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Mechanism of Ribosomal p70S6 Kinase Activation by Granulocyte Macrophage Colony-stimulating Factor in Neutrophils

2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês

10.1074/jbc.m300376200

1083-351X

Jason A. Lehman, Víctor Calvo, Julián Gómez-Cambronero,

NF-κB Signaling Pathways

We report here for the first time the detection of the ribosomal p70S6 kinase (p70S6K) in a hematopoietic cell, the neutrophil, and the stimulation of its enzymatic activity by granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF modified the V max of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr389 and Thr421/Ser424. The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr389 bands in Western blots, or as a downshift in the relative mobility of phospho-Thr421/Ser424 bands (consistent with the loss of phosphate), but not both simultaneously. The immunosuppressant FK506 failed to inhibit p70S6K activation, but was able to rescue the rapamycin-induced downshift, pointing to a role for the mammalian target of rapamycin (mTOR) kinase. Rapamycin also caused an inhibition (IC50 0.2 nm) of the in vitro enzymatic activity of p70S6K. However, the inhibition of activity was not complete, but only a 40–50%, indicating that neutrophil p70S6K activity has a rapamycin-resistant component. This component was totally inhibited by pre-incubating the cells with the mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 prior to treatment with rapamycin. This indicated that a kinase from the MEK/MAPK pathway also plays a role in p70S6K activation. Thus, GM-CSF causes the dual activation of a rapamycin-resistant, MAPK-related kinase, that targets Thr421/Ser424 S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr389, both of which are needed in cooperation to achieve full activation of neutrophil p70S6K. We report here for the first time the detection of the ribosomal p70S6 kinase (p70S6K) in a hematopoietic cell, the neutrophil, and the stimulation of its enzymatic activity by granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF modified the V max of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr389 and Thr421/Ser424. The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr389 bands in Western blots, or as a downshift in the relative mobility of phospho-Thr421/Ser424 bands (consistent with the loss of phosphate), but not both simultaneously. The immunosuppressant FK506 failed to inhibit p70S6K activation, but was able to rescue the rapamycin-induced downshift, pointing to a role for the mammalian target of rapamycin (mTOR) kinase. Rapamycin also caused an inhibition (IC50 0.2 nm) of the in vitro enzymatic activity of p70S6K. However, the inhibition of activity was not complete, but only a 40–50%, indicating that neutrophil p70S6K activity has a rapamycin-resistant component. This component was totally inhibited by pre-incubating the cells with the mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 prior to treatment with rapamycin. This indicated that a kinase from the MEK/MAPK pathway also plays a role in p70S6K activation. Thus, GM-CSF causes the dual activation of a rapamycin-resistant, MAPK-related kinase, that targets Thr421/Ser424 S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr389, both of which are needed in cooperation to achieve full activation of neutrophil p70S6K. p70S6K 1The abbreviations used are: p70S6K, ribosomal p70-S6 kinase; GM-CSF, granulocyte macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; EPO, erythropoietin; IL-8, interleukin-8; PI3K, phosphatidylinositol 3-kinase; PDK1, 3-phosphoinositide-dependent protein kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKi, MEK inhibitor PD-98059; mTOR, mammalian target of rapamycin; FKBP, FK506-binding protein; FRAP, FKBP12-rapamycin-associated protein; PP2B, protein phosphatase 2B. catalyzes the phosphorylation of the S6 protein (1Nemenoff R.A. Price D.J. Mendelsohn M.J. Carter E.A. Avruch J. J. Biol. 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Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar). The structural analog of rapamycin, FK506, competitively binds to FKBP and reverses the inhibition of mTOR by the former (31Chung J. Kuo C.J. Crabtree G.R. Blennis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1031) Google Scholar). Polymorphonuclear leukocytes (neutrophils) are recruited to sites of inflammation responding to chemoattractants that are secreted by several tissue cells in response to a physical or chemical insult, or by certain bacterial products in the case of a localized infection. The cytokine GM-CSF elicits the two components of cell migration in neutrophils: chemotaxis and chemokinesis. 