Am J Physiol Cell Physiol Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 292: C1013-C1023, 2007. First published November 1, 2006; doi:10.1152/ajpcell.00456.2006
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/C1013    most recent
00456.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Luo, Y.
Right arrow Articles by Harrington, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Luo, Y.
Right arrow Articles by Harrington, M. A.

RECEPTORS AND SIGNAL TRANSDUCTION

Phosphorylation of SIMPL modulates RelA-associated NF-{kappa}B-dependent transcription

Yong Luo,1,2 Hyung-Joo Kwon,2,3 Sherwin Montano,1 Millie Georgiadis,1 Mark G. Goebl,1,2 and Maureen A. Harrington1,2

1Department of Biochemistry and Molecular Biology, 2Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, Indiana; and 3Institute of Life Science and Biotechnology, Yonsei University, Seoul, South Korea

Submitted 25 August 2006 ; accepted in final form 31 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epidemiological data have implicated perturbations in the regulation of NF-{kappa}B activity to diseases that affect a large number of Americans today. Specifically, chronic activation of genes involved in the inflammatory response is associated with the progression of and complications in diabetes, arthritis, atherosclerosis, and cancer. Insight into the mechanisms governing the regulation of NF-{kappa}B transcriptional activity will provide the molecular link between NF-{kappa}B and these pathological states. SIMPL (signaling molecule that associates with mouse Pelle-like kinase) is a component of a signaling pathway through which tumor necrosis factor-{alpha} (TNF-{alpha}) induces NF-{kappa}B-controlled gene transcription. SIMPL interacts with the nuclear pool of the NF-{kappa}B subunit, p65, in a TNF-{alpha}-dependent manner to enhance p65-dependent gene transcription. How SIMPL activity is regulated is unknown. Under basal as well as TNF-{alpha}-stimulated conditions, SIMPL phosphopeptides were identified. SIMPL mutants lacking sites that are phosphorylated under basal conditions diminished p65 transactivation activity but had no effect on SIMPL nuclear localization. SIMPL mutants lacking sites of TNF-{alpha}-enhanced phosphorylation impaired nuclear localization and prevented TNF-{alpha}-induced p65 transactivation activity. Together, these studies reveal that phosphorylation of the SIMPL protein plays a critical role in SIMPL regulation by affecting both SIMPL subcellular localization and the p65 coactivator function of SIMPL.

cytokines; inflammation; signal transduction; nuclear factor-{kappa}B

THE INNATE IMMUNE RESPONSE is important for host defense against pathogens and is an integral component of the wound repair process (4, 10). Recent reports suggest the innate immune response can direct the nature of the subsequent acquired immune response, which involves B and T lymphocytes (29). Dysregulation of the innate immune response has been linked to several debilitating chronic inflammatory diseases, including rheumatoid arthritis and Crohn's disease, as well as the complications associated with diabetes and cancer (2, 23). In vivo, the cytokine TNF-{alpha} is required for activation of the innate immune response (26). Key transcriptional regulators of TNF-{alpha}-induced genes are the NF-{kappa}B/c-rel family of transcription factors (1).

The most prevalent form of NF-{kappa}B is the p50/p65 heterodimer. In its inactive state, NF-{kappa}B is bound by the inhibitor I{kappa}B{alpha} (inhibitor of NF-{kappa}B) and the complex shuttles between the cytoplasm and nucleus (18). In response to TNF-{alpha} stimulation, the IKK{alpha}/IKKbeta/IKK{gamma} (IKK) complex phosphorylates serines 32 and 36 of I{kappa}B{alpha} (8, 24, 27, 35, 37); phosphorylated I{kappa}B{alpha} is ubiquitinated, thus targeting I{kappa}B{alpha} for destruction by the proteasome (6). Removal of the I{kappa}B{alpha} causes nuclear localization signal (NLS) on NF-{kappa}B to be uncovered and allows NF-{kappa}B to translocate into the nucleus to regulate changes in gene transcription. Genetic analysis has revealed that TNF-{alpha}-dependent activation of NF-{kappa}B requires IKK complex activity, and in the absence of IKK{alpha} and IKKbeta, signal-dependent activation of NF-{kappa}B-controlled gene expression does not occur (17).

In a previous report (34), our laboratory identified a novel protein called SIMPL (signaling molecule that interacts with Pelle-like kinase) that we determined is required for TNF-{alpha} but not IL-1-dependent activation of NF-{kappa}B activity (34). Ectopic expression of SIMPL induces the activity of an NF-{kappa}B-controlled reporter construct. Furthermore, the induction of NF-{kappa}B activity is blocked in the presence of either catalytically inactive IKK{alpha} or IKKbeta. Intriguingly, a SIMPL mutant that blocks TNF-{alpha}-induced NF-{kappa}B activity also blocks ectopic IKK{alpha} or IKKbeta induction. SIMPL contains a carboxy-terminal nuclear localization signal, and nuclear localization of SIMPL is required for TNF receptor type I (TNF RI)-induced NF-{kappa}B activity (16). Nuclear SIMPL interacts with p65 in a TNF-{alpha}-dependent manner, enhancing p65 transactivation activity and leading to an increase in endogenous NF-{kappa}B-dependent gene expression (16). On the basis of these data, we proposed a model in which TNF-{alpha}-dependent activation of endogenous NF-{kappa}B-dependent gene expression requires at least two independent events: the nuclear localization of NF-{kappa}B and the nuclear localization of SIMPL.

How SIMPL activity is regulated is largely unknown. We originally identified SIMPL in a yeast two-hybrid system screen by using the first 500 amino acids of the serine/threonine kinase IRAK-1 (IL-1 receptor-associated kinase) as bait (34). The catalytic activity of IRAK-1, along with several other serine/threonine kinases (including the IKKs, Akt, and MEKKs), has been linked to TNF-{alpha}-dependent activation of NF-{kappa}B (17, 25, 33, 36). We therefore postulated that SIMPL regulation occurs, at least in part, by regulated phosphorylation. In this report we describe the identification of basal and TNF-{alpha}-enhanced sites of phosphorylation in SIMPL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasmids and antibodies. The Flag-SIMPL construct was described previously (16), as were the wild-type and catalytically inactive (IRAK-1D358N) myc-tagged IRAK-1 constructs (33). The IRAK-1 antisense construct was generated by inverting the IRAK-1 cDNA. The Gal4-p65 and Gal4-Luc constructs were kindly provided by Dr. Richard Gaynor (University of Texas Southwestern Medical Center, Dallas, TX). PCR-based mutagenesis was utilized to generate SIMPL mutants by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Primer sets 5'-CGTGAGGTCCACGTGGCCGGCGCGGCCGAGGTG-3' and 5'-CACCTCGGCCGCGCCGGCCACGTGGACCTCACG for SIMPLS55A and 5'-GGCGCGGCCGAGGTGGCCGCCGCCCCCGACCGAGCG-3' and 5'-CGCTCGGTCGGGGGCGGCGGCCACCTCGGCCGCGCC-3' for SIMPLS61,63A were sequentially used to generate the SIMPLS55,61,63A mutant. Primer sets 5'-CAACAGAAAATCAAAGCTGCAACCATCCATGCAG-3' and 5'-CTGCATGGATGGTTGCAGCTTTGATTTTCTGTTG-5' for SIMPLS234A; 5'-CAAAGCTGCAGCCATCCATGC-3' and 5'-GCATGGATGGCTGCAGCTTTG-3' for SIMPLT236A, 5'-CATCCATGCAGCTGCGAAGGTGTTCATTAC-3' and 5'-GTAATGAACACCTTCGCAGCTGCATGGATG-3' for SIMPLS241A, and 5'-CGAAGGTGTTCATTGCTTTTGAGGTAAAAGG-3' and 5'-CCTTTTACCTCAAAAGCAATGAACACCTTCG-3' for SIMPLT246A were sequentially used to generate SIMPLNLSMU (contains S234A, T236A, S241A, and T246A substitutions). The sequence of all mutants was confirmed using automated DNA sequencing in the Biochemistry Biotechnology Facility (Indiana University School of Medicine). The mouse monoclonal anti-Flag antibody was purchased (Sigma, St. Louis, MO). The mouse c-myc monoclonal antibody and goat anti-hemagglutinin (HA) antibody were purchased from Roche Biochemicals (Indianapolis, IN). Chromatographically purified mouse IgG was purchased from Zymed Laboratories (So. San Francisco, CA). The rabbit IKK{alpha}, IKKbeta, and IRAK-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit IRAK-4 antibody was purchased from Upstate (Charlottesville, VA).

