The 90-kDa Ribosomal S6 Kinase (pp90rsk) Phosphorylates the N-terminal Regulatory Domain of Ikappa Balpha and Stimulates Its Degradation in Vitro*

(Received for publication, January 3, 1997, and in revised form, May 20, 1997)

Lucy Ghoda Dagger §, Xin Lin § and Warner C. Greene par **

From the Dagger  University of Colorado Health Sciences Center, Department of Pharmacology, School of Medicine, Denver, Colorado 80262 and the  Gladstone Institute of Virology and Immunology and par  Departments of Medicine and Microbiology and Immunology, University of California, San Francisco, San Francisco General Hospital, San Francisco, California 94141-9100

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Nuclear factor kappa B (NF-kappa B) is a eukaryotic member of the Rel family of transcription factors whose biological activity is post-translationally regulated by its assembly with various ankyrin-rich cytoplasmic inhibitors, including Ikappa Balpha . Expression of NF-kappa B in the nucleus occurs after signal-induced phosphorylation, ubiquitination, and proteasome-mediated degradation of Ikappa Balpha . The induced proteolysis of Ikappa Balpha unmasks the nuclear localization signal within NF-kappa B, allowing its rapid migration into the nucleus, where it activates the transcription of many target genes. At present, the identity of the Ikappa Balpha kinase(s) that triggers the first step in Ikappa Balpha degradation remains unknown. We have investigated the potential function of the 90-kDa ribosomal S6 kinase, or pp90rsk, as a signal-inducible Ikappa Balpha kinase. pp90rsk lies downstream of mitogen-activated protein (MAP) kinase in the well characterized Ras-Raf-MEK-MAP kinase pathway that is induced by various growth factors and phorbol ester. We now show that pp90rsk, but not pp70S6K or MAP kinase, phosphorylates the regulatory N terminus of Ikappa Balpha principally on serine 32 and triggers effective Ikappa Balpha degradation in vitro. When co-expressed in vivo in COS cells, Ikappa Balpha and pp90rsk readily assemble into a complex that is immunoprecipitated with antibodies specific for either partner. While phorbol 12-myristate 13-acetate produced rapid activation of pp90rsk, in vivo, other potent NF-kappa B inducers, including tumor necrosis factor alpha  and the Tax transactivator of human T-cell lymphotrophic virus, type I, failed to activate pp90rsk. These data suggest that more than a single Ikappa Balpha kinase exists within the cell and that these Ikappa Balpha kinases are differentially activated by different NF-kappa B inducers.


INTRODUCTION

Nuclear factor kappa B (NF-kappa B)1 is a transcription factor whose function is regulated by a family of cytoplasmic inhibitors termed the Ikappa Bs (reviewed in Refs. 1 and 2). At present, nine Ikappa B family members have been identified (Ikappa Balpha , Ikappa Bbeta , Ikappa Bgamma , Ikappa Bdelta , Ikappa Bepsilon , p105, p100, Bcl-3, and Cactus), each distinguished by the presence of multiple ankyrin repeats. The prototypic and best studied of the Ikappa Bs is Ikappa Balpha (3), which binds to the heterodimeric NF-kappa B complex (p50/Rel A) (4), masks the nuclear localization signal present in Rel A (5, 6), and sequesters NF-kappa B in the cytoplasm (4-6). When appropriate inductive signals are delivered to the cell, phosphorylation of Ikappa Balpha ensues (7-10), followed by the conjugation of multiple ubiquitin molecules and the degradation of the ubiquitinated Ikappa Balpha phosphoprotein by the 26 S proteasome complex (11-13). Of note, Ikappa Balpha degradation proceeds while the inhibitor is still physically associated with the NF-kappa B heterodimer (10, 14-17). However, the NF-kappa B complex is ultimately liberated, allowing its rapid translocation into the nucleus, where it engages cognate enhancer elements and alters the transcriptional activity of various target genes.

Although phosphorylation of Ikappa Balpha is required for its proteolysis and the subsequent activation of NF-kappa B, the nature of the cellular protein kinase(s) mediating this reaction remains unknown. Signal-induced phosphorylation involves two serine residues located at positions 32 and 36 near the N terminus of Ikappa Balpha . Substitution of these serines with alanine residues generates a constitutively acting Ikappa Balpha repressor that readily binds to NF-kappa B but fails to undergo signal-induced phosphorylation and degradation (7, 8, 10, 18).

Studies in Drosophila have also yielded valuable insights into the biology of the Rel proteins and their control by the Ikappa Bs. In the dorsal-ventral signal transduction pathway of Drosophila, dorsalizing signals mediated through the receptor Toll (an IL-1 receptor homologue) target Cactus (a member of the Ikappa B family) for degradation and result in activation of Dorsal (a Rel family member) (19). In this pathway, Pelle, a serine/threonine protein kinase, regulates the degradation of Cactus through phosphorylation, although it is unknown whether Pelle acts directly or indirectly on Cactus (20). Recently, a human IL-1 receptor-associated kinase has been cloned that is homologous to Pelle (21). This kinase appears to participate in the IL-1-induced signaling pathway leading to NF-kappa B induction, but no evidence yet exists for its direct phosphorylation of Ikappa Balpha .

Casein kinase II (CKII) also phosphorylates Ikappa Balpha in vivo (22-24). However, phosphopeptide mapping of phosphorylated Ikappa Balpha has shown that residues within the C-terminal PEST region, rather than the N-terminal serines, are targeted by CKII. Phosphorylation of the C terminus of Ikappa Balpha by CKII or other kinases may play a role in the constitutive degradation of uncomplexed Ikappa Balpha . Additionally, CKII-mediated phosphorylation appears important for the accelerated turnover of Ikappa Balpha and the persistent induction of NF-kappa B observed following HIV infection of macrophages (24). Immunodepletion of CKII from these cell extracts results in an inhibition of Ikappa Balpha degradation in vitro (24).

