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Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in Raw264.7 macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene, macrophage inflammatory protein-1β. Consistent with these results, over-expression of GRK2 or 5 enhances TNFα-induced NFκB activity. In addition, we show that GRK2 and 5 interact with IκBα via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and 5 in vitro. Furthermore, we also find that GRK5 but not GRK2 phosphorylates IκBα at the same amino acid residues (Ser32/36) as that of IKKβ. Interestingly, associated with these results, knockdown of IKKβ in Raw264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and 5 are important and novel mediators of a non-traditional IκBα-NFκB signaling pathway.
Tumor necrosis factor-alpha (TNFα) is a pleiotropic cytokine secreted by immune cells, in particular by monocytes and macrophages and mediates a number of biological activities ranging from cell proliferation, differentiation and death to inflammation and innate and adaptive immune responses . Importantly, TNFα has been implicated in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and Crohn’s disease and several drugs targeting the TNFα system are already in clinical practice . TNFα mediates its diverse effects through two cell surface receptors: p55TNFR1 and p75TNFR2. TNFR1 is constitutively expressed in most nucleated cells whereas TNFR2 is expressed mainly in immune and endothelial cells. Stimulation of TNFR1 leads to the recruitment of several death domain containing adapter proteins such as TRADD (Tumor necrosis factor receptor 1 associated death domain protein) and RIP1 (Receptor interacting protein-1). This signaling complex interacts with adapter proteins such as TRAF2/5 (TNF receptor associated factor 2/5) and c-IAP1 (Inhibitor of apoptosis 1) which subsequently leads to the activation of signaling pathways that regulate many of the important biological activities of TNFα . Especially important from a physiological as well as pathological perspective is the major role of the “Nuclear factor κ B (NFκB)” signaling pathway in chronic inflammatory disease .
The NFκB family of transcription factors regulate genes involved in inflammation, innate and adaptive immune responses, cell proliferation, cell adhesion, programmed cell death (apoptosis), and cellular stress response and tissue remodeling (reviewed in ). The NFκB family members include p65 (RelA), p50, RelB, cRel and p52. These NFκB transcription factors are sequestered in the cytoplasm in the form of homo- or heteromeric complexes with the inhibitory proteins of the IκB family. The IκB family members include IκBα, IκBβ, Iκβε, p105 (NFκB1), and p100 (NFκB2). Activation of the NFκB pathway typically involves the phosphorylation of an IκB member primarily by the IκB kinase (IKK) complex. The phosphorylated IκB then undergoes ubiquitination and subsequent proteolysis leading to the release and translocation of NFκB into the nucleus where it affects gene transcription (reviewed in . Amongst the various IκB members, IκBα plays a critical role in the early activation of NFκB after ligand stimulation . IκBα binds to NFκB subunits p50 and p65 under unstimulated conditions. Upon stimulation IκBα undergoes rapid phosphorylation by IKKβ, followed by ubiquitination and degradation. IκBα degradation releases the NFκB subunits p50 and p65, which then translocate into the nucleus to evoke NFκB-dependent gene transcription (reviewed in . In addition to being physiological substrates for the IKK complex of enzymes, the IκB family members have also been shown to be substrates for other kinases . In this context, we demonstrated recently that p105 is a substrate for G-protein coupled receptor kinase-5 (GRK5) and that GRK5 phosphorylation of p105 regulates Toll-like receptor-4-induced p105 phosphorylation in macrophages .
G-protein coupled receptor kinases (GRKs) are serine/threonine kinases originally discovered for their role in the phosphorylation of G-protein coupled receptors (GPCRs) (reviewed in ). The seven mammalian GRKs are divided into three subfamilies based on sequence and functional similarities. The rhodopsin kinase subfamily (GRK1 and GRK7); the GRK2 subfamily (GRK2 and GRK3) and the GRK4 subfamily (GRK4, 5 and 6). All GRKs have a similar structural organization possessing N-terminal, catalytic and C-terminal domains. Interestingly, recent studies have shown that GRKs have functions that go beyond their role in GPCR phosphorylation. For example, GRKs have been shown to phosphorylate a number of cytoplasmic and nuclear proteins as well as additional classes of membrane-localized receptors[10–12] Moreover, yeast-two hybrid analysis identified NFκB1 p105 as a GRK2-interacting protein (http://www.signalinggateway.org/data/Y2H/cgi-bin/y2h.cgi) while we found that GRK5 but not GRK2 regulates p105 function in macrophages .