Cell culture and transient transfections. Human embryonic kidney (HEK)-293EBNA cells (overexpressing the Epstein-Barr virus nuclear antigen-1; Invitrogen, Carlsbad, CA) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 500 µg/ml G-418 (Cellgro, Herndon, VA). The C3H10T1/2 mouse embryo fibroblast cell line was maintained as described previously (33). Cells were plated 24 h before transfection and transfected using Fugene6 (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's recommendation. Twenty-four hours after transfection, cells were treated with TNF-{alpha} [recombinant human (rHu), 10 ng/ml; Sigma] for the indicated time before harvest.

Luciferase assay. Cells were harvested and lysed 24 h after transfection or after the indicated period of TNF-{alpha} treatment. Luciferase activities were determined using the luciferase assay system (Promega, Madison, WI) according to the manufacturer's specifications. Individual assays were normalized for protein content. Experiments were performed in triplicate and repeated two or three times with similar results. The statistics were done using Student's t-test, with P < 0.05 considered significant.

p65 Transactivation assay. Mammalian expression vectors encoding the SIMPL cDNA constructs were transfected together with a construct encoding the Gal4 DNA binding domain fused in-frame to the p65 transactivation domain and a reporter construct in which the firefly luciferase cDNA is placed under the control of the yeast upstream activator sequence (UAS-luciferase). Twenty-four hours later, cells were treated with rHuTNF-{alpha} (10 ng/ml) for 18 h. All cultures were harvested, collected by centrifugation, and lysed, and luciferase assays were performed as described above. Assays were performed in triplicate and normalized by protein content. Statistics were done using a Student's t-test, with P < 0.05 considered significant.

Western blotting analysis. Cell monolayers were rinsed twice with ice-cold PBS, harvested with rubber policemen, and collected by centrifugation. Lysis buffer (50 mM Tris·HCl, pH 7.4, 125 mM NaCl, 1% Triton X-100, 0.5% NP-40, 2 mM sodium vanadate, 0.1 µM okadaic acid, 10 mM beta-glycerophosphate, 50 mM NaF, and 1 mM sodium pyrophosphate) supplemented with a Complete protease inhibitor tablet (Roche); cells were incubated on ice for 30 min with occasional vortexing. Cellular lysates were centrifuged (15,000 g, 4°C, 10 min). The protein concentration of the supernatants was measured (Bio-Rad, Hercules, CA), and equal amounts of protein were subjected to separation on 10% SDS-polyacrylamide gels. Western blotting was performed as described previously (33).

Metabolic labeling and immunocomplexing. Metabolic labeling with 32PO4 was performed as described previously (8). In brief, 24 h after transfection with a mammalian expression vector encoding the Flag-SIMPL cDNA, HEK-293EBNA cell monolayers were rinsed with phosphate-free DMEM medium four times and preincubated (37°C, 5% CO2) in 5 ml of phosphate-free DMEM medium for 1 h, followed by incubation for 4 h in phosphate-free DMEM containing 1 mCi/ml [32P]orthophosphate (i.e., 3,000 Ci/mmol specific activity; MP Biomedicals, Irvine, CA) and 10% dialyzed-FBS. TNF-{alpha} (10 ng/ml) or IL-1 (20 ng/ml) was added 20 min before cultures were harvested. Cells were harvested, collected by centrifugation, and lysed as described directly above. Cell extracts were precleared by incubation with protein A coupled to Sepharose beads (100 µl per ml of extract) with end-over-end rotation at 4°C for 1 h. Supernatants were transferred to a fresh tube, together with 5 µg of Flag antibody (Sigma) and 150 µl of protein A beads. Mixtures were incubated overnight at 4°C. Immunocomplexes were collected by centrifugation and subjected to seven cycles of resuspension and pelleting until no significant radioactivity was detected in the wash buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and Complete protease inhibitor tablet). Immunocomplexed materials were denatured, subjected to SDS-PAGE (10% gel), and transferred to 0.45-µm pore size nitrocellulose (Schleicher & Schuell). Membranes were rinsed in double-distilled water, and autoradiograms were generated by exposure to X-ray film. Radioactive bands corresponding to Flag-SIMPL were excised, and the amount of radioactivity was determined (Cerenkov).

Two-dimensional phosphopeptide mapping and phosphoamino acid analysis. Tryptic digestion of SIMPL immobilized on the nitrocellulose membranes was performed as described previously (3, 9). In brief, excised portions of the membrane were incubated in 0.5% PVP-360 (polyvinyl-pyrrolidone) and 100 mM acetic acid for 30 min at 37°C. Membranes were washed five times with double-distilled water, followed by two washes with 50 mM ammonium bicarbonate. Digests were performed using 10 µg of N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (TPCK-trypsin) in 200 µl of 50 mM ammonium bicarbonate at 37°C for 2 h, followed by the addition of another 10 µg of TPCK-trypsin for an additional 2 h of incubation. Three hundred microliters of double-distilled water were added to the samples, and the liquid was transferred to a new tube. Samples were lyophilized and then incubated in 50 µl of performic acid [9 parts formic acid and 1 part 30 (wt/vol) hydrogen peroxide] on ice for 1 h to oxidize all methionine and cysteine residues. Samples were diluted with double-distilled water, lyophilized, and subjected to two additional cycles of resuspension in water and lyophilization. Samples were dissolved in pH 1.9 buffer [2.5% (vol/vol) formic acid, 7.8% (vol/vol) glacial acetic acid] and spotted onto a TLC plate (Merck; 20 x 20 cm). Before application to the TLC plates, the radioactivity of each sample was measured by Cerenkov counting, and the same amount of radioactivity of each sample was spotted onto the TLC plate. For two-dimensional phosphopeptide mapping, the first dimension (electrophoresis) was performed in pH 1.9 buffer by using an HTLE 7000 apparatus (CBS Scientific); the second dimension was performed in phosphochromatography buffer [37.5% (vol/vol) n-butanol, 25% (vol/vol) pyridine, 7.5% (vol/vol) glacial acetic acid]. The plates were dried and exposed to film at –80°C to generate autoradiograms. To determine which phosphoamino acid was phosphorylated, the cellulose containing the radiolabeled material was scraped off the plate and peptides were eluted from the cellulose with pH 1.9 buffer. The materials were lyophilized, resuspended in 100 µl of 6 N HCl, and incubated at 110°C for 60 min. Samples were lyophilized and rinsed several times with water. Two-dimensional phosphoamino acid analysis mapping was performed essentially as described previously (3).

