs in its carboxyl-terminal PEST domain that functions to inhibit NF-B DNA binding and is thought to be primarily responsible for the formation of latent IBß/NF-B complexes (58, 70). However, unlike IB/NF-B, IBß/NF-B complexes do not undergo nuclear-to-cytoplasmic shuttling (68) due to the addition of a linker region between ankyrin repeats 3 and 4 in IBß that binds B-Ras to efficiently mask the second NLS in the NF-B dimer complex (16, 17, 29, 57). Also, unlike IB (8, 49), mice lacking IBß were noted to have a mild phenotype (18), suggesting at first that these proteins are functionally distinct. However, knock-in expression of IBß under the control of the IB promoter rescued IB-associated lethality (18). This demonstrates that functional overlap between these proteins clearly exists, but given the overt phenotypic differences among respective IB knockouts, it also points to the importance of spatiotemporal expression of IB proteins in the regulation of NF-B.

We have made the observation that IBß levels are dramatically reduced in mouse embryonic fibroblasts (MEFs) null for the p65 subunit of NF-B. Although others have indirectly noted this phenomenon (9, 40), to date a detailed characterization of this regulation has not been performed. In this report, the specificity, mechanism, and physiological relevance of p65 regulation of IBß are described. Our results reveal a remarkable dependence of IBß for p65 in most, but not all, tissues. This dependence is mediated through the stabilization of IBß protein by p65 and its carboxyl terminus encompassing the TADs. Our results also show that IBß expression in p65-null MEFs has a severe impact on cell growth and viability. Interestingly, although p65 is considered constitutively expressed, studies have reported that p65 expression is in fact low or even undetectable in early development (21, 62) and in selective cell types (73). Based on such studies, as well as our own present findings, we suggest that the destabilization of IBß in cells lacking p65 is a regulatory process that might have emerged to ensure proper cell growth and viability.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials. Murine tumor necrosis factor alpha (TNF-) was purchased from Roche Biochemicals (Indianapolis, IN). Antibodies to IB (C21), IBß (C20, N20), Bcl-3, IKK, and p100 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); myosin heavy chain and -tubulin were from Sigma (St. Louis, MO); p65 was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); and hemagglutinin (HA) was from Covance (Princeton, NJ). MG-132, ALLN, and lactacystin were purchased from Calbiochem (San Diego, CA); cycloheximide was from Sigma (St. Louis, MO); and -phosphatase was from New England Biolabs (Beverly, MA). [³⁵S] Easy Tag protein labeling mix was purchased from NEN (Boston, MA), and methionine/cysteine-free Dulbecco's modified Eagle's medium (DMEM) was from Invitrogen (Carlsbad, CA). Materials required for immunohistochemical analysis were obtained from Vector Laboratories (Burlingame, CA).

Cell culture. All fibroblast cells were cultured in high-glucose DMEM containing 10% fetal bovine serum and antibiotics. For p50 and Bcl-3 MEFs, mice null for these proteins (Jackson Laboratories, Bar Harbor, Maine) were crossed with their respective wild-type strains. Resulting heterozygotes were then bred, and MEFs were prepared from embryos at day 13.5 postcoitus. In a similar manner, p65 MEFs were generated from embryonic day 13.5 (E13.5) p65^+/+, p65^+/–, and p65^–/– embryos. C2C12 myoblasts were cultured as previously described (35).

Plasmids. Full-length p65 [p65(FL)] and carboxyl truncation mutants [p65(534), p65(521), and p65(313)] were cloned into the pFLAG-CMV-2 expression plasmid as previously described (72). For the generation of p65(431), Flag-tagged p65(FL) was used as a template and a DNA fragment corresponding to amino acids 1 to 431 was amplified with primers 5'-GATCAAGCTTGACGAACTGTTCCCCCTCATC and 3'-GATCGATATCTCAAGCCTGGGTGGGCTTGGGG. The DNA was subsequently cloned into the HindIII/EcoRV sites of a pFLAG-CMV-2 plasmid. Construct p65(319) was generated in a similar manner using primers 5'-GATCAAGCTTGACGAACTGTTCCCCCTCATC and 3'-CGATATCTCAGCTGAAAGGACTCTTCTTCATG. Retroviral expression constructs for p65 and IBß were created by reverse transcription-PCR to amplify the respective cDNA from human fibroblasts and cloned into pBabepuro. The p65 construct was generated to produce the full-length protein. Wild-type IBß and IBß-SR (deleted in the signal response region, amino acids 1 to 54) were generated with an N-terminal HA epitope tag. pBabeIB-SR was generated by excising the cDNA of human IB-SR (mutated in Ser-32 and Ser-36 to alanines) from a pCMV4 expression plasmid (generously provided by D. Ballard, Vanderbilt University) with BglII/SmaI restriction enzymes and subcloned in BamH1/SnaB sites in pBabepuro. pCMVIBß(S19/23A) and pCMVIBß(S19/23E) were generated by site-directed mutagenesis from pCDNA3.1 and pHM6 expression plasmids containing IBß using the QuikChange site-directed mutagenesis kit (Stratagene) with primers S19/23A (5'-GAATGGTGCGACGCCGGCCTGGGCGCCCTGGGTCCG-3') and S19/23E (5'-CAGATGAATGGTGCGACGAAGGCCTGGGCGAGCTGGGTCCGGAC-3') (mutated residues are underlined). All clones were confirmed by DNA sequencing and Western blot analysis.

