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The Journal of Immunology, 1999, 163: 4527-4536.
Copyright © 1999 by The American Association of Immunologists

Activation of p90RSK and cAMP Response Element Binding Protein in Stimulated Neutrophils: Novel Effects of the Pyridinyl Imidazole SB 203580 on Activation of the Extracellular Signal-Regulated Kinase Cascade1

Jian P. Lian*, RiYun Huang{dagger}, Dwight Robinson{dagger} and John A. Badwey2,*,{ddagger}

* Boston Biomedical Research Institute, {dagger} Arthritis Unit, Massachusetts General Hospital, and {ddagger} Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutrophils stimulated with the chemoattractant FMLP or the phorbol ester PMA are known to exhibit activation of a 90-kDa renaturable protein kinase. Activation of this kinase was maximal at ~1–3 min after cell stimulation and the time course for activation was similar to that of the extracellular-regulated kinases (ERKs) and p38-mitogen activated protein kinase (p38MAPK). Compounds that block activation of ERK-1/2 (PD 98059) or that inhibit the activity of p38MAPK (SB 203580) blocked activation of this 90-kDa kinase. SB 203580 is a highly selective inhibitor of p38MAPK in vitro and is under intense study as a lead compound for developing novel anti-inflammatory agents. However, we demonstrate that SB 203580 at concentrations >=10 µM can also inhibit activation of ERK-1/2 in neutrophils. An Ab to the protein kinase p90RSK2 (also referred to as MAPKAP-K1b, or p90rsk) immunoprecipitated the active 90-kDa kinase from lysates of stimulated neutrophils. No activity was observed for this enzyme in immunoprecipitates obtained from unstimulated cells, and the amounts of activity were markedly reduced if the cells were treated with PD 98059 or SB 203580 before stimulation. Neutrophils stimulated with FMLP exhibited phosphorylation of the cAMP response element binding protein (CREB), and this reaction was inhibited by SB 203580 and PD 98059. These data establish that the renaturable 90-kDa protein kinase is p90RSK2 and that CREB may be a substrate for this enzyme in these cells. Novel effects of compound SB 203580 on stimulated neutrophils are also described.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils stimulated with various agonists exhibit activation of a large number of protein kinases. These include four kinases with molecular masses of ~69, 63, 49, and 40 kDa that undergo a dramatic and transient activation upon stimulation of these cells with FMLP. These enzymes can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed within a gel that corresponds to amino acid residues 297–331 of p47phox (1, 2, 3). This peptide contains several phosphorylation sites that consist of serine residues flanked by basic amino acids and lacks the consensus sequence recognized by mitogen-activated protein (MAP)3 kinases (e.g., ERK, p38MAPK) (2). The 69-, 63-, 49-, and 40-kDa kinases exhibited maximal activation within 15 s of cell stimulation followed by significant inactivation at 3 min (1, 2, 3). The 69- and 63-kDa kinases have been identified as p21-activated kinases (Paks) (4). This renaturation assay also revealed a 90-kDa kinase that underwent a pronounced activation at about 1–3 min after stimulation of the cells with FMLP or PMA (2, 3). Interestingly, stimulation of neutrophils with PMA did not trigger activation of the 69-, 63-, 49-, and 40-kDa kinases, but, in fact, reduced the basal activities of all four of these enzymes (2, 5). Similarly, antagonists of phosphoinositide 3-kinase (e.g., wortmannin, LY294002) did not affect activation of the 90-kDa kinase but blocked activation of the 69-, 63-, 49-, and 40-kDa kinases in FMLP-stimulated neutrophils (6). These data strongly suggest that the 69-, 63-, 49-, and 40-kDa kinases are regulated by a stimulatory pathway that is distinct from that which triggers activation of the 90-kDa kinase.

Stimulated neutrophils also exhibit a pronounced activation of the MAP-kinases p42-ERK and p38-MAPK (7, 8, 9, 10, 11, 12, 13). These "proline-directed" kinases recognize substrates that contain a minimum consensus sequence of -(S/T)P- (14). Compounds that block activation of ERK in cells (PD 98059) (15) or inhibit the activity of p38-MAPK (SB 203580) (16) inhibit ß2 integrin-dependent adhesion, chemotaxis, phagocytosis, degranulation, and 02- release by neutrophils (17, 18, 19, 20, 21, 22). Thus, uncovering substrates of the various MAP-kinase cascades in neutrophils should provide valuable insights into some of the key signal transduction steps in these cells. Substrates uncovered to date include serine residues 345 and 348 in p47phox, phospholipase A2, MAP kinase-activated protein kinase 2 (MAPKAP-K2), heat shock protein 27 (HSP 27), and lymphocyte-specific protein 1 (11, 13, 23, 24, 25, 26). Pyridinyl imidazoles such as SB 203580 have recently attracted considerable attention as lead compounds for developing new anti-inflammatory agents because of their ability to suppress the synthesis of proinflammatory cytokines in various models of acute and chronic inflammation (16, 27).