Mass spectrometry. Nonradiolabeled samples were prepared in parallel to those used to generate the phosphopeptide maps. Mass spectrometry was performed by the Indiana Centers for Applied Protein Sciences with support in part from the Indiana Genomics Initiative and the Indiana 21st Century Research and Technology Fund. Briefly, matrix-assisted laser desorption/ionization (MALDI) mass spectra were recorded in positive reflectron mode of the MALDI-TOF mass spectrometer (Micromass, Manchester, UK). The time of flight was measured using the following parameters: 3,400-V pulse voltage, 15,000-V source voltage, 500-V reflectron voltage, 1,950-V multichannel plate voltage, and low mass gate of 500 Da. Internal calibration was performed using autodigestion peaks of bovine trypsin [M+H+, mass-to-charge ratio (m/z) 842.5099 and m/z 2,211.1045] in the same series as the samples to be measured. The measured peptide mass profiles were then compared with the theoretical peptide masses by using the ProFound search engine and National Center for Biotechnology Information database for protein or peptide identity. A match was considered if the difference between the theoretical peptide mass and the observed peptide mass was <0.5. Masses were screened for the presence of SIMPL phosphopeptides by using the calculated tryptide-SIMPL peptide m/z plus 80, 160, and 240 (phosphorylation adds 80 Da to the mass) against the MALDI mass spectrometry data.

In vitro kinase assays. Immunocomplexes were generated and subjected to four cycles of pelleting and resuspension in kinase assay buffer (20 mM Tris·HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT). Bacterial SIMPL protein (1 µg) or SIMPL mutants generated by immunocomplexing were added in 20 µl of kinase assay buffer with 10 µM ATP plus 10 µCi of [{gamma}-32P]ATP (3,000 Ci/mmol) per reaction. The kinase assays were incubated at 37°C for 30 min, and reactions were terminated by addition of an equal volume of 2x loading buffer and boiled for 10 min before being subjected to SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes that were exposed to X-ray film to generate autoradiograms.

Cloning, expression, and purification of recombinant SIMPL. An NH2-terminally tagged SIMPL-hexa-His plasmid was constructed by cloning a PCR-generated fragment containing the SIMPL open reading frame into the NdeI and XhoI sites of pET15b. A stop codon (TAA) was introduced before the XhoI site. Positive clones were identified by restriction analysis and protein expression in Rosetta Escherichia coli cells (Novagen). Cells were grown at 37°C in Luria-Bertani medium containing ampicillin (50 µg/l) and chloramphenicol (34 µg/l) until the optical density reached 0.70 to 1.0. Protein expression was induced by the addition of isopropyl-beta-D-thiogalactopyranoside to a final concentration of 1 mM, and cells were allowed to grow for three more hours. Cells were harvested by centrifugation, and cell pellets were stored at –80°C until protein purification was initiated. Cell pellets were suspended in 50 mM NaH2PO4/Na2HPO4 (pH 7.8) buffer containing 0.3 M NaCl and 10 mM imidazole and then lysed in two passes through a French pressure cell at 1,000 lb./in.2. Crude extracts were obtained by centrifugation of the lysates at 35,000 rpm at 4°C. The lysates were filtered through 0.45-µm filters and passed over a Ni-NTA agarose column preequilibrated with the lysis buffer described above. The column was washed using 10 column volumes of the phosphate buffer containing 20 mM imidazole. The protein was then eluted using 250 mM imidazole.

Immunofluorescence confocal microscopy. Mouse embryo fibroblasts (C3H10T1/2 cell line) or the HEK epithelial cells containing the Epstein-Barr nuclear antigen-1 (HEK-293EBNA cell line) were plated directly on glass coverslips, and 24 h later cells were transfected with the indicated constructs. Twenty-four hours after transfection, cells were fixed in PBS containing 4% paraformaldehyde, permeabilized by incubation in PBS containing 0.2% Triton X-100 for 10 min, and blocked in PBS supplemented with 1% BSA and 0.2% Tween 20 for 1 h. When C3H10T1/2 mouse embryo fibroblasts were analyzed, Flag antibody was applied for 1 h, followed by a 1-h incubation with fluorescein-conjugated anti-mouse IgG. DNA staining (0.5 µg/ml Hoechst 33258; Sigma) was used to identify cell nuclei. For double-immunofluorescence staining of Flag-SIMPL and myc-IRAK-1, primary mouse antibody that recognizes the myc-epitope was detected with fluorescein-conjugated anti-mouse IgG, primary mouse antibody that recognizes the Flag-epitope was detected with Texas red-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories), and primary mouse antibody that recognizes the myc-epitope was detected with fluorescein isothiocyanate-conjugated anti-mouse IgG (Zymed). DNA staining (0.5 µg/ml Hoechst 33258; Sigma) was used to identify cell nuclei. Coverslips were mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Samples were scanned with a Zeiss LSM 510 laser scanning confocal device attached to an Axiovert 100 microscope using a C-Apochromat 40X/1.2W Corr objective (Zeiss). Hoechst 33258, fluorescein, or Texas red was excited with laser light at wavelengths of 351, 488, or 543 nm, respectively. Fluorescence acquisitions were performed with the 351-, 488-, or 543-nm laser lines to excite UV, fluorescein isothiocyanate, or Texas red, respectively. To avoid bleed-through effects in double staining experiments, we scanned each dye independently using the multitracking function of the LSM 510 unit. Images were electronically merged using the LSM 510 software and stored as TIFF files. Figures were assembled from TIFF files using Microsoft PowerPoint software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Two-dimensional phosphopeptide mapping identifies basal as well as TNF-{alpha}-induced SIMPL phosphopeptides. Previous work in our laboratory has shown that SIMPL is a component of a signaling pathway through which TNF-{alpha} induces NF-{kappa}B-controlled gene expression in a TNF RI-dependent manner (16, 33). Initiation of the TNF-{alpha}/TNF RI-dependent signaling pathway leading to activation of NF-{kappa}B involves several kinases. To determine whether SIMPL is modified, basally or in response to TNF-{alpha}, we performed phosphopeptide mapping studies. HEK-293EBNA cell cultures transfected with a construct encoding Flag-tagged SIMPL were metabolically labeled with [32P]orthophosphate. One set of cultures was left untreated, a second set was treated with TNF-{alpha} (rHu, 10 ng/ml), and, as a specificity control, a third set of cultures was treated with IL-1{alpha} (rHu, 20 ng/ml). Twenty minutes later, all cultures were harvested and collected by centrifugation. Cell lysates were generated, and immunocomplexes were isolated with the M2 Flag antibody. Immunocomplexed materials were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Autoradiography was used to localize the SIMPL protein that was then excised and subjected to phosphopeptide mapping as described in MATERIALS AND METHODS.

The total amount of the radioactivity in Flag-SIMPL isolated from control, TNF-{alpha}-, or IL-1{alpha}-treated cultures did not differ significantly (Fig. 1A). Two-dimensional phosphopeptide maps identified a heavily radiolabeled peptide present in SIMPL isolated from untreated (control), TNF-{alpha}-, and IL-1{alpha}-treated cultures (Fig. 1B, filled arrowheads). The phosphopeptide maps of SIMPL isolated from all sets of cultures were remarkably similar; however, in the TNF-{alpha}-treated sample, we detected a more intense spot, presumably reflecting a greater level of phosphorylation of a specific peptide (Fig. 1B, open arrowhead). These results indicate that under basal conditions, SIMPL is phosphorylated on several residues, most strikingly on the peptide corresponding to the spot labeled "basal." In response to a brief treatment with TNF-{alpha}, the amount of radioactivity associated with a single phosphopeptide was preferentially increased (Fig. 1B, open arrowhead).