Immunoblotting, EMSA, and kinase assay. Whole-cell extracts from cultured cells and immunoblotting were prepared as previously described (35). Extracts from mouse tissue were prepared by homogenization in lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris [pH 7.5], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, standard protease inhibitors). Protein detection was obtained by enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) and imaged either by using a Chemidoc gel documentation system (Bio-Rad Laboratories, Hercules, CA) or by exposing blots to film. All quantitation was performed using the ImageJ software (National Institutes of Health, Bethesda, MD). Electrophoretic mobility shift assays (EMSAs) and IKK kinase assays were performed as previously described (19, 25).

³⁵S labeling and immunoprecipitations. For labeling reactions, p65^+/+ and p65^–/– MEFs were cultured in DMEM lacking methionine or cysteine for 2 h and subsequently pulse-labeled with ³⁵S-labeled methionine and cysteine for up to 1 h. Whole-cell extracts were prepared in a standard radioimmunoprecipitation assay buffer. For immunoprecipitation, 500 µg of protein was precleared with rabbit immunoglobulin G (IgG) for 2 h and nonspecific complexes were precipitated by centrifugation using 25 µl of A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Supernatants were then incubated overnight at 4°C with 1 µg of either IgG or an IBß antibody. The following day, complexes were precipitated by centrifugation using 30 µl of A/G agarose beads, washed three times in lysis buffer, resuspended in gel loading buffer, and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were dried and visualized on X-OMAT film.

Transfections and retrovirus infections. Typically, 75% confluent MEFs were transfected in low-serum Opti-MEM medium using Lipofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Retrovirus production and infections were performed as previously described (35).

Immunohistochemistry. E13.5 embryos either wild type or null for p65 were fixed in 10% formalin overnight at 4°C and then dehydrated and paraffin embedded. Longitudinal sections of the entire embryo and cross sections through selective tissues were prepared. Sections were deparaffinized by heating at 60°C for 1 h, followed by treatment with xylene and rehydration. Slides were treated with 3% hydrogen peroxide to block any endogenous peroxidase activity that could interfere with the detection reaction. Sections were steamed for 30 min in antigen retrieval solution and then incubated in avidin and biotin solutions. Following blocking for 1 h in 5% goat serum diluted in phosphate-buffered saline, sections were incubated with primary antibody against p65 (for 1 h; 1:5,000 dilution in 5% goat serum), IBß (for 1 h; 1:300 dilution in 5% goat serum), or myosin heavy chain (for 30 min; 1:2,000 dilution in mouse on mouse [MOM] diluent). Sections were subsequently incubated with a biotinylated secondary antibody. For p65 and IBß, the secondary antibody used was goat anti-rabbit (for 30 min; 1:250 in 5% goat serum) and for myosin heavy chain it was goat anti-mouse (for 10 min; 1:250 in MOM diluent). Sections were developed by incubation with avidin/biotin-complexed peroxidase to recognize secondary antibody (ABC Elite) and using 3,3'-diaminobenzidine as the enzyme substrate (DAB kit).

Real-time PCR. RNA was prepared in TRIzol reagent (Invitrogen, Carlsbad, CA) as recommended by the manufacturer and further purified using RNeasy affinity columns (QIAGEN, Valencia, CA). cDNA was generated from 2 µg total RNA by reverse transcription with Superscript II (Invitrogen, Carlsbad, CA) according to the manufacturer's specifications. One microliter of cDNA was used as the template in a total reaction volume of 25 µl containing final concentrations of 1x iQ SYBR Green Super mix (Bio-Rad, Hercules, CA) and 0.5 µM each forward and reverse primers. The primer sequences used were as follows: IB, 5'-GGAGACTCGTTCCTGCACTTGG and 3'-AACAAGAGCGAAACCAGGTCAGG; IBß, 5'-ACACAGCCCTGCACTTGGCTG and 3'-GGTATCTGAGGCATCTCTTGGG; internal control GAPDH, 5'-GCAAATTCAACGGCACAGTCAAG and 3'-GTTCACACCCATCACAAACATGG. Data were read and collected on the Bio-Rad iCycler.

Mice and genotyping. Animals were housed in the animal facility at the Ohio State University Heart and Lung Research Institute under supersterile conditions maintaining constant temperature and humidity and fed a standard diet. Treatment of mice was in accordance to the institutional guidelines of the Animal Care and Use Committee. p65^–/– TNF-^–/– mice were generated as previously described (26). Briefly, p65^+/– TNF-^+/+ mice were crossed to p65^+/+ TNF-^–– mice (Jackson Laboratories, Bar Harbor, Maine). From this cross, resulting p65^+/– TNF-^+/– mice were crossed to obtain p65^+/+ TNF-^–/– and p65^–/– TNF-^–/– mice in the expected Mendelian ratios. Genotypes of p65, TNF-, NFKB1/p50, and Bcl-3 mice were confirmed by PCR analysis from prepared tail DNA.