2J. Gomez Cambronero, J. Horn, M. A. Baumann, and C. C. Paul, submitted manuscript. GM-CSF has a number of other functions on neutrophils and their bone marrow precursors, that involve the activation of two major signaling cascades: the JAK/STAT and Ras/MAPK pathways (reviewed in Ref. 33Gomez-Cambronero J. Veatch C. Life Sci. 1996; 59: 2099-2111Crossref PubMed Scopus (17) Google Scholar). GM-CSF-induced translocation of p42mapk (ERK2) to the cell nucleus and concomitant phosphorylation of the ribosomal kinase p90rsk is central in mitogenic events. Although it has been shown that G-CSF, a human hematopoietic factor related to GM-CSF, activates PI-3K/Akt(PKB) and promotes cell survival (34Hunter M.G. Avalos B.R. Blood. 2000; 95: 2132-2137Crossref PubMed Google Scholar), little information exists regarding activation of other members of this cell signaling cascade, particularly p70S6K or whether GM-CSF will mediate its physiological effects (notably cell migration) through mTOR-S6K. We have previously demonstrated that MAPK activation in response to GM-CSF is up-regulated in mature cells such as the neutrophil and plays a role in chemotaxis (35Lehman J.A. Paul C.C. Baumann M.A. Gomez-Cambronero J. Am. J. Physiol. Cell Physiol. 2001; 280: 183-191Crossref PubMed Google Scholar), and that a molecular connection between the MAPK and the p70S6K pathways exists (36Lehman J.A. Gomez-Cambronero J. Biochem. Biophys. Res. Commun. 2002; 293: 463-469Crossref PubMed Scopus (63) Google Scholar). Here we report that p70S6K is present in neutrophils, that GM-CSF causes an increase in phosphorylation of Thr389 and Thr421/Ser424 concomitantly to an increase in its enzymatic activity. We also show for the first time that the mechanism by which GM-CSF activates ribosomal S6K is through a combination of activation of two signaling pathways: mTOR and MAPK. Materials and Antibodies—GM-CSF was from Sandoz (East Hanover, NJ); G-CSF was from Amgen (Thousand Oaks, CA); IL-8 was from R&D Systems (Minneapolis, MN); fMet-Leu-Phe, PKA inhibitor, calphostin-C, PMA, anti-rabbit IgG (agarose beads), the cAMP-dependent kinase inhibitor TTYADFIASGRTGRRNAIHD, anti-p70S6 kinase polyclonal antibody used for immunoblotting, and phalloidin-FITC conjugate conjugate from Amanita phalloides, were from Sigma; FACS FLOW buffer was from Fisher (Hanover Park, IL); electrophoresis chemicals were from Bio-Rad Laboratories (Richmond, CA); [γ-32P]ATP (30 Ci/mmol) was from Amersham Biosciences; PD-98059 was from BioMol (Plymouth Meeting, PA); rapamycin, FK506 and alkaline phosphatase (purified from calf intestine) were from Calbiochem (La Jolla, CA); ion-exchange chromatography cellulose phosphate paper was from Whatman (Hillsboro, OR); anti-p70S6 kinase (C-18) polyclonal antibody used for immunoprecipitation was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-phospho(Thr389)-p70S6 kinase and anti-phospho(Thr421/Ser424)-p70S6 kinase antibodies (polyclonal) were from Cell Signaling (Beverly, MA); p70S6 kinase peptide substrate KKRNRTLTK and 3T3 fibroblasts cell lysates were from Upstate Biotechnology Inc. (Lake Placid, NY). Cells—Peripheral blood neutrophils were isolated based on a protocol described by English and Andersen (37English D. Andersen B.R. J. Immunol. Methods. 1974; 5: 249-252Crossref PubMed Scopus (578) Google Scholar). Between 50–55 ml of blood were collected from the antecubital vein of healthy individuals (who signed an Institutional Review Board-approved consent form) using sodium citrate as anticoagulant. Blood was mixed with 15 ml of 6% dextran, allowed to settle, and the plasma and buffy coat were removed and spun down at 800 × g for 5 min. The pellet was resuspended in 35 ml of saline and centrifuged again for 15 min at 10 °C in a Ficoll-Histopaque discontinuous gradient. Neutrophils were recovered and contaminating erythrocytes were lysed by hypotonic shock. Cells were washed and the purified neutrophil pellet was resuspended in Hanks Balanced Salt Solution (HBSS). Our experience has indicated that using this protocol, neutrophil aggregation (i.e. the hallmark for neutrophil activation) does not occur. Viability is usually >98 ± 2% as per trypan blue exclusion. Cells were resuspended at the concentration of 5 × 106 cells/ml in fresh Hanks Balanced Salt Solution (HBSS) or in RPMI at 2 × 106 cells/ml at the time of the experiment, and used within 2–3 h after isolation. Immunoprecipitation