To better understand the role of GRKs in NFκB signaling in macrophages and to identify the potential role of these kinases in the regulation of other IκB members, we tested the role of GRK2 and 5 in the regulation of IκBα in the context of TNFα signaling in macrophages. Surprisingly, in contrast to the regulation of p105, our studies reveal that both GRK2 and 5 regulate the TNFα- induced IκBα-NFκB pathway in Raw264.7 mouse macrophages. We further demonstrate that IκBα directly interacts with GRK2 and 5 and is differentially phosphorylated by GRK2 and 5. Our observations add new insight into NFκB signaling as well as demonstrate novel GPCR-independent roles for GRKs.
Mouse TNFα was from PeproTech Inc. (Rocky Hill, NJ). IRDye® 700 labelled NFκB oligonucleotide probes were from LI-COR Biosciences (Lincoln, NE). Protease inhibitor cocktail tablets were from Roche Diagnostics (Indianapolis, IN). Luciferase assay buffer and substrate were from Promega (Madison, WI). All other reagents were from Sigma unless otherwise noted.
Anti-P50, P65, Lamin B, Protein A/G PLUS-agarose beads, Actin-HRP and polyclonal IκBα antibody were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-mouse IκBα and phospho-IκBα antibodies were from Cell Signaling Technology (Boston, MA), HA monoclonal antibody was from Covance and HA polyclonal antibody from Sigma. GRK2 and 5 monoclonal antibodies were from Upstate Biotechnology.
pcDNAGRK2, pcDNAGRK2-K220R, pcDNAGRK5 and pcDNAGRK5-K215R have been described previously[13, 14]. HA-GRK2 was created by excising GRK2 from pRK5-GRK2 construct with EcoRI and SalI and then inserting into pHA-CMV vector. Expression plasmid pCMX-IκBα (Addgene plasmid 12331) was from Dr. I. M. Verma , HA-IKKβ (Addgene plasmid # 15470) from Dr. H. Nakano , Flag-IKKγ (Addgene plasmid # 11970) from Dr. J. D. Ashwell , and Flag-IκBα from Dr. G. Pei . Adenoviruses expressing GRK2 and GRK5 were kindly provided by Dr. W. J. Koch.
Raw 264.7 macrophages and HEK293T cells were obtained from ATCC and were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin (100 units/ml) and streptomycin (100 µg/ml) at 37° C in 5% CO2.
Control siRNA pool (against luciferase gene not expressed in macrophages), GRK2 siRNA pool (against mouse GRK2), GRK5 siRNA pool (against mouse GRK5) and IKKβ siRNA pool (against mouse IKKβ) were purchased from Dharmacon (Dharmacon Research Inc., Lafayette, CO). Raw264.7 macrophages were transfected with siRNA using Amaxa nucleofector (Program D-032) as described previously . The cells were analyzed for knockdown by Western blotting after 48 hours of transfection.
HEK293T cells were transfected with an NFκB promoter luciferase plasmid (pELAM luciferase plasmid [19, 20] kindly provided by Dr. E. Latz, University of Massachusetts) and LacZ-expression plasmid  (kindly provided by Dr. Philip B. Wedegaertner, Thomas Jefferson University) together with either vector (control) or GRK2/5 wild type or GRK2-K220R/GRK5-K215R kinase deficient expression plasmids. Cells were stimulated with TNFα for 8 hours, lysates prepared and analysed for NFκB luciferase activity using Promega Luciferase Reporter Assay kit according to manufacturer’s instructions. β-Gal activity was determined as described previously . NFκB luciferase activity was expressed as a ratio of luciferase to beta-galactosidase activity. All experiments were performed in triplicate and repeated at least 3–5 times.
Nuclear extracts were prepared as described in Nuclear extraction kit (Panomics, CA). Briefly, control or GRK2 knockdown cells in a 60 mm plate were lysed in 500 µl of the buffer A mix [buffer A (100 mM HEPES, pH 7.9, 100 mM KCl, 100 mM EDTA), 100 mM DTT, 10 µl protease inhibitor cocktail and 0.38% NP-40]. Sonication was done to disrupt the cell clumps. The lysate was then centrifuged at a maximum speed (15,000 × g) for 3 min at 4°C. Supernatants (cytosolic fraction) were collected and the pellet suspended in 100 µl of buffer B mix [buffer B (100 mM HEPES, pH 7.9, 2 M NaCl, 5 mM EDTA), protease inhibitor cocktail and 100 mM DTT). The lysates were kept on a rocking platform for ~2 hours, after which the lysates were centrifuged at high speed for 20 min and the supernatants (nuclear fraction) were collected for further analysis.
Electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts . Briefly, ~9 fmol of double stranded oligonucleotide probes corresponding to the human consensus NFκB sequence end labeled with IRDye® 700 were incubated with 5 µg of nuclear extracts for 20 min in the dark at room temperature. Samples were then subjected to electrophoresis using 6% non-denaturing polyacrylamide gels and then analysed on LI-COR’s odyssey.
HEK293T cells were transiently transfected using Fugene (Roche) with the indicated expression plasmids in 60-mm cell culture dishes. The cells were washed twice with ice-cold phosphate-buffered saline after 48 hours and were lysed in 400 µl of cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% NP-40 containing protease and phosphatase inhibitors). Lysates were then incubated with antibody and protein A/G PLUS-agarose beads for 1 hour at 4° C. The beads were washed three times with lysis buffer and the immune complexes eluted with 2X SDS sample buffer. The eluates were then run on SDS-PAGE and transferred to nitrocellulose. Western blotting was then performed as described previously with either fluorescently tagged secondary antibodies using Licor or with HRP-conjugated secondary antibodies using chemiluminescence .
GST (GE HealthCare Biosciences, NJ), GST-IκBα and GST-IκBα(Δ1–75)  (kindly provided by Dr. Junan Li, Ohio State University) in pGEX bacterial expression plasmids were expressed and purified as described by the manufacturer (Amersham Biosciences). Briefly, overnight bacterial cultures were diluted 1:100 in 100 ml of fresh LB-ampicillin, grown for 2–3 hours until the OD reached 0.5–0.9. The culture was then induced with IPTG (1 mM) and incubated for another 3 hours after which the cells were pelleted (6000×g for 10 min at 4° C) and resuspended in ice cold PBS. Suspended cells were then disrupted using a sonicator and solubilized in 1% Triton-X-100. The cells were then centrifuged at 12,000 × g for 10 min at 4° C and the supernatants collected. These supernatants were incubated with 2 ml of the 50% slurry of Glutathione Sepharose 4B equilibrated with PBS for 30 min and the resin was transferred to a column (Empty Disposable PD-10 column). The column was washed 3 times with PBS after which the fusion proteins were eluted by adding 1 ml of elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The purity and integrity of the fusion proteins were analysed and confirmed by SDS-PAGE and Coomassie blue staining.
Interaction between GST fusion proteins and GRK2 or 5 were determined using an overlay assay . Briefly, GST-fusion proteins (5 µg) were resolved on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membrane was then blocked with 5% w/v fat-free milk in TBS-T and incubated overnight at 4° C in lysates (~400 µg total protein) of HEK293T cells expressing vector, HA-GRK2, or GRK5. Blots were then washed three times with TBST (Tris- Buffered saline-Tween 20) and immuno blotting performed using appropriate antibodies. Blots were then stained with Ponceau to confirm equivalent amount of GST-fusion protein loading.
In vitro phosphorylation reactions were performed using purified GRK2 and GRK5 with IκBα as a substrate . Purified GST-IκBα or GST (~200 nM each) were incubated with 25 nM GRK2 or GRK5 at 30°C for 15 min in the presence or absence of 2 mg/ml soybean phosphatidylcholine and 60 nM purified Gβγ in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, 1–2 µCi of [32P]ATP in a final volume of 20 µl. Reactions were stopped by the addition of 5 µl of SDS sample buffer and incubation at room temperature for 30 min. Samples were then electrophoresed on a 10% SDS-polyacrylamide gel, the gel was dried, and 32P-labeled IκBα was visualized by autoradiography and quantified by excising the bands and scintillation counting.
RNA extraction and real-time Q-RT-PCR were performed as described previously . Briefly, total RNA was extracted using TRIzol reagent (Invitrogen). After sodium acetate–ethanol precipitation and several ethanol washes, the RNA integrity was verified by formaldehyde–agarose gel electrophoresis. Synthesis of cDNA was performed with reverse transcriptase (RT) with the total RNA using the superscript II kit with Oligo-dt (12–18) primers as recommended by the manufacturer (Invitrogen). cDNA was amplified by PCR in a final reaction volume of 25 µl using SYBR Green Supermix (Invitrogen) with 10 pmol of each primer for MIP1β and cyclophilin (for normalization) (primers obtained from IDT DNA Technologies, primer sequence available upon request). Real-time PCR was performed using MX3000P (Stratagene) thermocycler and data analyzed using MX3000P software.