Growth curves and flow cytometry. p65^–/– cells infected with pBabe vector, IBß-SR, or IB-SR were grown under puromycin selection. Cells (1 x 10⁴) were plated in triplicate in 12-well cell culture plates and counted on indicated days. Apoptosis was evaluated using Annexin V-fluorescein isothiocyanate staining according to the manufacturer's specifications (Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, 5 x 10⁵ cells were washed with cold phosphate-buffered saline and suspended in 50 µl of annexin V-fluorescein isothiocyanate staining solution. After 15 min of incubation at room temperature, cells were fixed in 10% formaldehyde and subsequently analyzed on a FACScalibur flow cytometer (Becton-Dickinson, Mountain View, CA). Fluorescence data were analyzed using CellQuest Pro software.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Regulation of IBß is specific for the loss of p65. MEFs null for p65 were utilized in an attempt to gain insight into the mechanisms of NF-B-dependent transcription. During the course of this analysis, we made the observation that IBß levels were strikingly lower in p65^–/– fibroblasts compared to their wild-type counterparts (Fig. 1A). Although IB is a transcriptional target of p65, its level of regulation was noticeably less than that for IBß. In addition, no detectable changes in IB, p100, or Bcl-3 were seen between p65^–/– and p65^+/+ cells, demonstrating that p65 does not function as a general regulator of IB proteins. To determine the specificity of this regulation, levels of IBß were compared in MEFs lacking other subunits in either the NF-B family or the NF-B signaling pathway. As opposed to the marked reduction of IBß in p65^–/– MEFs, no changes in IBß were observed in fibroblasts lacking c-Rel, RelB, p50, p52, IKK, IKKß, IKK, IB, or Bcl-3 (Fig. 1B), implying that regulation of IBß is specific to p65.

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FIG. 1. IBß is specifically regulated by p65 in fibroblasts. (A) Whole-cell extracts (WCEs) were prepared from immortalized MEFs either wild type or null for the p65 subunit of NF-B, and the levels of the indicated proteins were analyzed by Western blotting. (B) Western blot assays to probe for IB and IBß in MEFs either wild type or null for various components in the NF-B signaling pathway. Genotypes for all cell lines were confirmed by PCR analysis. (C) WCEs were prepared from vector or IB-SR C2C12 myoblasts, and Western blot assays were performed to probe for IB proteins. (D) Primary MEFs were isolated from E13.5 embryos and genotyped for p65 by PCR. WCEs were prepared from p65 wild-type, heterozygous, and null MEFs, and Western blot assays were performed to probe for p65, IB, and IBß. -Tubulin was used as a loading control. (E) Quantitation of IB proteins from two Western blot assays performed with WCEs obtained from two independent litters as described for panel D. Levels of IB proteins were normalized to -tubulin and compared to the expression of IB proteins obtained from wild-type cells, which was set to a value of 100%.

To determine whether regulation of IBß was due to the physical absence of p65 or simply loss of its activity, we verified IBß protein levels in myoblast cells devoid of NF-B transactivation function due to the stable expression of the degradation-resistant IB-SR mutant (36). In these cells, lack of NF-B activity maintained IBß protein levels (Fig. 1C), indicating that reduction of IBß results from the physical loss of p65.

Since decreases in IBß were detected using established fibroblasts, it was important to determine whether this regulation was a consequence of the immortalization process due to the absence of p65. Mice heterozygous for p65 were therefore bred and E13.5 MEFs were prepared. In comparison to wild-type cells, p65^+/– MEFs expressed approximately 50% less IBß and, strikingly, IBß was nearly undetectable in null cells (Fig. 1D and E). In contrast, IB expression was only slightly reduced in p65^+/– MEFs while approximately 35% remained in cells lacking p65. These data demonstrate that in fibroblasts, p65 regulation of IBß is not a phenomenon of cellular immortalization and the degree to which p65 controls IBß expression is significantly higher than that for IB.

p65 regulation of IBß is maintained in embryonic and postnatal development. To determine if this regulation occurred in cells other than MEFs, immunohistochemical analysis of IBß was performed with p65^+/+ and p65^–/– embryos at day 13.5, a time that precedes liver apoptosis and lethality of p65-null mice (9). As expected, no overt morphological defects were observed at this developmental stage in embryos lacking p65 (Fig. 2A). Results revealed, however, that IBß expression was generally reduced in p65^–/– embryos, with more apparent regulation occurring in the liver, lung, and brain (Fig. 2A and B). This implied that IBß regulation by p65 occurred in multiple cell types. To confirm that these findings were not due to staining artifacts, immunoblotting was performed from isolated fetal livers. In line with the immunohistochemistry data, IBß was found strongly repressed in p65^–/– liver cells (Fig. 2C). Immunoblots also revealed that, similar to embryonic fibroblasts, the downregulation of IB in p65^–/– livers was not nearly to the same extent as that for IBß, reaffirming the tight control of IBß expression by p65.

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FIG. 2. p65 regulation of IBß is not limited to fibroblasts. (A) Histological hematoxylin-and-eosin (H&E) and immunohistochemical staining of p65 and IBß from longitudinal sections of p65+/+ (a, b, c) and p65–/– (d, e, f) embryos (x1 magnification). (B) Immunohistochemical staining of IBß of liver (a, d), lung (b, e), and brain (c, f) tissues from p65+/+ and p65–/– embryos (x4 magnification). (C) Primary fetal liver cells were prepared from E13.5 embryos, and after genotypes were confirmed, Western blot assays were performed to probe for p65, IB, and IBß. -Tubulin was used as a loading control. (D) p65 and IBß immunohistochemical staining of heart tissue from p65+/+ and p65–/– embryos (x4 magnification). (E) p65 and IBß immunohistochemical staining of forelimbs from p65+/+ and p65–/– embryos. To confirm skeletal muscle staining, serial sections of forelimbs were separately stained for myosin heavy chain (MyHC; arrowheads denote muscle tracks; x4 magnification).