Recently, a novel ubiquitination-stimulated protein kinase has been identified that phosphorylates Ikappa Balpha in a serine 32/36-dependent manner (25). This kinase resides in a large 700-kDa multiprotein complex, and ubiquitination of some component of the complex results in increased Ikappa Balpha phosphorylating activity. Whether ubiquitination directly activates the kinase or, alternatively, acts indirectly to alter another component of the complex remains unresolved.

We have investigated the potential role of a known intracellular protein kinase in the Ras-Raf-MEK-MAP kinase signaling pathway as an Ikappa B kinase. This enzyme, the 90-kDa ribosomal S6 kinase, or pp90rsk, lies immediately downstream of MAP kinase in the phorbol ester and growth factor signaling pathway (26, 27). We now show that pp90rsk phosphorylates the N terminus of Ikappa Balpha principally on serine 32 and functionally induces Ikappa Balpha degradation in vitro. We further show that pp90rsk and Ikappa Balpha can physically associate in vivo. Finally, we show that only a subset of the known NF-kappa B-inducing signals leads to the activation of pp90rsk. These findings suggest that rather than a single Ikappa B kinase, a family of Ikappa B kinases may exist within the cell that are differentially activated by different inducers of NF-kappa B.


MATERIALS AND METHODS

cDNA Vectors and Expression of Proteins

The expression vector containing a hemagglutinin-epitope-tagged pp90rsk cDNA (pMT2-HA-RSK1) was provided by Dr. Joseph Avruch (Harvard University and Massachusetts General Hospital, Boston, MA). Wild type Ikappa Balpha cDNA provided by Dr. Al Baldwin (University of North Carolina, Chapel Hill, NC) was cloned into the HindIII and XbaI sites of the pCMV4 eukaryotic expression vector provided by Dr. Mark Stinski (University of Iowa, Iowa City, IA). For in vitro translation, wild type Ikappa Balpha or mutant Ikappa Balpha containing alanine for serine substitutions at position 32 and/or 36 (4) cloned into the HindIII and XbaI or XmaI sites of the pBluescript SK(+) vector (Stratagene). Biosynthetically radiolabeled Ikappa Balpha or its mutant analogues were synthesized by transcription-coupled in vitro translation in wheat germ extracts (Promega).

A bacterial expression plasmid encoding hexahistidine (His)-tagged Ikappa Balpha was constructed by cloning the Ikappa Balpha cDNA into the pTrcHisC vector (Invitrogen). The N-terminal Delta 1-36 Ikappa Balpha deletion mutant was cloned into the pRSETC vector (identical to the pTrcHis vector except in the promoter region). Wild type Ikappa Balpha and the S32/36A Ikappa Balpha mutant containing alanine for serine substitutions at both residues 32 and 36 were isolated from Escherichia coli lysates by purification on a nickel chelate column (Ni-NTA, Qiagen). Following an initial wash in high salt buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl), successive washes were performed with elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing increasing concentrations of imidazole (0, 10, 50, 100 mM). The His-tagged proteins were eluted in buffer containing 200 mM imidazole. The fractions containing the desired proteins were dialyzed overnight in 50 mM Tris-HCl, pH 7.5, and 2 mM DTT.

Cell Lines and Tissue Culture Conditions

Jurkat cells and Jurkat cells stably expressing either HTLV-I Tax or Tax antisense cDNA constructs and a neomycin resistance gene were maintained in RPMI 1640 supplemented with 10% fetal calf serum and penicillin/streptomycin at 37 °C in 5% CO2; 800 µg/ml G418 was added to the Jurkat-Tax and anti-Tax cell culture media. Cells were treated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) or TNF-alpha (50 ng/ml) for various periods of time. Vehicle controls corresponding to the amounts of added Me2SO for PMA and water for TNF-alpha were performed in parallel. The cells were washed once with ice-cold phosphate-buffered saline and lysed in ELB buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 0.2% Nonidet P-40, 5 mM EDTA, 0.5 mM DTT, 1.0 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture containing 0.75 µg/ml bestatin; 0.5 µg/ml each of aprotinin, antipain, leupeptin, and trypsin inhibitor; 0.4 µg/ml phosphoramidon; and 0.05 µg/ml pepstatin). The cell lysates were clarified by centrifugation at 4 °C for 15 min at 15,000 × g, and the supernatant was used for immunoprecipitation as described below.

In Vitro Assay of Protein Kinase Activity

Phosphorylation of bacterially expressed Ikappa Balpha (0.5 µg) was performed in 50-µl reaction mixtures incubated for 15 min at room temperature. For Xenopus laevis egg-derived MAP kinase, X. laevis egg-derived pp90rsk, and mouse pp70S6K, the reaction buffer contained 20 mM HEPES, pH 7.0, 10 mM MgCl2, 2 mM DTT, 100 µM EGTA, 0.1 mg/ml bovine serum albumin, 100 µM ATP, 25 µCi of [gamma 32P]ATP (specific activity 3000 Ci/µmol). Myelin basic protein (0.1 mg/ml, Sigma) or 40 S ribosomal subunits (0.05 mg/ml) generously provided by Dr. James Maller (Howard Hughes Medical Institute and University of Colorado Health Sciences Center, Denver, CO) were used as positive controls for these kinase reactions. Aliquots of the reactions were mixed in SDS-PAGE sample buffer, heated to 98 °C, and microcentrifuged at 15,000 × g for 2 min, followed by analysis of the supernatants by SDS-PAGE. Alternatively, Ikappa Balpha was immunoprecipitated from the reactions with peptide-specific polyclonal rabbit antisera (2.5 µl) recognizing the C-terminus of Ikappa Balpha . Immune complexes were reacted with formalin-fixed Staphylococcus aureus cells (Pansorbin A, 50 µl, Calbiochem) and collected by centrifugation. The Protein A-bound immune complexes were washed three times and similarly analyzed by SDS-PAGE and autoradiography. In the case of pp90rsk, three independently purified preparations were tested. The specific activities of these pp90rsk preparations were 4.6, 8.9, and 11.2 nmol of ATP incorporated/min/mg of protein assayed using Kemptide as substrate.