and Western Blotting Analyses—The procedure was based on our previous report (38Joseph D.E. Paul C.C. Baumann M.A. Gomez-Cambronero J. J. Biol. Chem. 1996; 271: 13088-13093Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) with some modifications, as follows. Neutrophils were resuspended in RPMI 1640 at 3 × 106 cells/ml density and were pretreated with rapamycin, FK506, or MEKi where appropriate and then stimulated with GM-CSF at 37 °C. Aliquots (1 ml) were taken, spun down (14,000 × g, 15 s) and pellets were resuspended in 0.2 ml of boiling SDS solution (1% SDS in 10 mm Tris-HCl, pH 7.4). Samples were boiled in a heat block for 10 min with frequent vortexing to achieved complete dissolution, taken to an ice bucket and mixed with 0.3 ml cold ddH2O and 0.4 ml of cold, Triton X-100-based, lysis buffer (12 mm Tris-HCl, pH 7.2, 0.75 mm NaCl, 100 μm sodium orthovanadate, 10 mm phenylmethylsulfonyl fluoride, 0.2 mm β-glycerophosphate, 5 μg/ml each of aprotinin, pepstatin A, and leupeptin, and 0.12% Triton X-100). The resulting 1 ml of total cell lysates were spun down (14,000 × g, 1 min, 4 °C) to remove any insoluble material and then used for immunoprecipitation. For this, the primary antibody (anti-p70S6K) was previously mixed at a final concentration of 2 μg/ml with anti-rabbit (IgG, whole molecule) antibody conjugated to agarose beads in lysis buffer for 4 h at 4 °C. The beads were then thoroughly washed and mixed with total cell lysates prepared as indicated above at a ratio agarose beads/cell lysates 1:8 (v/v). After a 2-h incubation period at 4 °C, immune complexes were recovered by centrifugation (7,000 × g, 1 min, 4 °C). Pellets were washed twice with lysis buffer, twice with buffer A (100 mm Tris-HCl, pH 7.4, 400 mm LiCl) and twice with buffer B (10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA). Immune complex beads were resuspended in a final volume of 60 μl with lysis buffer and mixed with 2×-SDS sample buffer (1:1 v/v) for subsequent protein gel electrophoresis/immunoblotting. Resulting gels were transferred onto polyvinylidene difluoride membranes and used for immunoblotting. In several experiments, parallel blots were probed with the same antibody used for immunoprecipitation, to confirm that protein loading was similar (kept at <5% by measuring protein in samples by Bradford assay before loading) and that the small, unavoidable, differences in protein per lane can not account for differences in phosphorylation seen with the anti-phosphoantibodies. Immunocomplex p70S6 Kinase Assay—Ribosomal p70S6K enzymatic activity was quantified by using an immunocomplex kinase assay as reported previously (35Lehman J.A. Paul C.C. Baumann M.A. Gomez-Cambronero J. Am. J. Physiol. Cell Physiol. 2001; 280: 183-191Crossref PubMed Google Scholar, 36Lehman J.A. Gomez-Cambronero J. Biochem. Biophys. Res. Commun. 2002; 293: 463-469Crossref PubMed Scopus (63) Google Scholar, 38Joseph D.E. Paul C.C. Baumann M.A. Gomez-Cambronero J. J. Biol. Chem. 1996; 271: 13088-13093Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) tailored to measure this particular kinase activity in human neutrophils. Neutrophils were resuspended in RPMI 1640 at 3 × 106 cells/ml density and were pretreated with rapamycin, FK506, or MEKi where appropriate and then stimulated with GM-CSF at 37 °C. Cells were spun down (14,000 × g, 15 s) and pellets were resuspended in 0.3 ml of ice-cold, TRIS-based, lysis buffer (see above for composition) and incubated on ice for 15 min with occasional vortexing. Lysates were obtained after centrifugation (7,000 × g, 1 min, 4 °C) in supernatants were mixed with the antibody conjugated to agarose beads as indicated above. Immune complex beads were resuspended in a final volume of 40 μl with of ice-cold lysis buffer (diluted 1:10) and used in an in vitro kinase assay. For this, the phosphoacceptor peptide substrate for this assay was 75 μm of the S6 kinase substrate peptide KKRNRTLTK in freshly prepared kinase buffer (13.4 mm HEPES, pH 7.3, 25 mm MgCl2, 30 μm Na2VO3, 5 mm p-nitrophenyl phosphate, 2 mm EGTA, 2 μm cAMP-dependent kinase inhibitor TTYADFIASGRTGRRNAIHD, 0.420 μCi [γ-32P]ATP (7 nm), and 68 μm unlabeled ATP). 