Next we asked whether p65 regulation of IBß could be maintained in adult mice. Although p65^–/– mice die between E14.5 and E15.5 (9), liver apoptosis and embryonic lethality can be rescued with the additional deletion of TNF- (27). Thus, p65^–/– TNF-^–/– double knockouts were generated and at approximately 4 weeks of age, p65^+/+ TNF-^–/– and p65^–/– TNF-^–/– mice were sacrificed and tissues were processed for immunoblot analysis. Except for a p65-reactive band that reproducibly appeared in brain tissue, the complete absence of p65 staining in all remaining tissues confirmed the null phenotype of these mice (Fig. 3A). In agreement with immunohistochemical data, IBß was also found generally repressed in p65^–/– tissues, suggesting that this regulation is maintained into adulthood (Fig. 3A). Of the p65^–/– tissues examined, spleen, thymus, and skin samples contained the least IBß, with less but still significant reduction observable in the brain, liver, and lung compared to wild-type mice (Fig. 3A and B). Although IB was also downregulated in tissues lacking p65, similar to cultured MEFs and liver cells, the qualitative difference in expression compared to wild-type tissues was not as significant as that for IBß.

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FIG. 3. p65 regulates various forms of IBß in postnatal development in a tissue-specific manner. p65+/– TNF-–/– mice were bred to generate p65+/+ TNF-–/– and p65–/– TNF-–/– progeny. (A) At 4 weeks of age, mice were sacrificed and tissue homogenates were prepared (skmc, skeletal muscle). Western blot assays were then performed to probe for p65, IB, IBß, and -tubulin. Each Western blot assay is representative of a total of four different blot assays derived from two sets of littermates. (B) Western blot assay to probe for IBß in p65+/+ TNF-–/– brain, skin, skeletal muscle, and heart tissues. Arrows denote IBß forms I through IV. (C) Similar extracts as in panel A were either left untreated or treated with phosphatase (PPase) enzyme, and Western blot assay was performed to probe for IBß. Arrows denote phosphorylated forms of IBß, and asterisks denote the shifted (dephosphorylated) forms of IBß. (D) The N-terminal IBß antibody (N20) was used in a Western analysis to verify IBß forms in p65+/+ TNF-–/– tissues.

IBß is expressed in alternative forms that are differentially regulated by p65. Interestingly, upon closer examination of immunohistochemical sections shown in Fig. 2, we observed that in heart and skeletal muscle, levels of IBß expression appeared almost comparable between p65^+/+ and p65^–/– embryos (Fig. 2D and E; note the colocalization of IBß and the skeletal muscle marker myosin heavy chain). This suggested that IBß regulation by p65 might also be tissue specific. Indeed, the immunoblot analysis in Fig. 3A showed significantly less reduction of IBß in skeletal muscle and heart compared to other tissues lacking p65. IBß levels in these mice were also largely retained in testis tissue, which is consistent with previous findings showing that IBß is particularly rich in this tissue (14). Of further interest was the identification that IBß produced from skeletal muscle and heart tissues migrated at a distinctly higher mobility compared to other tissues (Fig. 3A and B). In fact, as opposed to reports that murine cells produce only one form of IBß (39, 43), under our standard gel fractionation conditions, at least four forms of IBß were clearly discernible, which we refer to as forms I, II, III, and IV (Fig. 3B). IBß-I and IBß-IV represented the major and minor expressing forms in most tissues, respectively, and both were sensitive to p65 regulation, although IBß-I appeared more so then IBß-IV (Fig. 3A). However, in skeletal muscle and heart tissues, IBß-I and IV forms were less expressed, while IBß-II and IBß-III forms were readily detectable compared to other tissues. In addition, similar to what we had observed with IBß-I in testis tissue, the IBß-II and IBß-III forms also remained largely expressed in muscle tissues deficient in p65.

Since IBß is constitutively phosphorylated (58, 69, 70), tissue homogenates were phosphatase treated to further ascertain the characteristics of these various IBß-reactive polypeptides. This treatment caused a shift in IBß-I and -IV forms, promoting the appearance of a slightly faster-migrating IBß protein (Fig. 3C, denoted by asterisks). Based on these results, IBß-I is likely to represent the basally phosphorylated 45-kDa form of IBß that is most commonly described and whose stimulus-dependent hypophosphorylated state is associated with persistent activation of NF-B (66, 69). Although IBß-IV is a minor component, the data also demonstrate that IBß can exist in a hyperphosphorylated state. In comparison, IBß-II and IBß-III were completely resistant to phosphatase treatment, which suggests that these proteins either represent unique forms of IBß devoid of phosphorylation or are IBß-like polypeptides that may have cross-reacted with this IBß carboxyl terminus-specific antibody used in immunoblot and immunohistochemical analyses. To make this distinction, immunoblots were repeated with a second antibody generated instead to the amino terminus of IBß (referred to as N20). Consistent with our previous results, the N20 antibody also reacted with IBß forms I and IV, thus validating the expression of these IBß forms in murine tissues (Fig. 3D). However, in contrast to the carboxyl-terminal antibody, the N20 antibody was clearly reactive with IBß-I, but less so with IBß-IV, in skeletal muscle and heart tissues (Fig. 3D). In addition, this IBß antibody again recognized altered IBß forms in skeletal muscle and heart tissues (Fig. 3D), but these forms did not migrate to the same mobility as forms II and III. Collectively, these data imply that skeletal muscle and heart tissues may be capable of synthesizing distinct IBß-like proteins. This same logic could apply to p65 expression in brain tissue, where a polypeptide migrating with an approximate mobility of 65 kDa, as described above, was reproducibly detected in p65-null mice (Fig. 3A). In any regard, it is clear that a more detailed investigation of IBß in muscle tissues will be required to determine whether such forms derive from posttranslational modifications or alternative splicing events.