All values are represented as mean±SEM. Data were analyzed and statistics performed using GRAPHPAD PRISM software (San Diego, California) using student’s t-test (for comparing two groups) and ANOVA (for comparing three or more groups). P value of less than 0.05 was considered significant.
Previous studies have shown that of the seven GRKs, GRK2 and 5 are expressed at relatively high levels in immune cells including macrophages [25, 26]. Furthermore, recent studies have shown that GRKs can regulate GPCR as well as non-GPCR signaling in macrophages . Because TNF receptor-induced NFκB signaling plays a major role in macrophage biology and has been implicated in many chronic inflammatory diseases [4, 27], we explored the role of GRKs in TNFα-induced NFκB signaling in macrophages. For this, we first tested the effect of GRK2 knockdown using siRNA pool, on TNFα-induced IκBα phosphorylation and degradation. Treatment of control macrophages with TNFα caused a time-dependent increase in IκBα phosphorylation (at serines-32/36) and a subsequent decrease in IκBα levels (Fig 1). IκBα phosphorylation was maximal at 5 min after TNFα stimulation, whereas, the consequent decrease in IκBα levels was maximal at 15 min after stimulation in control cells. However, the TNFα-mediated increase in IκBα phosphorylation and decrease in IκBα levels were both significantly blocked in GRK2 knockdown macrophages. Compared to the maximal stimulation of 95±5% at 5 min in control cells, IκBα phosphorylation reached only 40±2% in GRK2 knockdown cells (Fig. 1B). Consistent with the effects on IκBα phosphorylation, IκBα degradation was also inhibited in GRK2 knockdown cells compared to control cells. At 15 min after treatment IκBα levels reached 62±7% of untreated levels (=100%) in control cells, whereas it reached only 94±5% in GRK2 knockdown cells (Fig 1C). Importantly, IκBα levels did not differ between control and GRK2 knockdown cells in the absence of TNFα treatment (0.136±0.057 in control cells v/s 0.134±0.057 in GRK2 knockdown cells), suggesting that the effect of GRK2 knockdown on IκBα levels is specific for stimulated conditions. Furthermore, to rule out the nonspecific effects of the siRNA, we repeated these experiments using individual siRNAs and obtained similar results (Supplementary Figure 1). In addition, siRNA oligos that did not knockdown GRK2 levels did not affect TNFα-induced IκBα phosphorylation (data not shown). Interestingly, the effect of GRK2 knockdown on IκBα was specific for TNFα-induced pathway because LPS-induced IκBα degradation was not inhibited by GRK2 knockdown (Supplementary figure 2).
Because IκBα phosphorylation and degradation leads to subsequent release and translocation of the NFκB subunits, primarily p50 and p65, we next examined the nuclear levels of the NFκB subunits in control and GRK2 knockdown cells before and after TNFα treatment. As with IκBα phosphorylation/degradation, TNFα-induced p50 and p65 nuclear translocation were significantly inhibited in GRK2 knockdown cells compared to control cells (Fig 1D). These effects of GRK2 are specifically due to a decrease in nuclear translocation and not a decrease in expression levels of these proteins since the cytosolic expression of p50 and p65 did not differ between control and GRK2 knockdown cells (data not shown). Associated with these findings, we also observed that the NFκB binding activity (assessed by EMSA) was inhibited in GRK2 knockdown macrophages (Fig 1E).
To further rule out the possibility of non-specific effects of RNAi, we tested the effects of GRK2 over-expression in TNFα-induced IκBα phosphorylation in macrophages. Raw264.7 macrophages were transfected with adenoviruses expressing vector or GRK2 and the effect of TNFα-induced IκBα phosphorylation tested as described earlier. As predicted, over-expression of GRK2 significantly enhanced TNFα-induced IκBα phosphorylation compared to vector controls (Fig 2).