V8 Digestion of in Vitro Phosphorylated Ikappa Balpha

Bacterially produced His-tagged Ikappa Balpha was phosphorylated with purified pp90rsk as described above and subjected to mild V8 proteolysis at room temperature for 10 min. The protease inhibitor Nalpha -p-tosyl-L-lysine chloromethyl ketone was added to the reaction mixture together with 100 µg/ml (final concentration) of bovine serum albumin to terminate proteolytic cleavage. Ikappa Balpha peptides containing the N-terminal His-epitope were immunoprecipitated from the digest using his-tag-specific antibodies. The immunoprecipitates were analyzed with Tris-Tricine gels by the method of Schägger and von Jagow (28).

In Vitro Degradation of Ikappa Balpha

In vitro synthesized [35S]-radiolabeled Ikappa Balpha was phosphorylated with pp90rsk in the presence of unlabeled ATP as described. The phosphorylated or control Ikappa Balpha proteins (25 µl) were incubated in 100 µl of reticulocyte lysates containing 5 mM DTT, 2.5 mM ATP, 1 mM creatine phosphate, and 200 µg/ml creatine phosphokinase. Samples were removed at various times and quickly frozen in liquid nitrogen. The samples were then immunoprecipitated with C-terminalspecific anti-Ikappa Balpha antibodies and analyzed by SDS-PAGE followed by autoradiography.

Coimmunoprecipitation Assays

Monkey kidney COS7 cells, maintained in complete Dulbecco's modified Eagle's medium, were transfected with pMT2-HA-RSK1 (encoding pp90rsk-1) or pCMV4-HA-PP2A (encoding the A regulatory subunit of protein phosphatase 2A) and CMV4-Ikappa Balpha using LipofectAMINE (Life Technologies, Inc.). After 48 h, the cells were starved for 1 h in methionine/cysteine-free Dulbecco's modified Eagle's medium and then metabolically radiolabeled with [35S]methionine and [35S]cysteine for 2 h. Whole-cell extracts were prepared by lysis in ELB buffer, followed by immunoprecipitation analyses as described above using either anti-HA-epitope antibody (BAbCo, Berkeley, CA) or anti-Ikappa Balpha antisera specific for the C terminus of this inhibitor. Nonradioactive COS cell lysates were also prepared and immunoprecipitated with the anti-HA or Ikappa Balpha antibodies followed by immunoblotting with anti-pp90rsk-specific antibodies. These immunoblots were developed with a horseradish peroxidase-conjugated secondary antibody and enhanced chemoluminescense (ECL) as described by the manufacturer (Amersham).

Immune Complex-associated Protein Kinase Assay

Cell lysates derived from about 5 × 106 Jurkat cells were precleared for 1 h and incubated with 10 µl of anti-pp90rsk antibody (Santa Cruz Biotechnology) at 4 °C for 1 h. 30 µl of Protein A-agarose (Boehringer Mannheim) was then added to the mixture, and the incubation was continued for an additional 1 h. The mixture was centrifuged at 4 °C, followed by washing of the Protein A-agarose resin three times in ELB buffer. Immune complexes were washed three times with kinase buffer (25 mM glycerol-2-phosphate, 20 mM HEPES, pH 7.4, 10 mM MgCl2, 4 mM NaF, 2 mM MnCl2, 1 mM dithiothreitol, 0.1 mM Na3VO4, and either 20 µM ATP (specific activity 3000 Ci/µmol for [32P]labeling of recombinant Ikappa Balpha ) or 100 µM ATP (for phosphorylation of [35S]-labeled Ikappa Balpha )). 30 µl of the kinase buffer containing 0.3 µg of bacterially expressed Ikappa Balpha or 2 µl of wheat germ-translated reactions of wild type or mutant Ikappa Balpha was added to the immune complexes, and the mixtures were incubated at 30 °C for 30 min for [32P]radiolabeling of Ikappa Balpha or 2 h for phosphorylation of [35S]-labeled Ikappa Balpha . The reaction products were then analyzed by SDS-PAGE and autoradiography.

Phosphatase Treatment of Phosphorylated Proteins

Following the phosphorylation reactions, Ikappa Balpha was immunoprecipitated with anti-Ikappa Balpha antiserum specific for the C-terminus and Protein A-agarose. The bound immune complexes were washed three times in dephosphorylation buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA) followed by the addition of 10 units of calf intestinal alkaline phosphatase (Boehringer Mannheim). The samples were then incubated at 37 °C for 30 min and analyzed by SDS-PAGE and autoradiography.


RESULTS

In Vitro Phosphorylation of Ikappa Balpha by pp90rsk

Phorbol ester is both a potent inducer of NF-kappa B and a known activator of the MAP kinase pathway. To determine whether kinases positioned along this or related pathways are capable of phosphorylating Ikappa Balpha , the ability of MAP kinase, pp70S6K, and pp90rsk to phosphorylate Ikappa Balpha in vitro was examined. These kinases phosphorylate residues that lie within specific consensus sequences. For example, MAP kinase phosphorylates serines and threonines that precede proline residues (proline-directed kinase consensus sequence), while both pp90rsk and pp70S6K phosphorylate serines or threonines within the consensus sequence Arg-X-X-Ser/Thr (29).