Absence of p65 promotes IBß degradation by the 26S proteasome independent of IKK and phosphoacceptor serines 19 and 23. Next, we examined the mechanism by which p65 regulates IBß. Although a consensus NF-B binding site is contained within the IBß promoter, overexpression of p65 has been shown to be incapable of stimulating IBß transcription (14), which argued, as others have before (69), that IBß is not a transcriptional target of p65. In line with these findings, we too could not detect any significant difference in steady-state levels of IBß mRNA between p65^+/+ or p65^–/– fibroblasts treated with TNF while, as expected, IB mRNA was readily induced by this cytokine in a p65-dependent manner (Fig. 4A).

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FIG. 4. IBß downregulation in p65–/– MEFs is regulated by the proteasome independent of classical IKK signaling. (A) MEFs wild type and null for p65 were treated with TNF-, and at indicated time points a real-time PCR was used to measure IB and IBß RNAs. (B) p65+/+ and p65–/– MEFs were incubated for 2 h in methionine- and cysteine-free DMEM and subsequently labeled with a [35S]methionine/cysteine mix for an additional hour. Whole-cell extracts were prepared, and IBß was immunoprecipitated either with IgG (control) or with an IBß-specific antibody. Complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by exposing dried gels to film for up to 3 days (arrow denotes IBß). (C) p65+/+ and p65–/– MEFs were treated with cycloheximide (CHX; 10 µg/ml), and at indicated times whole-cell extracts were prepared and Western blot assays performed to probe for IBß. Levels of IBß were quantitated from an average of three Western blot assays. (D) Similar conditions were used to treat p65+/+ and p65–/– MEFs with MG-132, ALLN, and lactacysteine (all at 10 µM). (E) Kinase assays (KA) were performed with IKK immunoprecipitates from p65+/+ and p65–/– MEFs incubated with glutathione S-transferase-IB as a substrate. Whole-cell extracts from p65+/+ and p65–/– MEFs used in kinase assay were analyzed by Western bloting to probe for IKK and p65. (F) Western blot assays to probe for IBß and tubulin in p65–/– MEFs transfected with vector control or catalytically inactive IKK or IKKß proteins (top). Inhibitory activity of IKK proteins was verified by luciferase NF-B reporter assays (bottom). (G) p65–/– MEFs were transfected with either HA-tagged wild-type (WT) IBß or HA-tagged IBß mutated at serines 19 and 23 to either alanine (S19/23A) or glutamic acid (S19/23E) residues. After 24 h, cells were treated with cycloheximide (10 µg/ml) for the indicated times. Whole-cell extracts were then prepared and Western blot assays performed to probe for HA and -tubulin.

The above findings suggested that p65 regulation of IBß was not transcriptionally mediated and therefore is likely to occur at the protein level, affecting either the synthetic rate or stability of IBß. To address these possibilities, p65^+/+ or p65^–/– fibroblasts were metabolically labeled with ³⁵S and IBß was subsequently analyzed by immunoprecipitation. Results showed similar expression levels of IBß following 10, 30, or 60 min of labeling (Fig. 4B and data not shown), indicating that loss of p65 does not affect the rate of IBß synthesis. To examine IBß stability, fibroblasts were treated with cycloheximide and IBß was analyzed over time. During an 8-h period, little destabilization of IBß was observed in p65^+/+ cells, whereas in p65^–/– cells, nearly a third of the protein was degraded after only 30 min of treatment (Fig. 4C). Further treatment of fibroblasts with MG-132 to inhibit proteasome activity demonstrated little increase in IBß stability in p65^+/+ cells, while levels of IBß protein increased steadily over time in cells lacking p65 (Fig. 4D). Similar results were obtained with additional proteasome inhibitors, ALLN and lactacysteine (Fig. 4D), demonstrating that absence of p65 leads to IBß destabilization mediated by the 26S proteasome complex.

To further investigate the mechanism of IBß turnover, we asked whether this regulation by the proteasome was also dependent on IKK activity and Ser-19 and Ser-23 that are phosphorylated in response to a classical NF-B-inducing signal. Interestingly, kinase assays revealed that p65^–/– MEFs exhibited substantially higher basal IKK activity compared to wild-type cells (Fig. 4E). However, transient overexpression of catalytically inactive IKK and IKKß subunits (Fig. 4F) or treatment with IKK inhibitor compounds (Bay11-7085 and PS1145; data not shown) did not restore IBß levels in p65^–/– MEFs. In addition, transient expression of HA-tagged IBß proteins mutated at Ser-19 and Ser-23 to alanine (S19/23A) or glutamic acid (S19/23E) residues had no significant effect on either the basal level or turnover rate of IBß in p65^–/– cells (Fig. 4G). Therefore, despite elevated levels of IKK activity in p65^–/– MEFs, IBß proteolysis in these cells does not appear to be regulated by the classical IKK signaling pathway.

The carboxyl terminus of p65 is required for IBß stability. To address whether p65 is a direct regulator of IBß stability, p65 was reconstituted in null fibroblasts. Results showed that IBß expression was indeed restored in these cells (Fig. 5A). To further test the specificity of this regulation, we also examined the levels of B-Ras, a Ras-like small GTPase recently shown to directly bind IBß and contribute to its stabilization in response to an NF-B-inducing signal (16, 29). We considered the possibility that loss of IBß could be mediated by the preceding destabilization of B-Ras resulting from the absence of p65. Our findings revealed, however, that B-Ras expression was generally unaltered in either p65^–/– MEFs or p65^–/– tissues (Fig. 5B and C). Similar results were obtained with another recently identified IBß-stabilizing protein, ß-arrestin (30) (Fig. 5B). Together, these findings support the notion that IBß stability is directly mediated by p65.