In this paper we identify the 90-kDa renaturable protein kinase that undergoes activation in stimulated neutrophils as p90RSK2. We report that the transcription factor CREB, a substrate for p90RSK2 (28), also undergoes phosphorylation in these cells. Phosphorylation/activation of p90RSK2 and CREB are shown to be blocked by compounds PD 98059 and SB 203580. These results are discussed in terms of the possible roles of p90RSK in the functional responses of neutrophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Compound PD 98059 and various pyridinyl imidazoles (SB 203580, SB 202190, SB 202474) were obtained from Calbiochem (San Diego, CA). An affinity-purified, rabbit polyclonal Ab raised against a peptide corresponding to amino acid residues 711–724 of human p90RSK2 kinase, an affinity-purified, sheep polyclonal Ab raised against a peptide corresponding to amino acid residues 310–325 of rabbit MAPKAP-K2, monoclonal antiphosphotyrosine Abs (clone 4G10) and active p90RSK2 partially purified from rabbit skeletal muscle were purchased from Upstate Biotechnology (Lake Placid, NY). A goat polyclonal Ab to MAPKAP-K2 (MAPKAP-K2(C-18) Ab) and goat polyclonal Abs raised against peptides corresponding to amino acid residues 716–735 of human RSK1, amino acid residues 722–740 of human RSK2, and amino acid residues 714–733 of human RSK3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human shock protein 27 (HSP 27) was purchased from StressGen (Victoria, BC, Canada). Affinity-purified, rabbit polyclonal Abs that recognize the active (doubly phosphorylated) forms of ERK (p44/ERK1 and p42/ERK2) and p38MAPK were purchased from Promega (Madison, WI). Affinity-purified, rabbit polyclonal Abs that recognize only the active (doubly phosphorylated) forms of MEK1 and MEK2 (phospho-MEK-1/2 (Ser217/Ser221) Ab), the phosphorylated form of CREB (phospho-CREB (Ser133) Ab) and I{kappa}B-{alpha} phosphorylated on Ser32 (phospho-specific I{kappa}B-{alpha} (Ser32) Ab) were obtained from New England Biolabs (Beverly, MA). Affinity-purified rabbit polyclonal Abs that recognize both the phosphorylated and nonphosphorylated forms of ERK (p44/42 (ERK-1/2) MAPK Abs), CREB, and I{kappa}B-{alpha} were also purchased from New England Biolabs. Goat anti-rabbit IgG labeled with HRP, goat anti-mouse IgG labeled with HRP, a Super Signal substrate Western blotting kit for luminol-enhanced chemiluminescence, and an ImmunoPure binding/elution buffer system for stripping and reblotting Western blots were purchased from Pierce (Rockford, IL). Sources of all other materials are described elsewhere (1, 2, 4).

Preparation of neutrophils

Guinea pig peritoneal neutrophils were prepared as described previously (29). These preparations contained >90% neutrophils with viabilities always >90%.

Detection of renaturable protein kinases (p90RSK2, Paks) in polyacrylamide gels

p90RSK2, Paks, and certain other protein kinases were detected directly in gels by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed within a gel that corresponds to amino acid residues 297–331 of p47phox. This technique was performed as described previously (1, 2).

Stock solutions of PMA (2.0 mg/ml), FMLP (4.0 mM), PD 98059 (20 mM), SB 203580 (20 mM), SB 202474 (20 mM), and SB 202190 (20 mM) were prepared in DMSO. All stock solutions were stored at -20°C and diluted with DMSO so that the final amount of solvent in each assay did not exceed 0.50% (v/v) (this includes the 0.25% added with the stimulus). These amounts of solvent did not cause any of the effects noted.