Recombinant His-tagged Ikappa Balpha was used as the substrate in these in vitro kinase assays. Following the kinase reaction, Ikappa Balpha was immunoprecipitated from the reaction using polyclonal anti-Ikappa Balpha antibodies (30). Immunoprecipitation was performed to ensure that the phosphorylated protein was indeed recombinant Ikappa Balpha . As shown in Fig. 1, pp90rsk (lanes 7 and 8), but not pp70S6K (lanes 3 and 4) or p42MAPK (lanes 11 and 12), phosphorylated recombinant His-tagged Ikappa Balpha in vitro. The failure of the pp70S6K and p42MAPK preparations to phosphorylate Ikappa Balpha was not due to inactivation of these enzymes during their purification, as each capably phosphorylated known protein substrates, including the 40S ribosomal subunit for pp70S6K (lanes 1 and 2) and myelin basic protein for p42MAPK (lanes 9 and 10). Of note, while both pp90rsk and pp70S6K recognize the same consensus phosphoacceptor site and phosphorylate the S6 protein of the 40S ribosome, only pp90rsk phosphorylated Ikappa Balpha . Another kinase, Ca2+-calmodulin-dependent protein kinase II (CaMKII) also shares the same consensus phosphoacceptor site as pp90rsk and pp70S6K (31). However, like pp70S6K, CaMKII failed to phosphorylate Ikappa Balpha in vitro, although it did phosphorylate one of its physiological substrates, synapsin II (data not shown). These data highlight the ability of pp90rsk to utilize Ikappa Balpha as a substrate for phosphorylation in vitro.


Fig. 1. pp90rsk, but not pp70S6K or MAP kinase, phosphorylates Ikappa Balpha in vitro. Reactions containing [gamma -32P]ATP and pp70S6K (lanes 1-4) or pp90rsk (lanes 5-8) were incubated with 40 S ribosomal subunits (40S, lanes 1, 2, 5, and 6) or wild type his-tagged Ikappa Balpha (lanes 3, 4, 7, and 8). Samples containing MAP kinase (lanes 9-12) were similarly incubated with myelin basic protein (MBP, lanes 9 and 10) or wild type his-tagged Ikappa Balpha (lanes 11 and 12). In vitro kinase reactions were performed as described under "Materials and Methods" and analyzed by SDS-PAGE and autoradiography. Migration of the various protein substrates is indicated at the right margin.
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Immunoprecipitation of a Phosphorylated N-terminal Fragment of Ikappa Balpha

To ascertain whether pp90rsk phosphorylates Ikappa Balpha at either of the two critical N-terminal serine residues located at positions 32 and 36, a 6xhis-Ikappa Balpha and a similarly epitope-tagged S32/36A Ikappa Balpha mutant in which both of these serines were substituted with alanine, was subjected to in vitro phosphorylation as described above. The protein was then subjected to mild V8 (endoprotease Glu C) proteolysis with a sequencing-grade protease. The cleaved proteins were immunoprecipitated with antiserum specific for the N-terminal his-epitope. This antiserum recognizes both wild type Ikappa Balpha and the S32/36A His-tagged mutants. Phosphorylation of Ikappa Balpha at either serine 32 or 36 should result in a fragment of 84/87 amino acids (including the epitope tag) when exposed to V8 protease, which cleaves at residue 48 or 51. Fig. 2A depicts the recombinant Ikappa Balpha protein showing the signal-dependent N-terminal phosphorylation sites in relation to the V8 cleavage sites. The smallest band in the immunoprecipitate shown in the leftmost two lanes of Fig. 2B, indicated by the arrow, migrates between the 8.16- and 10.6-kDa myoglobin fragment, consistent with the molecular size of the 84/87 N-terminal fragment of Ikappa Balpha . In contrast, the S32/36A Ikappa Balpha protein failed to yield a similarly sized band (Fig. 2, fourth and fifth lanes). The even smaller band detected with these samples was also detected in the absence of added V8 protease. These results thus demonstrate that pp90rsk is capable of phosphorylating the regulatory N terminus of Ikappa Balpha involving serine 32 and/or 36. 


Fig. 2. pp90rsk phosphorylates the regulatory N terminus of Ikappa Balpha . A, schematic of Ikappa Balpha protein including N-terminal serines 32 and 36, ankyrin repeats, and C-terminal PEST sequences. Endoprotease Glu C (V8) digestion of this His-tagged Ikappa Balpha protein substrate is predicted to yield N-terminal Ikappa Balpha fragments that are 84 and 87 amino acids long and span serines 32 and 36 and the N-terminal His-epitope tag. B, autoradiogram of a Tris-Tricine SDS-PAGE gel (10-20% gradient) containing anti-N-terminal Ikappa Balpha immunoprecipitations (first, second, fifth, and sixth lanes) performed on pp90rsk-phosphorylated and V8-proteolyzed wild type His-tagged Ikappa Balpha (duplicate samples in first and second lanes) or the Ikappa Balpha S32/36A mutant (fourth and fifth lanes). Proteolytic digestions were performed as described under "Materials and Methods." Undigested phosphorylated wild type and S32/36A Ikappa Balpha proteins are shown in the third and sixth lanes, respectively. The arrow indicates an appropriate-sized fragment for the N-terminal peptide uniquely generated with wild type Ikappa Balpha but not with the S32/36A mutant of Ikappa Balpha . The even smaller band obtained with Ikappa Balpha S32/36A likely corresponds to a spontaneous degradation product, as it is also present in the untreated control sample.
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pp90rsk-mediated Phosphorylation Promotes Ikappa Balpha Degradation in Vitro

Although a variety of kinases can phosphorylate Ikappa Balpha in vitro, the critical functional issue is whether these kinases promote Ikappa Balpha degradation. Using an in vitro degradation system, we studied whether pp90rsk-mediated phosphorylation of Ikappa Balpha triggers its destruction. The reticulocyte lysate degradation system employed in these experiments contains all of the component proteins and macromolecules necessary for ubiquitin-dependent and -independent 26S proteasome-mediated degradation (31). A variety of proteins have been shown to be degraded in this reticulocyte lysate system, including ornithine decarboxylase, antizyme, and the transcription factors Fos, Jun, and Myc (32-38).