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FIG. 5. IBß stability is regulated by the carboxyl terminus of p65. (A) p65–/– MEFs were infected with either vector control retrovirus vector or virus expressing wild-type p65. Whole-cell extracts were prepared from a mixed population of drug selection-resistant cells, and Western blot assays were performed to probe for p65 and IBß. (B) Western blot assays to probe for B-Ras and ß-arrestin in p65+/+ and p65–/– MEFs. -Tubulin was used as a loading control. (C) Western blot assays were performed with p65+/+ TNF-–/– and p65–/– TNF-–/– tissue homogenates to probe for B-Ras. (D) Illustration of full-length and C-terminal truncation mutants generated in p65. The mutants are named according to the fragment of p65 expressed. For example, p65(534) denotes p65 containing amino acids 1 to 534. (E) Western blot assays for IBß (top) and p65 (bottom) in p65–/– MEFs transfected with the indicated p65 truncation mutants. (F) Western blot assay for IBß in p65(313)-expressing brain, skeletal muscle (skmc), heart, and testis cells from a p65+/+ TNF-–/– mouse. Arrows denote forms IBß-I and IBß-IV. (G) Identical lysates as described in panel E were either treated or not treated with phosphatase (PPase), and Western blot assays were subsequently performed to probe for IBß. (H) p65–/– MEFs were transfected with p65(431) or p65(313) and subsequently treated with MG-132 or not treated. Western blot assay was then performed to probe for IBß.

Next, p65 deletion mutants were generated (Fig. 5D) and subsequently expressed in p65^–/– MEFs in order to map the region in p65 responsible for IBß stability. Since IBß binding is known to occur through the RHD of NF-B monomers, intuitively we did not consider the possibility that amino acids carboxyl to the NLS of p65 would contribute to this stability. Although deletion of the first 17 amino acids from the carboxyl terminus (534) restored IBß to equivalent levels as that of wild-type p65(FL), to our astonishment, further deletion of the TA1 domain (521) was sufficient to cause a minor but reproducible reduction of IBß (Fig. 5E). MG-132 treatment of p65(521)-expressing cells restored IBß to wild-type levels, indicating that the reduction in IBß observed in p65(521) cells was due to IBß destabilization (data not shown). Additional destabilization of IBß occurred when residues mapping to the second TA domain of p65 were removed (431), and still further loss of IBß was observed upon deletion of residues lying just proximal to the RHD (319). However, further deletion of residues to the NLS (313) reproducibly had little to no further effect on IBß stability (Fig. 5E). Moreover, reconstitution of p65^–/– MEFs with only carboxyl-terminal residues 313 to 551 of p65 was unable to restore IBß expression over that of vector control cells (data not shown), demonstrating that both the RHD and carboxyl residues 319 to 521 of p65 are critical to sustained IBß expression.

Also intriguing was the observation that destabilization of IBß due to carboxyl-terminal deletions of p65 led to the increased expression of a higher-molecular-weight form of IBß that appeared similar to the IBß-IV form that we had earlier identified in mouse tissues. Direct comparison of IBß forms from tissue and p65-reconstituted fibroblasts showed that the higher-molecular-weight form of IBß in fibroblasts expressing p65(313) migrated to the same apparent molecular weight as IBß-IV from brain and testis tissues (Fig. 5F) and, like IBß-IV in tissues, was sensitive to phosphatase treatment (Fig. 5G). In addition, in contrast to IBß-I, MG-132 treatment was unable to further increase the expression of IBß-IV (Fig. 5H), suggesting that this form of IBß is resistant to 26S proteasome activity. This result is consistent with our previous observation that IBß-IV regulation appeared to be less dependent on p65 compared to IBß-I (Fig. 3A) and was unchanged in p65^–/– MEFs treated with various proteasome inhibitors (Fig. 4D). Taken together, these results demonstrate that IBß dependence on p65 occurs due to the stabilization of IBß protein mediated largely through the p65 carboxyl terminus encompassing the TADs and that this regulation is specific to IBß-I.

Expression of IBß but not IB causes defects in cellular growth and survival. In the final analysis of this study, an attempt was made to understand the physiological relevance underlying the regulation of IBß by p65. Although it is widely believed that p65 is ubiquitously expressed, evidence suggests that p65 expression can be quite low or even undetectable during pregastrula development (21, 62) or in specific cell types in late embryogenesis (73). Based on these findings, as well as our present data, we asked if there was a reason why cells would need to degrade IBß under conditions where p65 expression is either low or absent. To address this question, an HA-tagged IBß retrovirus was generated in order to infect p65^–/– MEFs. Attempts to stably express wild-type IBß or an S19/23A mutant by this system or by conventional cytomegalovirus expression plasmids proved unsuccessful. MG-132 treatment partially restored recombinant IBß expression, demonstrating that the inability to express IBß in p65^–/– cells resulted from ongoing proteolysis (data not shown). We were, however, able to readily express a truncated form of IBß lacking the first 54 amino acids (Fig. 6A). This suggested that other determinants in the N terminus aside from Ser-19 and Ser-23 are required to mediate IBß proteasome-mediated degradation in p65^–/– cells. We refer to this nondegradable mutant as an IBß superrepressor (pBabeIBß-SR).