1 μg of cAMP-dependent kinase inhibitor inhibits 2,000–6,000 phosphorylating units of PKA (equivalent to the transference of 2–6 nmol of phosphate from ATP). To initiate the phosphotransferase reaction, aliquots (20 μl) of kinase buffer containing the appropriate substrates were mixed 1:3 (v/v) with the cell lysates or immunocomplex beads. The reaction was carried out at 37 °C for 20 min in a rotator and terminated by blotting 40 μl of the reaction mixture onto P81 ion exchange chromatography cellulose phosphate papers. Filter squares were washed, dried, and counted for radioactivity. Controls were run in parallel with no S6 kinase substrate peptide. Counts were subtracted from samples. In some experiments, ribosomes were used as the natural p70S6K substrate following the in vitro kinase assay just indicated. Protein S6 is part of a multiprotein-rRNA complex that is multiphosphorylated on Ser residues in vitro in response to mitogenic stimulation. 40 S ribosomal subunits were prepared from Xenopus laevis at the concentration of 0.1–0.25 mg/ml following the procedure described in Ref. 11Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (590) Google Scholar. Alkaline Phosphatase Treatment—SDS-boiled samples as indicated in the immunoprecipitation and Western blotting analyses section above were diluted with 0.3 ml of cold H2O and 0.4 ml cold, Triton X-100-based, lysis buffer. The alkaline phosphatase enzyme (calf intestine-purified) was added in a 160 μl of total volume of freshly prepared, cold, alkaline phosphatase buffer (25 mm Tris-HCl, pH 7.6, 1mm MgCl2, and 0.1 mm ZnCl2). To initiate the phosphate removal reaction, 48 μl of 3 m Tris-base, pH 12.5 were added to each sample to achieve a favorable reaction pH of 10.0 ± 0.5. Samples were lightly vortexed and incubated at 37 °C for 45 min with slight agitation. The reaction was stopped by placing the samples on an ice bucket and adding to each reaction tube a small volume of 10 m Tris-HCl, pH 3.0 in order to bring the reaction pH to 7.0. Samples were used immediately for immunoprecipitation using anti-p70S6K antibodies, as indicated above. F-actin Measurement by Flow Cytometry—Neutrophils were stained with phalloidin-FITC as described (39Egger G. Burda A. Glasner A. Virchows Arch. 2001; 438: 394-397Crossref PubMed Scopus (12) Google Scholar) with some modifications. Briefly, F-actin polymerization was initiated in vivo by the addition of GM-CSF to a neutrophil cell suspension (5 × 106 cells/ml) for 5 min at 37 °C. After this, 0.2-ml aliquots were taken and mixed with 1 ml of pre-chilled fixing solution (two parts of double-concentrated phosphate buffer, pH 7.4, one part of 20% formaldehyde and one part of 75% glycerol in water). Samples were stored at –80 °C until ready for flow cytometry. At that time, samples were thawed and spun down for 5 min at 600 × g in a refrigerated Eppendorf microcentrifuge. Pellets were resuspended in freshly prepared F-actin staining solution (35 μlofa3.3 mg/ml methanol FITC-phalloidin stock plus 315 μl of H2O, and stained in the dark for 30 min at room temperature. Samples were centrifuged as above, and pellets were resuspended in 1 ml of FACS FLOW. They were then analyzed by flow cytometry on a FACSCAN Becton & Dickinson flow cytometer at 488 nm excitation wavelength. Data was analyzed using Cell Quest software and expressed as fluorescence intensity. Human Neutrophils Express p70S6K and GM-CSF Increases Its Activity and Phosphorylation Status—We demonstrate here that neutrophils express this protein in sufficient amounts to be clearly detected by immunoblotting using antibodies directed against the ribosomal p70S6K (Fig. 1A). A major band shows the predicted molecular weight for the mammalian S6K1 isoform. We were also able to detect its enzymatic activity in vitro in whole cell lysates and in anti-p70S6K immunoprecipitates. The detection of p70S6K activity is presented in Fig. 1B, with GM-CSF causing a significant and reproducible increase of total S6K activity. Accurate confirmation of ribosomal p70S6K activity is given by the fact that 32P incorporation into the substrate peptide KKRNRTLTK and ribosomal 40 S subunits (containing the natural substrate protein S6), was observed after anti-p70S6K immunoprecipitation. GM-CSF increased basal activity by ∼3-fold in immunocomplexes. The complete biochemical characterization of p70S6K in neutrophils is presented in Fig. 2, A–D. A ∼3-fold activation due to GM-CSF is consistently seen when activity is expressed as a function of time, protein concentration and substrate concentration (Fig. 2, A–C). Moreover, there is a considerable change in V max (from 7.2 to 20.5 pmol/min/mg protein) and also in the K m (from 26.1 to 15.4 μm) by GM-CSF stimulation (Fig. 2D) that makes GM-CSF a bona fide p70S6K physiological stimulator in neutrophils.Fig. 2Biochemical characterization of neutrophil p70S6K. Neutrophils were challenged with GM-CSF and treated as