[35S]-Radiolabeled wild type Ikappa Balpha (Fig. 3, upper panel) and the S32/36A Ikappa Balpha mutant (lower panel) were synthesized in vitro and preincubated with Xenopus egg-derived pp90rsk (lanes 1-4), mammalian pp70S6K (lanes 5-8), or control kinase buffer lacking an added kinase (lanes 9 and 10). The Ikappa Balpha proteins were then incubated in a degradation-competent reticulocyte lysate that had specifically not been pretreated with RNase or hemin. Hemin is known to inhibit the 26S proteasome but is often added to reticulocyte lysates to improve translation since it prevents premature peptide chain termination (39). Degradation of [35S]-Ikappa Balpha was monitored by immunoprecipitation with specific anti-Ikappa Balpha antisera over the course of a 120-min incubation conducted in the presence of an ATP-regenerating system. As shown in lanes 1-4 of the upper panel, pp90rsk-treated wild type Ikappa Balpha was significantly degraded during the 2-h incubation. However, Ikappa Balpha treated with pp70S6K (lanes 5-8) or control buffer (lanes 9 and 10) was not degraded. In contrast to wild type Ikappa Balpha , the S32/36A Ikappa Balpha mutant was not degraded when incubated with pp90rsk (lower panel, lanes 1-4), indicating that Ikappa Balpha degradation in this in vitro system is dependent on the presence of these N-terminal regulatory serines, as it is in vivo. Together, these results suggest that pp90rsk-mediated phosphorylation of Ikappa Balpha is biologically relevant since it leads to serine 32- and/or serine 36-dependent degradation of wild type Ikappa Balpha protein in vitro.


Fig. 3. pp90rsk, but not pp70S6K, promotes serine 32- and/or serine 36-dependent degradation of wild type Ikappa Balpha in vitro. Degradation reactions were performed in vitro in reticulocyte lysates containing [35S]methionine/cysteine metabolically radiolabeled wild type Ikappa Balpha (upper panel) or the S32/36A double- substitution mutant Ikappa Balpha (lower panel) in the presence of active pp90rsk (lanes 1-4), active pp70S6K (lanes 5-8), or control buffer (lanes 9 and 10). Samples were removed from the reaction mixture at the indicated times and immunoprecipitated with anti-Ikappa Balpha antibodies, followed by SDS-PAGE and autoradiography.
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pp90rsk Phosphorylates Ikappa Balpha by pp90rsk Principally on Serine 32

Previous in vivo studies have shown that both S32 and S36 at the N terminus of Ikappa Balpha are required for signal-induced phosphorylation and degradation of this inhibitor (7-10, 18). Additionally, both of these serine residues are directly phosphorylated in vivo by an unknown kinase(s) following cellular stimulation with PMA or TNF-alpha (45). Phosphorylation at these sites in vivo results in a slower electrophoretic mobility for the Ikappa Balpha protein that is readily detectable on SDS-PAGE gels. In contrast, no mobility shift is observed when cells containing the S32/36A Ikappa Balpha mutant is similarly induced. To assess whether pp90rsk mediates phosphorylation on S32, S36, or both sites, wild type or mutant Ikappa Balpha proteins altered at one or both of these N-terminal serines were used as substrates in the in vitro kinase reactions. Activated pp90rsk was obtained by immunoprecipitating this kinase from PMA-stimulated HeLa cells. As shown in Fig. 4A, wild type Ikappa Balpha displayed a mobility shift when incubated with activated pp90rsk (lane 2), while the S32/36A Ikappa Balpha mutant containing alanine substitutions at both positions 32 and 36 did not (lane 4). The S36A single-substitution mutant of Ikappa Balpha also exhibited a significant change in mobility in the presence of pp90rsk (lane 8); however, the S32A Ikappa Balpha mutant displayed only a minimal change in migration (lane 6). These data suggest that the principal site of pp90rsk-mediated phosphorylation in vitro corresponds to serine 32. Assuming that in vitro and in vivo degradation requirements are the same for Ikappa Balpha , this finding suggests either that phosphorylation at this single site is sufficient for in vitro degradation or, alternatively, that a second kinase present in the reticulocyte lysate may act in concert with pp90rsk to modify serine 36 and thus complete the phosphorylation requirements for degradation. Alternatively, pp90rsk phosphorylation of serine 32 may facilitate subsequent modification of serine 36 by pp90rsk.


Fig. 4. pp90rsk principally phosphorylates Ikappa Balpha on serine 32. [35S]Methionine/cysteine metabolically radiolabeled Ikappa Balpha proteins (wild type, lanes 1 and 2; S32/36 mutant, lanes 3 and 4; S32A mutant, lanes 5 and 6; and S36A mutant, lanes 7 and 8) were incubated in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of activated pp90rsk immunoprecipitated from PMA-stimulated HeLa cells in the presence of unlabeled ATP (1 mM). Samples were analyzed for altered Ikappa Balpha electrophoretic mobility on SDS-PAGE gels. B, wild type Ikappa Balpha (lanes 1-4) or S32/36A Ikappa Balpha (lanes 5-8) was phosphorylated with activated pp90rsk (lanes 3, 4, 7, and 8) or buffer control (lanes 1, 2, 5, and 6) as described for A and then treated with calf intestinal alkaline phosphatase (CIP) (even-numbered lanes) or buffer alone (odd-numbered lanes). C, [35S]-labeled wild type Ikappa Balpha proteins were treated with activated pp90rsk (even-numbered lanes) or kinase buffer alone (odd-numbered lanes) in the presence (lanes 5-8) or absence (lanes 1-4) of cotranslated Rel A.
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Phosphatase Treatment of Phosphorylated Ikappa Balpha Reverses the Shift in Mobility on SDS-PAGE

To confirm that the observed mobility shifts reflect phosphorylation of Ikappa Balpha , pp90rsk-treated wild type and mutant S32/36A Ikappa Balpha proteins were incubated with calf intestinal alkaline phosphatase (Fig. 4B). The retarded mobility of pp90rsk-treated wild type Ikappa Balpha (lane 3) was lost following treatment with phosphatase (lane 4). In contrast, phosphatase treatment of the S32/36A Ikappa Balpha mutant, which did not display a mobility shift in the presence of pp90rsk, did not alter its electrophoretic mobility (compare lanes 7 and 8). These results confirm that pp90rsk-mediated phosphorylation of Ikappa Balpha is responsible for the altered migration of the wild type Ikappa Balpha proteins.