Figure 1
View larger version (71K):
[in this window]
[in a new window]

  		Fig. 1. Basal and TNF-{alpha}-induced SIMPL (signaling molecule that associates with mouse Pelle-like kinase) phosphopeptides. HEK-293EBNA cell cultures were transfected with a mammalian expression vector encoding SIMPL (5 µg/plate). Twenty-four hours later, cell monolayers were rinsed 4 times with phosphate-free DMEM and then incubated in phosphate-free DMEM supplemented with [32P]orthophosphate for 4 h. Cultures were either left untreated or were treated with TNF-{alpha} (recombinant human, rHu; 10 ng/ml) or IL-1{alpha} (rHu, 20 ng/ml) for 20 min. Cells were harvested, and Flag antibody was used to generate immunocomplexes. Individual samples, normalized by the amount of radioactivity, were subjected to SDS-PAGE (10%) and transferred to nitrocellulose membrane. A: autoradiography of the membranes was used to identify the location of the radiolabeled SIMPL protein. B: tryptic digests of excised band corresponding to SIMPL were subjected to 2-dimensional peptide mapping. The location of the phosphopeptides visualized by autoradiography is indicated (filled arrowhead, major basal; open arrowhead, induced). C: phosphoamino acid analysis of the basal peptides. Location of the phosphorylated serine (pSer), threonine (pThr), and tyrosine (pTyr) standards is indicated.

 
To identify the type of amino acid residues phosphorylated in the major basal phosphopeptide, this major basal phosphopeptide from each sample was recovered from the cellulose plate and subjected to acid hydrolysis (described in MATERIALS AND METHODS). Results of these experiments revealed that only serine residues were phosphorylated in the major basal peptide (Fig. 1C). The limiting amount of material recovered for the TNF-{alpha}-induced phosphopeptide precluded phosphoamino acid analysis of the induced peptide.

To identify the tryptic peptides in SIMPL phosphorylated under basal conditions and in response to TNF-{alpha}, we generated two-dimensional phosphopeptide maps with nonradiolabeled material as well as radiolabeled Flag-SIMPL isolated from duplicate sets of control and TNF-{alpha}-treated cultures. The autoradiogram of the tryptic peptides generated with the radiolabeled Flag-SIMPL protein was used as a guide to isolate the corresponding material on the TLC plate containing the nonradiolabeled tryptic peptides. The tryptic peptides corresponding to the major basal and induced spots (Fig. 1B) were recovered from the cellulose plates and subjected to MALDI-TOF mass spectrometry (described in MATERIALS AND METHODS). The putative phosphopeptides with their corresponding m/z ratios are shown in Table 1. More than one peptide was identified in the mass spectrometry analysis and is most likely the result of isolating adjacent tryptic fragments. The autoradiogram of the radiolabeled samples was used as a guide for isolating the nonradiolabeled sample, which contained ninefold more material, thus the position of each spot was most likely not identical. In each case (major basal and induced), we focused on the putative phosphopeptide for which the selected ion count was the highest.


View this table:
[in this window]
[in a new window]

  		Table 1. Putative phosphopeptides

 
Basal sites of phosphorylation on SIMPL. Consistent with the phosphoamino acid analysis of the radiolabeled basal tryptic peptide in which only phosphoserine residues were detected, three serine residues and no threonine or tyrosine residues were detected in the peptide spanning amino acid residues 51–66 (EVHVSGAAEVSASPDR). To determine whether the phosphopeptide corresponding to this putative major basal site of phosphorylation in SIMPL was correctly identified, we used site-directed mutagenesis to convert all three serine residues to alanine residues, generating SIMPLS55,61,63A. Ectopic expression of the Flag-tagged SIMPLS55,61,63A mutant in HEK-293EBNA cells revealed that it was expressed as well as Flag-SIMPL (Fig. 2A, WB). To determine whether the alanine substitutions decreased the basal level of SIMPL phosphorylation, we performed metabolic labeling studies with cultures of HEK-293EBNA cells transfected with either Flag-SIMPL or Flag-SIMPLS55,61,63A. Analysis of the metabolically labeled SIMPL proteins revealed an overall decrease in the phosphorylation of the SIMPLS55,61,63A mutant compared with wild-type SIMPL (Fig. 2A, metabolic labeling). A comparison of the two-dimensional phosphopeptide maps of metabolically labeled SIMPLS55,61,63A mutant and wild-type SIMPL proteins revealed a loss of the major radiolabeled phosphopeptide (Fig. 2B, compare with wild type). There also was a decrease in the abundance of several more faintly detectable phosphopeptides in the SIMPLS55,61,63A mutant compared with wild-type SIMPL. The latter changes may reflect either a requirement for phosphorylation at the "basal sites" before modification at other sites, and/or the introduced mutations may influence the structure of the SIMPL such that other sites of phosphorylation are not recognized by their cognate kinase(s). Two-dimensional phosphopeptide mapping of a fragment of SIMPL spanning amino acids 1–90 revealed a single radiolabeled fragment (Fig. 2C). These data lend further support to our hypothesis that serines 55, 61, and/or 63 are the residues modified in SIMPL under basal conditions.


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 2. Serines 55, 61, and 63 are phosphorylated under basal conditions. HEK-293EBNA cells were transfected with a mammalian expression vector encoding either wild-type (WT) SIMPL or SIMPLS55/61/63A. Twenty-four hours later, cell monolayers were rinsed 4 times with phosphate-free DMEM and then incubated in phosphate-free DMEM supplemented with [32P]orthophosphate for 4 h. Cells were harvested and lysed, and Flag antibody was used to isolate SIMPL proteins that were subjected to SDS-PAGE and transferred to nitrocellulose membranes. A: autoradiography was used to localize the radiolabeled SIMPL; membranes were reprobed with Flag-antibody to confirm that equal amounts of SIMPL protein were isolated. WB, Western blot. B: tryptic digests of radiolabeled, immunocomplexed Flag-SIMPL proteins were separated and subjected to 2-dimensional peptide mapping, and radiolabeled peptides were visualized using autoradiography. C: a SIMPL mutant spanning residues 1–90 was expressed in HEK-293EBNA cells and subjected to metabolic labeling and 2-dimensional peptide mapping as described in B.