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FIG. 6. MEFs stably expressing IBß exhibit a growth defect and increased apoptosis. p65–/– MEFs were infected with either a pBabe vector retrovirus or a virus expressing a degradation-resistant form of IBß tagged with an HA epitope (pBabeIBß-SR). (A) Extracts from p65–/–-expressing vector or HA-IBß-SR cells were prepared, and Western blot assays were performed to probe for HA and -tubulin. (B) p65–/– cells expressing either the vector control or IBß-SR were treated with TNF-, and at the indicated times nuclear extracts were prepared for EMSA. A supershift EMSA was performed with antibodies against p50, p65, or c-Rel to confirm the composition of NF-B complexes (arrowheads denote supershifted subunits). (C) p65–/– MEFs were infected with a vector control or IBß-SR-expressing virus. Following 4 days of cell expansion under 1-µg puromycin selection, the cell number was determined in both cell lines. Cell number was normalized to vector control cells, which was set to a value of 100% growing cells. (D) Growth curves of p65+/+ or p65–/– cells expressing either a vector control or IBß-SR. (E) Growth curves identical to those performed in panel B from p65–/– cells infected with a vector control, IB-SR, or IBß-SR pBabe retrovirus. Cell number was normalized to the vector, which was set to a value of 100%. (F) Cells infected as in panel E were expanded under puromycin selection for 2 days and subsequently stained for Annexin V.

To address whether IBß-SR was functional, p65^–/– vector or IBß-SR-expressing cells were treated with TNF- and NF-B activity was monitored by EMSA. Results showed that even in the absence of p65, an NF-B complex was induced by TNF- that by supershift analysis was found to contain p50 and c-Rel subunits (Fig. 6B). Expression of the IBß-SR transgene reduced this activation, which confirmed the inhibitory property of the IBß protein. But despite this function, IBß-SR expression was not found to increase the incidence of TNF--mediated killing over that of vector control cells, which suggested that IBß destabilization in p65^–/– cells does not simply result to allow compensatory antiapoptotic function from c-Rel-containing complexes.

Clearly observable, however, was that p65^–/– MEFs expressing IBß-SR, while under puromycin selection, grew at a considerably slower rate than vector control cells (Fig. 6C). Although not as evident, this growth defect was maintained even in the absence of antibiotic treatment (data not shown). To determine whether this effect was dependent on p65, IBß-SR retroviral infections were repeated in both p65^+/+ and p65^–/– MEFs. Over time, a growth reduction was also observed in p65^+/+ MEFs, but not to the same extent as in p65^–/– cells (Fig. 6D). To further examine the specificity of this phenotype, p65^–/– MEFs were infected with viruses expressing nondegradable versions of either IB or IBß and growth rates were monitored compared to vector control cells. Results showed little growth difference between control and IB-SR-expressing cells, while again IBß-SR cells exhibited a clear growth defect (Fig. 6E). To address whether this defect was related to viability, cells were stained with Annexin V to monitor for apoptosis. Results showed that levels of apoptosis were nearly equivalent between vector control- and IB-SR-expressing cells. In contrast, apoptosis was increased approximately 65% in cells expressing IBß-SR (Fig. 6F). Collectively, these data demonstrate that IBß expression is linked to cellular growth defects, which provides at least one rationale to explain why IBß downregulation would be required in p65-deficient cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In contrast to IB, much less is known regarding the regulation of IBß by NF-B. The present study was performed based on the observation that p65^–/– MEFs contained dramatically lower levels of IBß. Although our group is not the first to note such a phenomenon (9, 40), a comprehensive study as to how p65 regulates IBß had yet to be undertaken. We have now performed such an analysis, which we believe provides fresh insight into the specificity, mechanism, and biological significance of this regulation.

One question we sought to address was the specificity of IBß regulation by p65. By using fibroblasts null for various components of NF-B and its signaling pathway, we determined that regulation of IBß only occurred in cells lacking p65. By this genetic criterion, the data strongly support the idea that IBß regulation is specific to this NF-B subunit. In addition to p65, IBß can also associate with c-Rel. It thus remains possible that the inability to detect IBß regulation in c-Rel^–/– MEFs may be due to underrepresentation of c-Rel in mouse fibroblasts. Nonetheless, to the best of our knowledge, regulation of IBß in tissues from c-Rel^–/– mice has not been reported, which further supports the specificity of p65 in this regulation.

Our findings also revealed the differences in the degree to which IB and IBß expression is dependent on p65. IB is the prototypical IB protein whose basal and stimulated expression is controlled by p50/p65 DNA binding sites within its promoter (24, 44, 51). From this perspective, it stands to reason why IB has long been considered to be the most highly regulated IB protein by NF-B, specifically by the p65 subunit. Our present data, however, challenge this thinking. We found that IB levels were indeed downregulated in p65^–/– primary MEFs and fetal liver cells, but only to about 60% of that of wild-type cells (Fig. 1). Such data support the role of p65 in regulating IB transcription and/or protein stability (64), but they also highlight the requirement for other factors in this regulation. In comparison, IBß expression was almost completely absent in these same cells, accentuating this protein's dependency on p65. These data provide compelling evidence that of these two IB proteins, IBß is the one most tightly regulated by the p65 subunit of NF-B.