Phosphorylation of Ikappa Balpha in the Presence or Absence of Rel A

Under basal conditions, Ikappa Balpha is normally complexed with NF-kappa B in the cytosol. The Rel A subunit of NF-kappa B is directly associated with Ikappa Balpha in this complex. Studies by Chen et al. (11) suggest that Ikappa Balpha is not only phosphorylated, but also ubiquitinated and degraded while still complexed to NF-kappa B. To investigate whether the presence of Rel A affects the pattern of phosphorylation of Ikappa Balpha , the pp90rsk-mediated in vitro kinase assays were performed with Ikappa Balpha in both the presence and absence of in vitro cotranslated Rel A. These proteins assembled in vitro as indicated by the ability of anti-Rel A antibodies to coimmunoprecipitate Ikappa Balpha from the translation mix (data not shown). As shown in Fig. 4C, Ikappa Balpha displayed the same mobility shift when incubated with pp90rsk in the presence or absence of Rel A (compare lanes 2 and 6). No changes in mobility were obtained when the S32/36A Ikappa Balpha mutant was incubated with pp90rsk in the absence of Rel A, suggesting that Rel A did not occlude additional pp90rsk phosphorylation sites whose modification would affect Ikappa Balpha mobility (data not shown).

pp90rsk Physically Associates with Ikappa Balpha in Vivo

Proteins that participate in a linear pathway of signaling may sometimes physically associate with each other. Some protein kinases associate with their substrates under circumstances where the kinase is inactive or where ATP is limiting. If the association between a protein kinase and its substrate is sufficiently avid, their interaction may be detected by coimmunoprecipitation of the two proteins. To assess whether Ikappa Balpha can physically associate with pp90rsk in vivo, COS cells were cotransfected with expression vectors encoding HA-tagged pp90rsk or control HA-tagged protein phosphatase-2A A regulatory subunit (HA-PP2A) and Ikappa Balpha . Following transfection, proteins were metabolically radiolabeled with [35S]methionine and cysteine, and the resultant cell lysates were immunoprecipitated with nonspecific preimmune, anti-HA, or anti-Ikappa Balpha antibodies. Immunoprecipitation of HA-PP2A and Ikappa Balpha -transfected cells with anti-HA antibodies revealed a major band corresponding to HA-PP2A but no coimmunoprecipitation of Ikappa Balpha (Fig. 5A, compare lanes 1 and 2). Similarly, addition of anti-Ikappa Balpha antibody immunoprecipitated Ikappa Balpha but not PP2A (compare lane 3 to lanes 1 and 2). In contrast, cotransfection of cells with HA-pp90rsk and Ikappa Balpha led to coimmunoprecipitation of both of these molecules using either the HA- or Ikappa Balpha -specific antibodies (lanes 5 and 6). The in vivo association of pp90rsk and Ikappa Balpha was confirmed in experiments involving initial immunoprecipitation with anti-HA or anti-Ikappa Balpha followed by immunoblotting with an anti-pp90rsk antibody (Fig. 5B). As shown in lanes 2 and 3, anti-Ikappa Balpha coimmunoprecipitated significant amounts of pp90rsk in these cotransfected cells, while nonspecific preimmune sera yielded no detectable pp90rsk (lane 1). Together, these results indicate that Ikappa Balpha can physically associate with pp90rsk in vivo and thus provide further support for the possibility that pp90rsk functions as a physiologically relevant Ikappa Balpha kinase.


Fig. 5. Ikappa Balpha and pp90rsk physically associate in vivo. A, COS cells were cotransfected with expression vectors encoding Ikappa Balpha (all lanes) and HA-PP2A (lanes 1-3) or HA-pp90rsk (lanes 4-6) followed 48 h later by radiolabeling with [35S]methionine/cysteine. Cell lysates were immunoprecipitated with nonspecific control antibodies (lanes 1 and 4), anti-HA antibodies (lanes 2 and 5), or anti-Ikappa Balpha antibodies (lanes 3 and 6). Samples were analyzed by SDS-PAGE and autoradiography. B, combined immunoprecipitation and anti-pp90rsk immunoblotting were performed using nonradioactive COS cell lysates transfected as described above. Immunoprecipitations were performed using nonspecific (lane 1), anti-HA (lane 2), or anti-Ikappa Balpha (lane 3) antisera. Immunoblotting was performed using an anti-pp90rsk-specific antibody and developed using a second antibody and ECL.
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Induction of pp90rsk Kinase Activity by Phorbol Ester but Not by TNF-alpha or HTLV-I Tax

Studies were next performed to assess whether pp90rsk is activated by various well known inducers of NF-kappa B in vivo (Fig. 6). Activation of pp90rsk was monitored either by a very small but perceptible change in its electrophoretic mobility, reflecting autophosphorylation (Fig. 6A), or by its ability to phosphorylate recombinant Ikappa Balpha when the latter was added as an exogenous substrate to an in vitro kinase assay performed with anti-pp90rsk immunoprecipitates (Fig. 6B). PMA stimulation of Jurkat cells for 5 or 15 min resulted in the rapid activation of pp90rsk (panel A, lanes 3 and 4) and phosphorylation of Ikappa Balpha (panel B, lanes 3 and 4). In contrast, two other recognized inducers of NF-kappa B, TNF-alpha and HTLV-I Tax, did not significantly activate pp90rsk autophosphorylation (panel A, lanes 5-8) or induce phosphorylation of Ikappa Balpha in the in vitro kinase assay (panel B, lanes 5-8). However, this particular preparation of TNF-alpha (see panel C, lanes 5-7) and HTLV-I Tax (data not shown) stimulated phosphorylation and degradation of Ikappa Balpha . These findings indicate that only a subset of the known inducers NF-kappa B leads to the activation of pp90rsk, suggesting that other kinases are likely activated by different NF-kappa B inducers, such as TNF-alpha and HTLV-I Tax. This result argues against the notion of a single cellular Ikappa Balpha kinase.