 
SIMPLS55,61,63A has impaired activity. Ectopic expression of wild-type SIMPL increases endogenous NF-{kappa}B-controlled gene expression (34) by enhancing the transactivation activity of p65 (16). Thus it was of interest to determine whether the SIMPLS55,61,63A mutant retained this activity. Constructs encoding wild-type SIMPL, SIMPLS55,61,63A, or a SIMPL mutant lacking the SIMPL nuclear localization signal (NLS; aa 239–259), a domain known to be required for SIMPL activity (16), were cotransfected into mouse embryo fibroblasts with a construct encoding the Gal4 DNA binding domain fused in-frame to the p65 transactivation domain (Gal4-p65TAD) and a luciferase reporter under control of the yeast Gal4 transcription factor. Twenty-four hours later, cultures were harvested, cell lysates were generated, and luciferase activities were measured. Consistent with previous studies, ectopic expression of a SIMPL mutant lacking the NLS had no effect on p65 transactivation activity (Fig. 3). Compared with wild-type SIMPL, the activity of the SIMPLS55,61,63A mutant was reduced by about 30% (Fig. 3; P < 0.01). Results of these studies reveal that the SIMPLS55,61,63A mutant is functional but that phosphorylation of amino acid residues 55, 61, and/or 63 is required for full activity.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 3. Impaired induction of p65 transactivation by SIMPLS55,61,63A. C3H10T1/2 mouse embryo fibroblasts were transfected with 100 ng of the indicated constructs together with 10 ng of mammalian expression vector encoding the Gal4 DNA binding domain-p65 transactivation domain fusion protein and a firefly luciferase cDNA under the control of Gal4. Twenty-four hours later, cells were harvested and luciferase activity was quantitated (described in MATERIALS AND METHODS). SIMPL{Delta}NLS, SIMPL mutant lacking the SIMPL nuclear localization signal. *P < 0.01.

 
Identification of TNF-{alpha}-induced sites of phosphorylation in SIMPL. Analysis of phosphopeptides increased in response to TNF-{alpha} by mass spectrometry identified three potential phosphopeptides (Fig. 1B and Table 1). The phosphopeptide spanning amino acid residues 234–242 (SATIHAASK) flanking the carboxy-terminal NLS (aa residues 239–259) in SIMPL had the highest selected ion count. Nuclear localization of SIMPL is required for SIMPL induction of NF-{kappa}B-dependent gene transcription, and ectopic expression of SIMPL{Delta}NLS inhibits TNF-{alpha}-induced activation of the NF-{kappa}B-dependent IL-8 gene promoter (16). Analysis of other nuclear proteins has revealed that phosphorylation in or near the NLS can modulate nuclear localization (13, 32). Therefore, we pursued the peptide spanning amino acid residues 234–242 and included in the analysis threonine 246, because it is a potential site of phosphorylation located in the NLS of SIMPL. Site-directed mutagenesis was used to convert serines 234 and 241 and threonines 236 and 246 to alanine residues to generate SIMPLS234,241A,T236,246A. To determine whether these phosphorylation sites preferentially modified in response to TNF-{alpha} could be detected in SIMPLS234,241A,T236,246A, we performed two-dimensional phosphopeptide mapping. Duplicate sets of HEK-293EBNA cells were transfected with either Flag-tagged wild-type SIMPL or Flag-tagged SIMPLS234,241A,T236,246A, and 24 h later all cultures were incubated in medium supplemented with [32P]orthophosphate for 4 h. Twenty minutes before harvest, one set of cultures was treated with TNF-{alpha} (rHu, 10 ng/ml). Cultures were harvested, and Flag antibody was used to generate immunocomplexes that were subjected to SDS-PAGE. Autoradiography of the polyacrylamide gel revealed no detectable difference in the total amount of radioactivity incorporated into the SIMPLS234,241A,T236,246A mutant compared with wild-type SIMPL (Fig. 4A). Analysis of the phosphopeptide maps generated with wild-type SIMPL and SIMPLS234,241A,T236,246A revealed that the abundance of the preferentially phosphorylated peptide was undetectable (Fig. 4B).


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 4. Identification of the TNF-{alpha}-induced phosphopeptide. HEK-293EBNA cells were transfected with a mammalian expression vector encoding Flag-tagged SIMPL or SIMPLS234,241A,T236,246A. Twenty-four hours later, cells were washed in phosphate-free DMEM and then incubated in DMEM supplemented with [32P]orthophosphate for 4 h. One set of each transfectant was treated with TNF-{alpha} (rHu, 10 ng/ml) for 20 min before harvesting. Cultures were harvested and lysed, and Flag antibody was used to generate immunocomplexes that were subjected to SDS-PAGE and transferred to nitrocellulose membranes. A: autoradiography of immunocomplexed SIMPL proteins. B: tryptic digests of immunocomplexed SIMPL protein were separated on cellulose plates, and the phosphopeptides were visualized using autoradiography (open arrowhead indicates location of induced phosphopeptide).

 
SIMPLS234,241A,T236,246A blocks TNF-{alpha}-induced NF-{kappa}B transactivation. To determine whether phosphorylation of the SIMPL peptide SATIHAASKVFIT affects the ability of SIMPL to induce NF-{kappa}B activity, we examined the ability of SIMPLS234,241A,T236,246A to enhance p65 transactivation activity. In one set of experiments, construct encoding wild-type SIMPL, a SIMPL mutant lacking the NLS (SIMPL{Delta}NLS), or SIMPLS234,241A,T236,246A was cotransfected with constructs encoding a Gal4 DNA binding domain-p65 transactivation domain fusion protein and a luciferase reporter construct under the control of Gal4. Twenty-four hours later, cells were treated with TNF-{alpha} (10 ng/ml) for 18 h. As shown previously, ectopic expression of SIMPL enhances p65 transactivation activity (Fig. 5A). The ability of the SIMPLS234,241A,T236,246A mutant compared with wild-type SIMPL to enhance the activity of the p65 transactivation domain was significantly reduced (Fig. 5A). Consistent with the reports of others (28), in response to TNF-{alpha} there was a modest (3-fold) increase in the activity of the Gal4-p65 fusion protein, which was inhibited in cultures expressing the SIMPL mutant lacking its NLS or the SIMPLS234,241A,T236,246A. Thus phosphorylation of SIMPL is required for TNF-{alpha}-induced activation of p65 transactivation activity. Next, we examined the requirement for SIMPL phosphorylation in TNF-{alpha} induction of the IL-8 gene promoter, which is a complex promoter composed of binding sites for several transcription factors in addition to NF-{kappa}B, including activator protein 1 and CCAAT/enhancer binding protein-beta. Previous studies linked the SIMPL activity to NF-{kappa}B-dependent changes in gene expression (16). As shown previously, if cultures overexpress SIMPL{Delta}NLS instead of wild-type SIMPL, the TNF-{alpha}-induced increase in IL-8 promoter activity decreases to a level below that detected when cultures are treated with TNF-{alpha} alone (Fig. 5B); in parallel to this result and those obtained in the assays presented directly above, overexpression of SIMPLS234,241A,T236,246A decreased TNF-{alpha}-induced IL-8 promoter activity. Previous reports also have demonstrated that NF-{kappa}B activation is elicited through overexpression of signaling pathway components such as Akt and MEKK3 in the absence of TNF-{alpha} (25, 36). However, in each case, expression of a dominant inactivating mutant blocked TNF-{alpha} activation of NF-{kappa}B-dependent gene expression.


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 5. SIMPLS234,241A,T236,246A blocks TNF-{alpha}-dependent p65 transactivation activity. Duplicate sets of mouse embryo fibroblasts (C3H10T1/2 cells) were transfected with the indicated SIMPL constructs (50 ng) and a construct encoding a Gal4-controlled firefly luciferase cDNA (10 ng) and a Gal4DBD-p65TAD construct (10 ng) (A) or a construct encoding the luciferase cDNA under the control of the IL-8 gene promoter (B). Twenty-four hours later, one set of cultures was treated with TNF-{alpha} (rHu, 10 ng/ml). Eighteen hours later, cultures were harvested and luciferase activity was quantitated. *P < 0.01; **P < 0.01.