We next tested whether other GRKs can regulate IκBα phosphorylation/degradation. We especially focused on GRK5 since we previously showed that GRK5 inhibits LPS-induced p105 phosphorylation in macrophages . Interestingly, similar to the effects of GRK2, knockdown of GRK5 using siRNA pool (Fig. 3A) significantly inhibited TNFα-induced IκBα phosphorylation and degradation (Fig 3B, C & D). TNFα-stimulated IκBα phosphorylation (Ser32/36) in GRK5 knockdown cells reached only 27±5% of the maximal response after 5 min compared to 100% in control cells (Fig 3C). Similarly, IκBα levels after 15 min of TNFα stimulation reached 62±6% of untreated levels in control cells but only 99±5% in GRK5 knockdown cells (Fig 3D). Unlike GRK2 knockdown, basal levels of IκBα were somewhat elevated after GRK5 knockdown (0.361±0.080 in control vs. 0.563±0.071 in GRK5 knockdown cells). Similar to GRK2 siRNAs, individual siRNAs against GRK5 also gave similar results to that of the pool (Supplementary Figure 3). Interestingly, over-expression of GRK5 using adenovirus only modestly enhanced IκBα phosphorylation (Supplementary Figure 4).
In addition to IκBα phosphorylation, TNFα treatment also induces NFκB1 p105 (another member of the IκB family) phosphorylation at Ser932 . Previous studies have shown that GRK2 and 5 can interact with p105 and that GRK5 negatively regulates LPS-induced p105 phosphorylation . To determine whether the observed effects of GRK2/5 knockdown are specific for TNFα-induced IκBα phosphorylation or whether p105 phosphorylation can also be regulated in a similar manner, we examined TNFα-induced p105 phosphorylation in control and GRK2/5 knockdown macrophages. Interestingly, unlike IκBα phosphorylation, GRK2/5 knockdown did not affect TNFα-induced p105 phosphorylation (at Ser932) (Supplementary Figure 5 and Supplementary Figure 6). This suggests that the role of GRK2/5 is specific for TNFα-induced IκBα-NFκB pathway.
IκBα-NFκB pathway was recently shown to be critical for the expression of MIP1β (macrophage inflammatory protein 1β), one of the major chemokines expressed by macrophages . Therefore, we tested whether the role of GRK2 and 5 on the TNFα-induced NFκB pathway extended downstream of NFκB activation to MIP1β mRNA expression. Treatment of control macrophages with TNFα induced MIP1β mRNA expression by ~4–5-fold. This increase in MIP1β expression was significantly blocked in both GRK2 and 5 knockdown macrophages (Fig 4A and 4B). These results demonstrate that GRK2 and 5 regulate TNFα-induced IκBα-NFκB pathway as well as its physiological gene expression target in macrophages.
To further explore the biochemical mechanism by which GRK2 and 5 mediate TNFα-induced NFκB activity, we next tested whether the kinase activity is essential for the observed effects of the GRKs. For this, we over-expressed vector or wild type or kinase dead GRK2 or GRK5 in HEK293T cells along with an NFκB reporter plasmid (pELAM luciferase) and LacZ (for transfection normalization). Forty-hours after transfection, cells were serum starved (~3 hours) and were stimulated (or not) with TNFα for 8 hours and luciferase and β-galactosidase activity determined as described in the methods. In vector transfected cells, TNFα stimulation significantly increased NFκB-luciferase activity (Fig 5). As predicted, over-expression of wild type GRK2 or GRK5 significantly enhanced TNFα-induced NFκB activity while over-expression of kinase-inactive GRK2 or GRK5 (GRK2-K220R or GRK5-K215R) had no effect (Fig 5A and 5B). This demonstrates that the kinase activities of GRK2 and 5 are required for the observed effects of GRKs in TNFα-induced NFκB signaling. Interestingly, as observed with IκBα phosphorylation in macrophages, over-expression of GRK2 but not GRK2-K220R enhanced NFκB-luciferase activity even in the absence of ligand stimulation (1.0 ± 0.51 activity in vector cells compared to 2.35 ± 1.07 in GRK2 and 0.70 ± 0.32 in GRK2-K220R expressing cells). In contrast, over-expression of GRK5 or GRK5-K215R did not significantly affect basal NFκB activity (1 ± 0.14 activity in vector cells compared to 1.51 ± 0.17 in GRK5 and 0.86 ± 0.04 in GRK5-K215R expressing cells).