Fig. 6. Activation of pp90rsk by some but not all inducers of NF-kappa B. A, immunoprecipitates from Jurkat cells (lanes 1-8) were analyzed by immunoblotting with anti-pp90rsk antibodies. Immunoprecipitations were performed using either nonspecific control serum (lane 1) or anti-pp90rsk antiserum (lanes 2-8) with untreated cells (lanes 1, 2, 9, and 10) or cells treated with PMA for 5 min (lane 3) or 15 min (lane 4), TNF-alpha for 5 min (lane 5) or 15 min (lane 6), or with Jurkat cells stably expressing HTLV-I Tax (lane 7) or a control antisense Tax cDNA (as Tax, lane 8). B, anti-pp90rsk-associated protein kinase activity assayed by adding Ikappa Balpha as an exogenous substrate. Lanes correspond precisely to the treatment conditions described in A. C, Western blot analysis using anti-Ikappa Balpha antibodies to test Ikappa Balpha degradation in extracts of untreated Jurkat cells (lane 1) or cells treated with PMA for 5 min (lane 2), 15 min (lane 3), or 30 min (lane 4), or with the same lot of TNF-alpha used in A and B for 5 min (lane 5), 15 min (lane 6), or 30 min (lane 7).
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DISCUSSION

Phosphorylation and dephosphorylation represents a general strategy frequently employed for the dynamic regulation of eukaryotic transcription factor function. The enhancer-binding protein is often the specific target of such post-translational modifications that lead to an alteration in its DNA binding or functional activity. However, in the NF-kappa B system, primary regulation is exerted through phosphorylation of Ikappa Balpha , an ankyrin-rich inhibitor that sequesters the NF-kappa B complex in the cytoplasm. Phosphorylation targets Ikappa Balpha for ubiquitination and subsequent degradation by the 26 S proteasome. Although a necessary step in the activation of transcription, phosphorylation alone does not result in the release of Ikappa Balpha from the NF-kappa B complex and thus is insufficient for activation of NF-kappa B-mediated transcription. Thus far, the identity of the kinase(s) responsible for coupling cellular activation to phosphorylation of Ikappa Balpha or other members of the Ikappa B family has remained elusive.

In the current paper, we have explored the possible function of pp90rsk as a stimulus-coupled Ikappa Balpha kinase. In quiescent cells, inactive pp90rsk resides in the cytoplasm and is partially complexed with its upstream regulator, p42/44MAPK (41). Cellular activation mediated by various growth factors operating through the Ras-Raf-MEK-MAPK pathway or phorbol ester leads to the activation of MAP kinase, the phosphorylation and activation of pp90rsk, and the import of these kinases into the nucleus. Activated forms of pp90rsk have already been implicated in the regulation of various nuclear transcription factors, including c-Fos (40) and Nur77 (42, 43). Recently, pp90rsk has been reported to produce both positive and negative effects on another family of inducible transcription factors, the cyclic AMP response element-binding proteins (CREB). Specifically, pp90rsk2, one of three closely related rsk genes (rsk1, rsk2, rsk3), has been shown to function as a stimulus-coupled CREB kinase modulating CREB activity by phosphorylating this factor on a key regulatory serine located at position 133 (44). Conversely, pp90rsk appears to oppose CREB action by inducibly but stably associating with the CREB-binding protein and blocking the interaction of this co-activator with CREB (45). Of note, the enzymatic function of pp90rsk is not required for these latter inhibitory effects. Together, these various results provide an experimental precedent for the participation of pp90rsk as a regulatory interface between the signals induced by the ligation of various growth factor receptors on the membrane and specific transcription factors that modulate the activity of target genes within the nucleus.

Using a purified activated enzyme preparation in initial in vitro kinase assays, we found that pp90rsk mediates phosphorylation of Ikappa Balpha . Furthermore, based on V8 protease analysis, this phosphorylation involves the N-terminus of Ikappa Balpha , where two critical residues for signal-induced phosphorylation, Ser-32 and Ser-36, reside. pp90rsk-mediated phosphorylation of Ikappa Balpha proved biologically relevant since this post-translational modification stimulated proteasome-dependent degradation of Ikappa Balpha . In contrast, the S32/36A double-substitution mutant of Ikappa Balpha was not degraded in the presence of activated pp90rsk. The stoichiometry of Ikappa Balpha phosphorylation by pp90rsk appeared quite high, as assessed by the ability of pp90rsk to elicit a gel mobility shift for Ikappa Balpha . By this criterion, one-third to one-half of the Ikappa Balpha molecules displayed an altered mobility in the presence of pp90rsk. Interestingly, the closely related S6 kinase, pp70S6K, which recognizes the same consensus phosphoacceptor site, Arg-X-X-Ser/Thr, as pp90rsk is incapable of phosphorylating Ikappa Balpha . Likewise, CaMKII, a calmodulin-dependent kinase that also phosphorylates within the same consensus phosphoacceptor site as pp90rsk and pp70S6K, fails to phosphorylate Ikappa Balpha . These findings indicate that the recognition of Ikappa Balpha by these protein kinases involves determinants beyond the consensus phosphoacceptor site. It is likely that the overall three-dimensional structures of Ikappa Balpha and the kinase play a pivotal role in the effectiveness of this protein-protein interaction.