 
SIMPLS234,241A,T236,246A localizes in the cytoplasm. The loss of the ability of SIMPLS234,241A,T236,246A to enhance the p65 transactivation activity could reflect either an inability to undergo nuclear localization or a loss of biological activity. Since the sites in SIMPL preferentially phosphorylated in response to TNF-{alpha} were located near the SIMPL NLS, we examined the subcellular localization of the SIMPLS234,241A,T236,246A mutant. Duplicate sets of mouse embryo fibroblasts were transfected with constructs encoding either wild-type SIMPL or various SIMPL mutants. Twenty-four hours later, cultures were fixed and immunofluorescence microscopy (described under MATERIALS AND METHODS) was used to determine their subcellular localization. As shown previously (16), ectopically expressed wild-type SIMPL can be found in the nucleus independently of TNF-{alpha} treatment, whereas SIMPL{Delta}NLS is predominantly cytoplasmic. Ectopically expressed SIMPLS234,241A,T236,246A is also preferentially located in the cytoplasm (Fig. 6). These results indicate that phosphorylation of residues adjacent to or within the NLS are required for SIMPL nuclear localization.


Figure 6
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 6. Cytoplasmic localization of SIMPLS234,241A,T236,246A. Mouse embryo fibroblasts (C3H10T1/2 cells) were transfected with the indicated SIMPL constructs (600 ng). Twenty-four hours later, cultures were fixed and processed for processed for immunofluorescence with Flag-specific antibody (Flag). Nuclei were stained with Hoechst 33258 (DAPI).

 
IRAK-1 phosphorylates SIMPL in vitro. SIMPL was originally identified as a binding partner of IRAK-1 in yeast two-hybrid screen (34). Since IRAK-1 is a serine/threonine kinase required for full TNF-{alpha} activation of NF-{kappa}B-dependent signaling (30, 34), we examined whether SIMPL is a substrate for IRAK-1. IRAK-1 also has been found in complexes containing IRAK-4 (5, 19) and in complexes containing IKK{alpha} and IKKbeta (17). IRAK-4, IKK{alpha}, and IKKbeta are serine/threonine kinases, as is IRAK-1. Thus we examined whether SIMPL could serve as a substrate for any of these kinases. To address this question, we generated immunocomplexes by using antibodies specific for each of these kinases (IRAK-1, IRAK-4, IKK{alpha}, and IKKbeta) and performed in vitro kinase assays by using bacterially expressed SIMPL as a substrate. In previous studies using immunocomplexed IKK{alpha}, IKKbeta, IRAK-4, or IRAK-1, substrates were successfully phosphorylated in the absence of cellular stimulation to simply experimental interpretations (7, 19, 20). We therefore used the same approach in characterizing SIMPL kinases. Preliminary in vitro kinase assays performed with IRAK-1 revealed that within 20 min, the maximal amount of phosphate incorporation had been reached. Immunocomplexed IRAK-1 and IKKbeta, but not IRAK-4 or IKK{alpha}, phosphorylate SIMPL. Based on the amount of radioactivity incorporated, SIMPL appears to be a better substrate for IRAK-1 (Fig. 7A). To confirm that phosphorylation of SIMPL was dependent on IRAK-1 or IKKbeta catalytic activity and not a copurifying kinase, we repeated the in vitro kinase assays, using SIMPL as a substrate for catalytically inactive IKKbeta or catalytically inactive IRAK-1. In the assays with the catalytically inactive proteins, SIMPL was not phosphorylated. We next examined whether the SIMPL mutants containing alanine substitutions at the basal sites of phosphorylation (SIMPLS55,61,63A) and/or alanine substitutions at the sites preferentially phosphorylated in response to TNF-{alpha} (SIMPLS55,61,63,S234,241A,T236,246A) were substrates for IRAK-1. Wild-type SIMPL or the SIMPL mutants were expressed in HEK-293EBNA cells, and immunocomplexes prepared with Flag-antibody were generated. A separate culture of HEK-293EBNA cells was used to generate immunocomplexes with IRAK-1 antisera. The immunocomplexed IRAK-1 was combined with the SIMPL-complexed immunocomplex, and in vitro kinase assays were performed. These assays revealed that the ability of IRAK-1 to phosphorylate SIMPLS55,61,63A or SIMPLS55,61,63,S234,241A,T236,246A was diminished compared with wild-type SIMPL (Fig. 7B). These data further support the hypothesis that SIMPL is an IRAK-1 substrate.


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 7. SIMPL is an IL-1 receptor-associated kinase (IRAK)-1 and IKKbeta substrate. A and B: HEK-293EBNA cell lysates were used to generate immunocomplexes with a control IgG or antisera to indicated kinases. In vitro kinase assays were performed with the indicated immunocomplexed kinases by using recombinant SIMPL protein as a substrate. Kinase assays were denatured, subjected to SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were exposed to X-ray film to generate autoradiograms and reprobed for the indicated proteins: lane 1, catalytically inactive IRAK-1; lane 2, wild-type IRAK-1 (A) or lane 1, IgG; lane 2, IRAK-1; lane 3, IRAK-4; lane 4, IKK{alpha}; lane 5, catalytically inactive IKK{alpha}; lane 6, IKKbeta; lane 7, catalytically inactive IKKbeta (B). C: HEK-293EBNA cells were transfected with myc-IRAK-1 and wild-type SIMPL, SIMPLS55,61,63A, or SIMPLS55,61,63,234,241A,T236,246A. Twenty-four hours later, cultures were harvested and immunocomplexes were generated with myc- or Flag-antibody. Immuncomplexed materials were used in in vitro kinase assays. Assays were denatured, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were exposed to X-ray film to generate autoradiograms or reprobed for the indicated proteins: lane 1, wild-type SIMPL; lane 2, SIMPLS55,61,63A; lane 3, SIMPLS55,61,63,234,241A,T236,246A.

 
IRAK-1 catalytic activity required for SIMPL nuclear localization. As a further test of whether IRAK-1 catalytic activity is required for nuclear accumulation of SIMPL, the subcellular localization of SIMPL was determined in cells expressing wild-type IRAK-1, catalytically inactive IRAK-1, or an antisense IRAK-1 construct. In HEK-293EBNA cells overexpressing wild-type IRAK-1, SIMPL is found predominantly as a nuclear protein (Fig. 8A). In HEK-293EBNA cells expressing a catalytically inactive IRAK-1 construct that is known to block TNF-{alpha}-induced NF-{kappa}B activity (31), SIMPL is predominantly cytoplasmic (Fig. 8B). In Fig. 8, A and B, HEK-293EBNA cells were engineered to overexpress IRAK-1. To determine whether loss of endogenous IRAK-1, which is expressed at a high level in HEK-293EBNA cells (unpublished observation) would preclude nuclear localization of SIMPL, we used an antisense approach. This also enabled us to confirm that the predominantly cytoplasmic localization of SIMPL was not due to trapping of SIMPL by catalytically inactive IRAK-1. A decrease in the steady-state level of endogenous IRAK-1 protein also leads to a predominantly cytoplasmic localization of SIMPL (Fig. 8, D and E). Together, these data and our group's previous report (33) demonstrating that IRAK-1 protein is required for SIMPL-dependent activation of NF-{kappa}B activity support a model in which nuclear accumulation of SIMPL is IRAK-1 dependent and is necessary for TNF-{alpha}-induced activation of NF-{kappa}B.