To define the biochemical mechanisms that mediate GRKs’ actions, we first tested whether GRKs affect IKKβ expression or activity. For this purpose, we knocked down and over-expressed GRK2/5 in Raw264.7 macrophages and HEK293T cells respectively and examined the levels of IKKβ. Neither knockdown nor over-expression of GRK2/5 affected IKKβ levels in the presence or absence of TNFα (data not shown). To further rule out the effect of GRKs on IKKβ activity, we performed an in vitro IKKβ kinase assay using IKKβ immunoprecipitated from cells over-expressing GRK2/5. In vitro phosphorylation of GST-IκBα by immunoprecipitated IKKβ was not affected by over-expression of either GRK2 or GRK5 (data not shown). Similarly, the interaction of IκBα and IKKβ was not affected by over-expression of GRKs in HEK293 cells (data not shown). Based on these results we hypothesized that GRKs might mediate their effects via directly interacting with and phosphorylating IβBα. To first examine if GRK2 or 5 can interact directly with IκBα, we tested the ability of GST or GST-IκBα to bind GRK2 and 5 using an overlay blot assay. For this, bacterially expressed and purified GST or GST-IκBα or GST- IκBα(76–302) were run on SDS-PAGE, and transferred to nitrocellulose. The membranes were then incubated with HEK293T lysates over-expressing either vector, GRK2 or GRK5 and tested for the ability of GRKs to specifically bind to GST-IκBα. As hypothesized, both GRK2 (Fig 6A and B) and GRK5 (Fig 7A and B) bound to GST-IκBα with no significant binding to GST. In addition, neither GRK2 (Fig 6A) nor GRK5 (Fig 7A) interacted appreciably with an N-terminal deletion mutant of IκBα [IκBα(76–302)], suggesting that both GRKs primarily interact with IκBα at the N-terminus. We also tested the ability of GRKs to interact with IκBα in intact cells and found that immunoprecipitation of IκBα co-immunoprecipitated both GRK2 and 5 (Fig 6C and Fig 7C in HEK293 cells; Fig 6D in human monocytic cells THP1). Taken together, these results suggest that IκBα is a direct interaction partner for both GRK2 and GRK5.
Experiments using kinase-dead mutants of GRK2 and 5, as well as the experiments described above, suggest that IκBα may be a substrate for GRKs in TNFα-induced NFκB signaling. To directly test this, we performed in vitro phosphorylation assays using purified GRK2 and 5 with IκBα as the substrate. GRK5 effectively phosphorylated IκBα to a stoichiometry of ~0.75 mol/mol (Fig. 8A) and interestingly, phosphorylation was effectively attenuated by the addition of phospholipids, which normally activate GRK5 (Fig. 8B) . In contrast, IκBα was a relatively poor substrate for GRK2 (Fig 8A), although the phosphorylation was enhanced by the addition of Gβγ subunits and phospholipids, known activators of GRK2 (Fig. 8B) . Taken together, these results demonstrate that IκBα is an in vitro substrate for both GRK2 and GRK5.
To identify the GRK phosphorylation sites in IκBα and to examine if IκBα is differentially phosphorylated by GRK2 and 5, we first tested whether the known IKKβ phosphorylation sites (Ser32/36) are also phosphorylated by GRK2 or GRK5. Indeed, previous studies have shown that these two residues are targeted by additional kinases such as ribosomal S6K and CK II, especially in NFκB pathways that are largely IKKβ-independent [32, 33]. To test this, we assessed the ability of GRK2 and GRK5 to phosphorylate a GST-IκBα S32/36A mutant. Our results show that GRK2 mediated phosphorylation of wild type and mutant GST-IκBα was comparable, suggesting that these sites are not phosphorylated by GRK2 (Fig 8C). In contrast, GRK5 mediated phosphorylation of the IκBα mutant was decreased ~60% compared to wild type IκBα (Fig. 8C), suggesting that GRK5 phosphorylates one or both of these sites. This result was confirmed using an antibody that recognizes phosphoSer32 in IκBα. For these studies, GST-IκBα was initially phosphorylated in vitro using GRK2, GRK5 or IKKβ and the samples were run on SDS PAGE and immunoblotted using anti-IκBα-phospho-Ser32. These results show that Ser32 is selectively phosphorylated by GRK5 but not GRK2 and that GRK5 mediated phosphorylation of IκBα is comparable to that seen with IKKβ (Fig 8D). Overall, these results reveal that Ser32 is phosphorylated by GRK5 and that GRK2 and GRK5 phosphorylate distinct residues.