indicated above. The kinase assay was performed against the KKRNRTLTK (p70S6K peptide substrate). Activity was measured as a function of incubation time of the in vitro enzyme assay (A); protein concentration (B), and substrate concentration at 0.5–0.7 mg/ml protein (C). From C, a Lineweaver-Burk plot was derived (D) from which the V max and K m values were calculated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After protein, activity and kinetic measurements, we next investigated whether GM-CSF was able to induce the phosphorylation of p70S6K in human neutrophils. As seen in Fig. 3A, GM-CSF induces a rapid (3 min) phosphorylation of p70S6K on Thr389, which is one of the residues critical for S6K activation. The migration of the phosphorylated band in SDS gels very well coincides with that of the 3T3 fibroblast controls. Also, GM-CSF induces phosphorylation of p70S6K on two other residues, Thr421/Ser424 (Fig. 3B), that are also key for conferring enzyme activity. The effect is dose-dependent, clearly noticeable with GM-CSF concentration as low as 0.5 nm. Fig. 4 shows that the increase in both phosphorylation and enzyme activity elicited by GM-CSF are time-dependent. A maximum phosphorylation at 3 min is seen for phospho-Thr421/Ser424 (Fig. 4A) as well as for phospho-Thr389 (Fig. 4B). Phosphorylation of Thr389 is seen as an increase in density of the immunoprecipitated p70 band. The Thr421/Ser424 dual phosphorylation is seen at 3–5 min post GM-CSF, and is demonstrated by both a robust increase in density of the immunoprecipitated p70 band and by an upward mobility shift (Fig. 4A). The figure also shows results of immunoprecipitation with anti-p70S6K antibody and immunoblotting with the same antibody to demonstrate equal loading (Fig. 4C). In vitro kinase activity experiments also reveal a time-dependent increase due to GM-CSF (Fig. 4D). Maximal activity is reached at 5 min and declines slightly thereafter, and as such, the biphasic pattern of phosphorylation seen in Fig. 4, A and B correlated with enzymatic activity. Thus, the results presented in Figs. 1, 2, 3, 4 demonstrate that p70S6K is expressed in human neutrophils, that GM-CSF increased its enzymatic activity in a time and dose-dependent fashion, changes the V max for its peptide substrate, and that this cytokine induces robust phosphorylation in 3 key residues: Thr389, Thr421, and Ser424. mTOR Is Involved in GM-CSF-activated p70S6K in Neutrophils—The next series of experiments were aimed at investigating what the mechanism that accounts for the observed increases in both phosphorylation and activity, was. Our first approach was the use of the immunosuppressant drug rapamycin, a well known inhibitor of mTOR, one of the several upstream regulators of p70S6K (17Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In neutrophils, rapamycin inhibited GM-CSF-stimulated p70S6K Thr389 phosphorylation in a concentration-dependent manner (Fig. 5A). The intensity of phospho-Thr389 signal all but disappears at 10 nm. The effect of rapamycin on the dual phosphorylation Thr421/Ser424 was also very profound, but manifested itself differently and warranted close examination. Fig. 6A shows that rapamycin causes a dramatic downward mobility shift in the phospho-Thr421/Ser424 band observed in the presence of GM-CSF. In this study, for the sake of clarification, we have labeled the lower band in GM-CSF + rapamycin as a and the upper band in GM-CSF alone as b. A shift from the upper b to lower a band observed with rapamycin treatment, is consistent with p70S6K dephosphorylation, as a less phosphorylated species runs faster in SDS-PAGE. Also shown in Fig. 6A (to the right) is a representative Western blot of fibroblast lysates to provide yet one more relative mobility comparison. The upper b band in GM-CSF-treated neutrophils has a M r similar to that of serum-treated fibroblasts. Fig. 6A also shows that, in contrast to what was observed with phospho-Thr389 where the intensity signal disappeared with rapamycin + GM-CSF (Fig. 5A), a positive signal is still present to anti-phospho-Thr421/Ser424 antibodies, indicating that phosphorylation of Thr421/Ser424 was not completely affected by rapamycin. To ascertain if the signal to Thr421/Ser424 antibodies still remained simply because there was not enough rapamycin to inhibit it, we analyzed the status of this dual phosphorylation in response to GM-CSF with a range of rapamycin concentrations. Fig. 6B confirms that concentrations of rapamycin as low as 0.5 nm had already increased the mobility of p70S6K, and this ef