Figure 8
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 8. IRAK-1 catalytic activity is required for SIMPL nuclear localization. HEK-293EBNA cells were transfected with vectors encoding myc-tagged IRAK-1 and Flag-tagged SIMPL (A), myc-tagged catalytically inactive IRAK-1 (ciIRAK-1) and Flag-tagged SIMPL (B), Flag-tagged SIMPL (C), or Flag-tagged SIMPL and a vector encoding an antisense IRAK-1 cDNA (D). Twenty-four hours later, cultures were fixed and processed for indirect immunofluorescence using epitope-specific antibody, and images were visualized using confocal microscopy (for details see MATERIALS AND METHODS). In nuclei images, cultures were stained with Hoechst 33258. E: Western blot of cultures expressing antisense IRAK-1 (asIRAK-1).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Protein phosphorylation plays a critical role in regulating the activity of intracellular signal transduction pathways and in controlling transcription factor activity (for review, see Refs. 12, 14). In this article, we provide evidence that two distinct phosphorylation events regulate SIMPL coactivator function and subcellular localization, respectively. A combination of phosphopeptide mapping and mass spectrometry identified basal as well as sites of phosphorylation preferentially increased by TNF-{alpha} on SIMPL. The basal sites of phosphorylation on SIMPL include at least serines 55, 61, or 63. A SIMPL mutant containing alanine substitutions at these sites of phosphorylation was compromised in its ability to stimulate p65-dependent transactivation activity. Serines 234 and 241 and threonines 236 and 246 were identified as potential sites of TNF-{alpha}-induced phosphorylation. These residues are located adjacent to (S234, T236, and S241) or within (T246) the NLS in SIMPL, a domain required for SIMPL induction of NF-{kappa}B activity (16). Ectopic expression of a SIMPL mutant containing alanine substitutions at the sites of phosphorylation in SIMPL preferentially increased by TNF-{alpha} blocks TNF-{alpha} induction of p65 transactivation activity. Results of in vitro kinase assays revealed that wild-type SIMPL is an IRAK-1 substrate and that a SIMPL mutant lacking the site(s) of phosphorylation preferentially increased by TNF-{alpha} does not serve as an IRAK-1 substrate. These data are consistent with previous studies in which IRAK-1 catalytic activity has been linked to the activation of TNF-{alpha}-dependent signaling transduction pathways in vitro (15, 16, 34) and to TNF-{alpha}-dependent responses in vivo (30, 31). Thus control of SIMPL activity may require changes in phosphorylation status that can be mediated by IRAK-1. The observation that overexpression of SIMPL or IRAK-1 (33) in the absence of TNF-{alpha} stimulation leads to activation of NF-{kappa}B-controlled responses parallels what is seen with other activators of NF-{kappa}B-controlled responses, including Akt, MEKK3, and IKKbeta (22, 25, 36). Furthermore, ectopic expression of IKK{alpha}, which is responsible for basal NF-{kappa}B activity, does not respond in a like manner (22).

Ectopically expressed SIMPL is located in the cytoplasm as well as the nucleus (16). In response to TNF-{alpha}, SIMPL-p65 complexes form and induction of endogenous NF-{kappa}B-controlled gene expression occurs. Results of the present studies suggest that nuclear localization of SIMPL is regulated in part by changes in IRAK-1-dependent phosphorylation. The SIMPL mutant lacking IRAK-1-dependent sites of phosphorylation is a weak substrate for IRAK-1, and when ectopically expressed in cells, it preferentially accumulates in the cytoplasm. In cells either overexpressing catalytically inactive IRAK-1 or with a decreased level of endogenous IRAK-1, SIMPL is preferentially located in the cytoplasm. Together, these data argue that IRAK-1-dependent phosphorylation is required for SIMPL nuclear localization. SIMPL is also an IKKbeta substrate. SIMPL also has been detected in IKKbeta-containing complexes (34), and IKKbeta is required for TNF-{alpha}-induced NF-{kappa}B activity (17); therefore, IKKbeta also may modulate SIMPL phosphorylation.

Phosphorylation governs nuclear localization of SIMPL. However, which aspects of SIMPL function are governed by phosphorylation is less clear. Phosphorylation may change the conformation of SIMPL, enabling the NLS signal to be exposed to the nuclear transport machinery, or phosphorylation may be required for the recognition of SIMPL by the nuclear transport machinery. Alternatively, phosphorylation may be required for an interaction between SIMPL and the transcriptional machinery. In the absence of phosphorylation, the interaction between SIMPL and the transcriptional machinery may be diminished, thus SIMPL may not be retained in the nucleus and may appear to be localized predominantly in the cytoplasm. The relationship between the basal and induced sites of phosphorylation on SIMPL also is not completely clear. Compared with wild-type SIMPL, the SIMPLS55,61,63A mutant is a poorer substrate for IRAK-1 in in vitro kinase assays. Since phosphorylation of these residues (S55, 61, and 63) occurs under basal conditions and not in response to TNF-{alpha}, a hierarchy of phosphorylation events may regulate SIMPL activity. Analysis of the SIMPL protein sequence has revealed that the first 90 amino acid residues of SIMPL are largely unstructured (28). Serines 55, 61, and 63 lie in this largely unstructured domain; thus phosphorylation of these residues may enable SIMPL to adopt a conformation facilitating IRAK-1 and/or IKKbeta access to residues located near/in the NLS. Alternatively, the phosphorylation in the unstructured domain may affect the binding affinity of SIMPL for either IRAK-1 and/or IKKbeta.

The I{kappa}B kinase (IKK) complex is a key regulator of NF-{kappa}B-controlled gene expression (for review, see Ref. 11). Analysis of IKK complex components has revealed that IKK{alpha} is required for maintaining the basal levels of NF-{kappa}B-controlled gene expression, whereas IKKbeta is required for induction of signal-specific changes in NF-{kappa}B-controlled gene expression. A key issue in the regulation of NF-{kappa}B-controlled gene expression is how signal-specific gene expression is achieved if the IKK complex plays an integral role in numerous intracellular signaling pathways. One mechanism through which signal-specific changes in gene expression can be achieved is via signal-specific transcriptional coactivators activated in parallel (38). On the basis of our findings, SIMPL is a transcriptional coactivator required for full activation of TNF-{alpha}-dependent NF-{kappa}B-controlled gene expression (16, 34). The identification of SIMPL, as a component of the TNF-{alpha} signaling pathway provides a mechanism through which a specific cytokine (or other stimuli) induces changes in NF-{kappa}B-controlled gene expression. In our studies, we have detected a novel phosphopeptide in SIMPL, preferentially phosphorylated in TNF-{alpha}-treated cultures. In this context, SIMPL provides an attractive therapeutic target because of its potential specificity for NF-{kappa}B activity associated with the TNF-{alpha} signaling pathway.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Indiana University School of Medicine-Biomedical Research Fund.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Harrington, Dept. of Biochemistry and Molecular Biology, Indiana Univ. School of Medicine, 635 Barnhill Drive, MS 4071, Indianapolis, IN 46202-5122 (e-mail: mharrin{at}iupui.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Baldwin AS Jr. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 14: 649–683, 1996.[CrossRef][ISI][Medline]

2. Baldwin AS Jr. Series introduction: the transcription factor NF-{kappa}B and human disease. J Clin Invest 107: 3–6, 2001.[CrossRef][ISI][Medline]

3. Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol 201: 110–149, 1991.[ISI][Medline]