Similar to GRK5, other kinases such as casein Kinase II and ribosomal S6 kinase have been shown to phosphorylate IκBα at Ser32/36. Interestingly, these kinases were shown to selectively regulate IκBα-NFκB pathway in an IKKβ-independent manner. Therefore, we hypothesized that because of the role of GRK2 and 5 as “IκBα kinases”, IKKβ may be dispensable in Raw264.7 cells, particularly for TNFα-induced IκBα phosphorylation. To test this hypothesis, we knocked down IKKβ in Raw264.7 macrophages (Fig 9) and tested the effect of TNFα on IκBα phosphorylation (Ser32/36). As predicted, we found that TNFα-induced IκBα phosphorylation is not significantly affected by knockdown of IKKβ, suggesting that IKKβ may be redundant in this system (Fig 9). However, it is possible that the level of knockdown is not sufficient to inhibit IκBα phosphorylation because of the residual IKKβ kinase activity present. To rule out this possibility, we further tested the ability of IKKβ knockdown to inhibit LPS-induced IκBα phosphorylation. Interestingly, our results demonstrate that LPS-induced IκBα phosphorylation (at Ser32/36) is inhibited by IKKβ knockdown and this effect was particularly evident at later time points (Supplementary figure 7). These results suggest that the IKKβ plays a crucial role in LPS-induced IκBα phosphorylation, but not in TNFα-induced IκBα-NFκB pathway in Raw264.7macrophages.
Taken together, our results demonstrate a critical role for GRK2 and 5 in the regulation of TNFα-induced IκBα-NFκB pathway in Raw264.7 macrophages and suggest that IκBα phosphorylation by GRKs might be an essential step in this regulation.
G-protein coupled receptor kinases were first discovered for their role in GPCR phosphorylation and desensitization [34, 35]. Recent studies, however, have revealed a number of non-GPCR substrates for GRKs [8, 10, 11, 30, 36, 41]. Although GRKs mediate their cellular effects for the most part through their catalytic activity, recent studies have also proposed a kinase-independent role for GRKs in cellular signaling (via protein-protein interaction with the RH domain). In this regard, GRK2 has been shown to interact with MEK1 and regulate ERK activation in a kinase-independent manner . GRK2 has also been shown to interact with other proteins such as PI3K , Akt , and GIT  and regulate a number of cell biological effects. In addition to the receptor and cytosolic substrates, GRKs, have also been found to phosphorylate nuclear proteins. A physiologically important role for the nuclear localized GRK5 was recently identified by Martini et al  who showed that the nuclear GRK5 is a HDAC kinase the mediates the epigenetic regulation of gene expression in cardiomyocytes. Taken together these studies suggest that the role of GRKs is much broader than previously appreciated.
Studies have just begun to emerge on the potential role for GRKs in the regulation of various components of the NFκB pathway. In this regard, we demonstrated that GRK5 stabilizes LPS-stimulated p105 levels in macrophages . More recently, while this manuscript was in preparation, a similar stabilizing role for GRK5 in maintaining IκBα levels in endothelial cells was shown . Surprisingly in the present study we find that GRK2 and GRK5 are important regulators of IκBα-NFκB signaling and mediate TNFα-induced IκBα phosphorylation. In addition, in contrast to the findings of Sorriento et al  in endothelial cells, our results demonstrate that GRK2 and GRK5 mediate TNFα-induced NFκB-dependent gene transcription in macrophages. Our results further suggests that even with in a given cell type, the role of GRKs is selective for a particular ligand.