Since a "purified" pp90rsk preparation was used in these in vitro studies, we considered the possibility that the Ikappa Balpha phosphorylation might be due to a contaminating kinase. However this possibility is unlikely because: 1) each of three independently purified pp90rsk preparations displayed the same Ikappa Balpha kinase activity in at least three assays; 2) overloading of an SDS-PAGE gel with the pp90rsk preparation followed by silver staining revealed only pp90rsk and no other bands; and 3) autophosphorylation reactions with the pp90rsk preparation revealed no other bands. The observed Ikappa Balpha kinase activity in the pp90rsk preparations thus appears to reflect the activity of pp90rsk rather than a contaminant.

Since serines 32 and 36 located near the N terminus of Ikappa Balpha are key regulatory sites that must be directly phosphorylated to trigger subsequent ubiquitination and degradation of this inhibitor (7-10, 18, 46), potential phosphorylation of these sites by pp90rsk was studied. Serine 32 is embedded within a sequence that conforms to the consensus phosphoacceptor site for phosphorylation by pp90rsk; however, serine 36 is not. With wild type Ikappa Balpha or the S32A and S36A single-substitution mutants of this inhibitor as substrates, pp90rsk was shown to readily phosphorylate Ikappa Balpha proteins containing serine 32. In contrast, as judged by mobility shift, serine 36 functioned as a very poor substrate for pp90rsk. Since both serine 32 and serine 36 must be directly phosphorylated for subsequent degradation (46), these findings suggest that the action of pp90rsk alone may not be sufficient to trigger the subsequent ubiquitination and degradation of Ikappa Balpha . It is possible that a second, yet unidentified, kinase present in the reticulocyte lysate mediates phosphorylation at serine 36, thus promoting Ikappa Balpha degradation. Although unproven, it is intriguing to consider the possibility that phosphorylation in vivo at the first serine site by one kinase may facilitate sequential phosphorylation at the second serine site by a different kinase. Alternatively, pp90rsk phosphorylation at serine 32 may enhance its activity at serine 36, producing a polarity to the sequence of modifications. Precedence for regulation by such sequential phosphorylation is found in the case of both pp90rsk and pp70S6K phosphorylation of S6 peptide (47). Finally, degradation of Ikappa Balpha in vitro may proceed with phosphorylation at serine 32 only.

To test the potential in vivo relevance of pp90rsk as an Ikappa Balpha kinase, we investigated whether these proteins can physically associate within a cell. Using COS cells for cotransfection with HA epitope-tagged pp90rsk and Ikappa Balpha expression vectors, we demonstrated that these two proteins, but not similarly epitope-tagged control proteins, are coimmunoprecipitated using either anti-HA or anti-Ikappa Balpha -specific antibodies. Using mutants of Ikappa Balpha to study this interaction further, our preliminary results indicate that the N terminus of Ikappa Balpha spanning the regulatory serines at positions 32 and 36 is not required for Ikappa Balpha binding to pp90rsk. In contrast, deletion of ankyrin repeats 1-5 of Ikappa Balpha severely impairs the interaction of Ikappa Balpha with pp90rsk.

Our final series of studies explored what NF-kappa B-inducing signals operate through pp90rsk-mediated phosphorylation of Ikappa Balpha . These studies revealed that PMA induced activation of pp90rsk, phosphorylation of Ikappa Balpha , and induction of NF-kappa B. In sharp contrast, neither TNF-alpha nor the Tax trans-activator protein of HTLV-I activated pp90rsk. However, both of the latter inducers potently stimulated phosphorylation and degradation of Ikappa Balpha and activated nuclear NF-kappa B expression. This result clearly indicates that not all NF-kappa B inducers operate through pp90rsk activation. These results predict the likely existence of multiple Ikappa B kinases that are differentially coupled to various signaling pathways. Thus, the critical kinase(s) ultimately responsible for phosphorylating serines 32 and 36 may vary, depending on the particular NF-kappa B-inducing signal. We propose that pp90rsk represents one such kinase in a larger set of enzymes that regulate Ikappa Balpha phosphorylation.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants P30 AI27763-08 Supplement (to W. C. G.) and 5RO1 GM49055, American Cancer Society Research Award VM-91, a Junior Faculty Research Award of the American Cancer Society (to L. G.), and Grant P30 AI27763 from the National Institutes of Health/University of California, San Francisco, Center for AIDS Research.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.
§   These authors contributed equally to this work.
**   To whom correspondence should be addressed: Gladstone Institute of Virology and Immunology, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-695-3800, Fax: 415-826-1817; E-mail: Warner_Greene{at}quickmail.ucsf.edu.
1   The abbreviations used are: NF-kappa B, nuclear factor kappa B; PMA, phorbol 12-myristate 13-acetate; IL, interleukin; CKII, casein kinase II; MEK, MAP/ERK kinase kinase; MAP, mitogen-activated protein; HA, hemagglutinin; DTT, dithiothreitol; HTLV, human T-cell lymphotrophic virus; TNF-alpha , tumor necrosis factor alpha ; PAGE, polyacrylamide gel electrophoresis; CaMKII, Ca2+-calmodulin-dependent protein kinase II; CREB, cyclic AMP response element-binding protein; PP2A, protein phosphatase 2A.

ACKNOWLEDGEMENTS

We thank Dr. Joseph Avruch, Dr. Al Baldwin, Dr. Dean Ballard (Vanderbilt University, Nashville, TN), Dr. James Maller and Dr. Michael Browning (University of Colorado Health Sciences Center, Denver, CO), and Dr. Mark Stinski for reagents, Dr. Dean Ballard and Dr. James Maller for helpful scientific discussions, and Mark Dettle for preparation of the manuscript. We also thank Richard Ruhlen and David Brand for technical assistance.


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