Analysis into the mechanism of IBß downregulation revealed the regulation by the proteasome complex. Unlike stimulus-induced activation of NF-B that requires IKK-dependent phosphorylation of serine residues and subsequent polyubiquitination in the N terminus, we found that proteasome-mediated degradation of IBß was independent of both IKK and serines 19 and 23. However, deletion of the first 54 amino acids stabilized IBß, suggesting that other determinants within the signal response element are required for IBß turnover in cells lacking p65. This regulation is highly reminiscent of the mechanism controlling IB degradation in response to UV treatment, which also requires the N terminus but not IKK activity or phosphorylation of N-terminal serines (10, 52). Similar to UV-induced degradation of IB (10), it remains to be seen whether polyubiquitination in the signal response element of IBß is critical for its decay in p65^–/– cells.

The facts that the expression of p65 could completely restore IBß in p65^–/– MEFs and that B-Ras and ß-arrestin levels were unaffected in these cells suggested that p65 was a direct regulator of IBß stabilization (Fig. 5). Data derived from the IBß/p65 crystal structure have shown that much of the binding from the IBß inhibitor ankyrin repeats occurs in the RHD of p65, between residues 191 and 319 (57). Because these structures lacked the TADs of p65, it has not been possible to formally conclude whether residues in the carboxyl half of the RHD participate in IBß binding. To our surprise, however, it is precisely this region of p65, between residues 319 and 534, that was found to be required for maximal IBß stability. Based on these findings, we predict that IBß stabilization is directly regulated by contributions from both the RHD and the carboxyl-terminal half of p65. This carboxyl terminus is likely to adopt a particular conformation when p65 is activated and involved in interactions with the basal transcription machinery and transcriptional coactivators in the nucleus. As an inactive cytoplasmic complex, we foresee that the carboxyl half takes on a different structure as a result of direct or indirect contacts with IBß. Interestingly, S. Ghosh and colleagues had previously proposed that p65 bound to IBß in the cytoplasm exists as an intramolecular structure that, when phosphorylated on serine 276, undergoes a conformational change that is necessary for nuclear CBP/p300 interaction and p65 TAD function (76). In an analogous fashion, we reason that the carboxyl-terminal half of p65 within this intramolecular structure plays a critical role in mediating IBß stability.

Our results also revealed that destabilization of IBß due to removal of the p65 carboxyl half gave way to increased expression of an IBß variant which co-migrated with IBß-IV in tissues and existed in a hyperphosphorylated state (Fig. 5). Intriguingly, unlike IBß-I, which was stabilized by proteasome inhibition in p65^–/– MEFs, IBß-IV levels did not increase under these same conditions, nor were levels stabilized by proteasome inhibition in p65^–/– cells expressing carboxyl deletion mutants of p65. This implies that hyperphosphorylation of IBß-IV may be a contributing factor in the stability of this IBß form. While phosphorylation is generally considered to target proteins for degradation by the 26S proteasome, studies have also demonstrated the requirement of phosphorylation for protection from the proteasome (28). So perhaps deletion of the p65 carboxyl terminus can lead to IBß hyperphosphorylation, resulting in a protein now refractory to 26S proteasome activity. It will be interesting to ascertain in future studies whether stability associated with IBß-IV is a result of phosphorylation and whether this minor form of IBß that exists in tissues possesses a functional activity distinct from the predominantly expressed IBß-I form.

In the final aspect of our study, we attempted to address the physiological significance underlying the tight control of IBß by p65. Our data revealed that p65^–/– MEFs stably expressing a nondegradable form of IBß exhibited an extreme growth defect that could at least partially be attributed to apoptosis (Fig. 6). A similar but less severe phenotype was seen in wild-type cells, which argues that this defect is p65 dependent but also reveals the general susceptibility of cells to IBß expression. Importantly, our data demonstrated that this effect on growth and survival was specific to IBß since a comparable phenotype was not observed in cells expressing a nondegradable form of IB. Taken together, these results provide a rationale for why IBß may undergo such strong proteolysis in cells lacking p65 and the more pronounced regulation of IBß compared to IB. Our results indicate that even in p65-containing cells, proper regulation of IBß turnover is likely to be required to circumvent growth defects associated with stable expression of this protein. Although p65 is widely considered to be a constitutively expressed transcription factor, studies have shown that certain development stages early in embryogenesis (21, 62), or specific cell compartments of the thymus (73), do in fact possess low levels of p65. Such regulation of p65 has also been observed in selective neurons throughout development and adulthood (K. Pahan, personal communication). Based on our findings, we predict that it is precisely under such conditions that IBß proteolysis would be required in order to allow the proper development and maintenance of tissue homeostasis.

 
We thank U. Siebenlist and F. Weih for MEF knockout cell lines, A. Beg for RelA/p65 mice, S. Ghosh for B-Ras antibody, W. Miller for ß-arrestin antibody, M. Karin for cell lines and expression plasmids, and A. Baldwin for plasmids and helpful discussion. We also thank S. Acharyya and M. Carathers for technical assistance and the rest of the Guttridge laboratory for support and insight throughout the course of this study.

This work was supported by NIH grant CA97953 and startup funds to D.C.G. and the OSU Up on the Roof Fellowship to J.W.

 
* Corresponding author. Mailing address: Division of Human Cancer Genetics, 420 W. 12th Avenue, The Ohio State University College of Medicine, Columbus, OH 43210. Phone: (614) 688-3137. Fax: (614) 688-4006. E-mail: guttridge-1{at}medctr.osu.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, June 2005, p. 4956-4968, Vol. 25, No. 12
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