Our data further demonstrate that the role of GRK2 and GRK5 on TNFα-induced NFκB signaling is dependent on the kinase activities because the kinase-deficient GRKs failed to mediate NFκB activation. Also, in vitro kinase assays indicate that IκBα may be differentially phosphorylated by GRK2 and 5. Our results show that the presence of Gβγ subunits and liposomes significantly enhances the phosphorylation of IκBα by GRK2. Gβγ subunits have clearly been demonstrated to be important in the translocation of GRK2 to the plasma membrane for GPCR phosphorylation. Whether a similar role for Gβγ subunits in TNFα signaling exists, is presently not known. However, Kawamata et al  showed recently that TNFα signaling is mediated by activation of G-proteins in adipocytes. In addition, TNFα treatment of THP-1 monocytic cells has been shown to mediate GRK2 translocation to the membrane and affect β-adrenergic receptor desensitization , suggesting possible regulation of TNFα-induced GRK2 activity by G-proteins. In contrast to the role of lipids in GRK2 activity, GRK5 phosphorylation of IκBα appears to be effectively inhibited in the presence of lipids even though previous studies have clearly shown that lipids activate GRK5 . Thus it is possible that GRK5 phosphorylation of IκBα is regulated by biochemical mechanisms that are distinct from its phosphorylation of other substrates such as synucleins  as well as from that of IκBα phosphorylation by GRK2. Whether these differences in the phosphorylation of IκBα by GRK2 and 5 translate into regulation of IκBα in different sub-cellular environments in not known and will be tested in future studies. Other studies have clearly shown that IκBα-NFκB complexes can be present in different sub-cellular environments [45, 46] and therefore, these complexes could be potentially regulated by GRK2 or 5 depending on the local cellular environment.
IKKβ has been identified as the primary kinase that phosphorylates IκBα. However, there is now extensive evidence that other kinases including IKKα, CK II and ribosomal S6K can phosphorylate IκBα at the same sites as that of IKKβ. This redundancy can in part be explained by the receptor- and cell type-specific regulation of IκBα-NFκB pathways [7, 32, 47]. For example, studies have shown that UV light-induced IκBα degradation is mediated by phosphorylation of IκBα by CK II . Also, PMA-induced IκBα phosphorylation has been shown to involve ribosomal S6K . Similarly, recent studies have shown IKKβ-dependent and –independent pathways that regulate IκBα-NFκB pathway in human macrophages in response to specific ligands . Our studies clearly suggest that GRKs while necessary for TNFα-induced IκBα-NFκB pathway, they are not involved in LPS-induced IκBα-NFκB signaling. Interestingly, knockdown of IKKβ does not affect TNFα-induced IκBα phosphorylation (at Ser32/36), but does inhibit LPS-induced IκBα phosphorylation, suggesting that in Raw264.7 macrophages, GRKs play the role of IκBα kinases selectively for TNFα signaling. Our in vitro kinase reactions further support our findings in macrophages in that, GRK5 is able to phosphorylate some residues in IκBα that are similar to that of IKKβ. Thus it is possible that GRK5 specifically might function in a similar capacity to that of IKKβ. If GRK5 can function as an IκBα kinase, then what is the role of GRK2? It appears from our macrophage experiments that GRK2 is also necessary for TNFα-induced IκBα phosphorylation (Ser32/36). However, in vitro GRK2 does not phosphorylate Ser32/36 and therefore appears to phosphorylate a different set of residues. In cells, GRK2 phosphorylation of these unidentified residues appears to be necessary for phosphorylation of Ser32/36 since knockdown of GRK2 inhibits IκBα phosphorylation at these two sites. It is also possible the level of IKKβ knockdown obtained might not be sufficient to define IKKβ-independent regulation because of the residual kinase activity present in the knockdown cells. If this is the case, GRKs might work co-operatively with IKKβ in the phosphorylation of IκBα. In this regard, although Ser32/36 in IκBα are the well-characterized IKK phosphorylation sites, there is strong evidence that IKK also phosphorylates less well-characterized sites at the c-terminus . Therefore, in our experiments, we cannot rule out the role of IKKβ on phosphorylation of these other sites. Our results, however, indicate that IKKβ knockdown can certainly inhibit LPS-induced IκBα phosphorylation (on Ser32/36) and therefore, suggest that LPS and TNFα signal to NFκB activation via different mechanisms in Raw264.7 macrophages.
In conclusion, our studies unravel important biochemical roles for GRK2 and 5 in TNFα-induced IκBα phosphorylation and NFκB signaling. Because regulation of the NFκB pathway appears to be receptor-specific as well as cell type-specific, further broad and unbiased proteomic approaches are necessary to identify macrophage-specific and TNFR-specific signaling complexes that mediate NFκB activation. These studies will undoubtedly identify therapeutic targets for inhibiting TNFα signaling in chronic inflammatory diseases.
We would like to thank Dr. Christina Pao and Ms. Michelle Kratz for preparing purified GRKs. This work was funded in part by grants from National Institutes of Health AR055726 and HL095637 (to N.P.) and GM44944 (to J.L.B.) and grant-in-aid from the Midwest affiliate of American Heart Association (to N.P.).