4. Brennan CA, Anderson KV. Drosophila: the genetics of innate immune recognition and response. Annu Rev Immunol 22: 457–483, 2004.[CrossRef][ISI][Medline]

5. Burns K, Janssens S, Brissoni B, Olivos N, Beyaert R, Tschopp J. Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J Exp Med 197: 263–268, 2003.[Abstract/Free Full Text]

6. Chen ZJ, Parent L, Maniatis T. Site-specific phosphorylation of I{kappa}B{alpha} by a novel ubiquitination-dependent protein kinase activity. Cell 84: 853–862, 1996.[CrossRef][ISI][Medline]

7. Delhase M, Hayakawa M, Chen Y, Karin M. Positive and negative regulation of I{kappa}B kinase activity through IKKbeta subunit phosphorylation. Science 284: 309–313, 1999.[Abstract/Free Full Text]

8. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M. A cytokine-responsive I{kappa}B kinase that activates the transcription factor NF-{kappa}B. Nature 388: 548–554, 1997.[CrossRef][Medline]

9. Hardie G. Protein Phosphorylation: A Practical Approach. New York: IRL, Oxford Univ. Press, 1993.

10. Janeway CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 20: 197–216, 2002.[CrossRef][ISI][Medline]

11. Hayden MS, Ghosh S. Signaling to NF-{kappa}B. Genes Dev 18: 2195–2224, 2004.[Abstract/Free Full Text]

12. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80: 225–236, 1995.[CrossRef][ISI][Medline]

13. Jans DA. The regulation of protein transport to the nucleus by phosphorylation. Biochem J 311: 705–716, 1995.

14. Karin M, Hunter T. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol 5: 747–757, 1995.[CrossRef][ISI][Medline]

15. Kim JA, Yeh DC, Ver M, Li Y, Carranza A, Conrads TP, Veenstra TD, Harrington, MA, Quon MJ. Phosphorylation of Ser24 in the pleckstrin homology domain of insulin receptor substrate-1 by mouse Pelle-like kinase/interleukin-1 receptor-associated kinase: cross-talk between inflammatory signaling and insulin signaling that may contribute to insulin resistance. J Biol Chem 280: 23173–23183, 2005.[Abstract/Free Full Text]

16. Kwon HY, Breese EH, Vig-Varga E, Luo Y, Lee Y, Goebl MG, Harrington MA. Tumor necrosis factor {alpha} induction of NF-{kappa}B requires the novel coactivator SIMPL. Mol Cell Biol 24: 9317–9326, 2004.[Abstract/Free Full Text]

17. Li Q, Estepa G, Memet S, Israel A, Verma IM. Complete lack of NF-{kappa}B activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation. Genes Dev 14: 1729–1733, 2000.[Abstract/Free Full Text]

18. Li Q, Verma IM. NF-{kappa}B regulation in the immune system. Nat Rev Immunol 2: 725–734, 2002.[CrossRef][ISI][Medline]

19. Li S, Strelow A, Fontana EJ, Wesche H. IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase. Proc Natl Acad Sci USA 99: 5567–5572, 2002.[Abstract/Free Full Text]

20. Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M, Johnson R, Karin M. The IKKbeta subunit of I{kappa}B kinase (IKK) is essential for nuclear factor {kappa}B activation and prevention of apoptosis. J Exp Med 189: 1839–1845, 1999.[Abstract/Free Full Text]

21. Mayo MW, Madrid LV, Westerheide SD, Jones DR, Yuaj XJ, Baldwin AS Jr, Whang YE. PTEN blocks tumor necrosis factor-induced NF-{kappa}B-dependent transcription by inhibiting the transactivation potential of the p65 subunit. J Biol Chem 277: 11116–11125, 2002.[Abstract/Free Full Text]

22. McKenzie FR, Connelly MA, Balzarano D, Muller JRR, Geleziunas R, Marcu KR. Functional isoforms of I{kappa}B kinase {alpha} (IKK{alpha}) lacking leucine zipper and helix-loop-helix domains reveal that IKK{alpha} and IKKbeta have different activation requirements. Mol Cell Biol 20: 2635–2646, 2000.[Abstract/Free Full Text]

23. Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 343: 338–344, 2000.[Free Full Text]

24. Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li JW, Young DB, Barbosa M, Mann M, Manning A, Rao A. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF-{kappa}B activation. Science 278: 860–866, 1997.[Abstract/Free Full Text]

25. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pferrer LM, Donner DB. NF-{kappa}B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401: 82–85, 1999.[CrossRef][Medline]

26. Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNF{alpha}-deficient mice: a critical requirement for TNF{alpha} in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med 184: 1397–1411, 1996.[Abstract/Free Full Text]

27. Regnier CH, Song HY, Gao X, Goeddel DV, Cao Z, Rothe M. Identification and characterization of an I{kappa}B kinase. Cell 90: 373–383, 1997.[CrossRef][ISI][Medline]

28. Romero P, Obradovic Z, Dunker AK. Natively disordered proteins: functions and predictions. Appl Bioinformatics 3: 105–113, 2004.[CrossRef][Medline]

29. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2: 947–950, 2001.[CrossRef][ISI][Medline]

30. Thomas JA, Allen JL, Tsen M, Dubnicoff T, Danao J, Liao XC, Cao Z, Wasserman SA. Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. J Immunol 163: 978–984, 1999.[Abstract/Free Full Text]

31. Thomas JA, Haudek SB, Koroglu T, Tsen MF, Bryant DD, White DJ, Kusewitt DF, Horton JW, Giroir BP. IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction. Am J Physiol Heart Circ Physiol 285: H597–H606, 2003.[Abstract/Free Full Text]

32. Vandromme M, Gauthier-Rouviere C, Lamb N, Fernandez A. Regulation of transcription factor localization: fine-tuning of gene expression. Trends Biochem Sci 21: 59–64, 1996.[CrossRef][ISI][Medline]

33. Vig E, Green M, Liu Y, Donner DB, Mukaida N, Goebl MG, Harrington MA. Modulation of tumor necrosis factor and interleukin-1-dependent NF-{kappa}B activity by mPLK/IRAK. J Biol Chem 274: 13077–13084, 1999.[Abstract/Free Full Text]

34. Vig E, Green M, Liu Y, Kang-Yeol K, Kwon HJ, Tian J, Goebl M, Harrington MA. SIMPL is a TNF RI-mPLK/IRAK-dependent NF-{kappa}B activator. J Biol Chem 276: 7859–7866, 2001.[Abstract/Free Full Text]

35. Woronicz JB, Gao Z, Cao Z, Rothe M, Goeddel DV. I{kappa}B kinase-beta: NF-{kappa}B activation and complex formation with I{kappa}B kinase-{alpha} and NIK. Science 278: 866–869, 1997.[Abstract/Free Full Text]

36. Yang J, Lin Y, Guo Z, Cheng J, Huang J, Deng L, Liao W, Chen Z, Liu Z, Su B. The essential role of MEKK3 in TNF-induced NF-{kappa}B activation. Nat Immunol 2: 620–624, 2001.[CrossRef][ISI][Medline]

37. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The I{kappa}B kinase complex (IKK) contains two kinase subunits, IKK{alpha} and IKKbeta, necessary for I{kappa}B phosphorylation and NF-{kappa}B activation. Cell 91: 243–252, 1997.[CrossRef][ISI][Medline]

38. Ziegler EC, Ghosh S. Regulating inducible transcription through controlled localization. Sci STKE (284): re6, 2005.