Immunoblotting/detection of activated ERKs, p38MAPK, MEK, and CREB in stimulated neutrophils

Neutrophils (7.5 x 106/ml) were stimulated and lysed as described (1). Aliquots of these samples were separated by SDS-PAGE (35 µg/lane) on 9.0% (w/v) polyacrylamide slab gels and transferred electrophoretically to Immobilon-P membranes as described (1). Activated ERK, p38MAPK, and MEK were assayed by Western blotting with Abs that recognized only the activated (doubly phosphorylated) forms of these kinases (30). Activated CREB was detected with an Ab that recognized this protein only when it was phosphorylated on Ser133. The activated kinases and phosphorylated CREB were visualized with a luminol-enhanced chemiluminescence detection system (Pierce) which monitored the activity of HRP bound to the secondary Ab (31). Ab dilutions and conditions for Western blotting are detailed in Huang et al.(32).

In certain experiments (see Figs. 5Go and 8Go), the substrate for the chemiluminescence detection system was removed by washing the membranes two times (10 min/wash) with TBST (20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 0.01% (v/v) Tween 20). These blots were then reprobed with a different Ab as described above so that both Ags could be visualized simultaneously (see Figs. 5Go and 8Go). At the end of these experiments, both the immunodetection system and the bound Abs were removed from the blot by incubating the membranes with ImmunoPure elution buffer (Pierce) for 30–60 min at room temperature followed by two washes with TBST. The blots were then stained with an Ab that recognized both the phosphorylated and nonphosphorylated forms of ERK or CREB (New England Biolabs) to confirm that equal amounts of cellular protein were present in each lane of the gel.



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  		FIGURE 5. Time course for the activation of p42ERK, p44ERK, and p38MAPK in neutrophils stimulated with FMLP. Effects of compounds PD 98059 and SB 203580. Activation of ERK (A) and p38MAPK (B) was monitored in neutrophils stimulated with 1.0 µM FMLP by Western blotting with Abs that recognized only the activated (doubly phosphorylated) forms of these kinases. C shows the membrane of A that was first blotted with the Ab to activated ERKs and then reblotted with the Ab to activated p38MAPK. The blots shown in A–C were from cells treated with 0.25% (v/v) DMSO for 15 s (lane a), FMLP for 15 s (lane b), FMLP for 30 s (lane c), FMLP for 1 min (lane d), FMLP for 3 min (lane e), FMLP for 5 min (lane f), FMLP for 7 min (lane g), FMLP for 10 min (lane h), and 0.25% (v/v) DMSO for 10 min (lane i). D and E show the effects of compounds PD 98059 and SB 203580 on the activation of the ERKs and p38MAPK in FMLP-stimulated neutrophils. The membrane was first blotted with the Ab to activated ERKs (D) and then reblotted with the Ab to activated p38MAPK (E). Neutrophils were stimulated with 1.0 µM FMLP for 3 min. The Western blots shown in D and E are for cells treated for 30 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (lane a), 0.25% DMSO followed by FMLP (lane b), 50 µM PD 98059 followed by FMLP (lane c), and 50 µM SB 203580 followed by FMLP (lane d). Stimulation of the cells and Western blotting were performed as described under Materials and Methods. p42ERK and p44ERK are designated by a solid arrow and an open arrowhead, respectively. p38MAPK is designated by a broken arrow. PD, PD 98059; and SB, SB 203580.

 


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  		FIGURE 8. Time-course for the activation of the ERKs, p38MAPK, and MEK in neutrophils stimulated with PMA. Effects of compounds PD 98059 and SB 203580. Activation of ERKs (A; solid arrow and open arrowhead), p38MAPK (B; broken arrow), and MEK (C, closed arrowhead) was monitored in neutrophils stimulated with 50 nM PMA by Western blotting with Abs that recognized only the activated (doubly phosphorylated) forms of these kinases. The membrane was first blotted with an Ab to activated ERKs (A), reblotted with an Ab to activated p38MAPK (B), and then reblotted again with an Ab to activated MEK (C). The blots shown in A–C were from cells treated with 0.25% (v/v) DMSO for 15 s (lane a), PMA for 15 s (lane b), PMA for 30 s (lane c), PMA for 1 min (lane d), PMA for 3 min (lane e), PMA for 5 min (lane f), PMA for 7 min (lane g), PMA for 10 min (lane h), and 0.25% (v/v) DMSO for 10 min (lane i). D shows the effects of compounds PD 98059, SB 203580, and SB 202474 on the activation of the ERKs, p38MAPK, and MEK in PMA-stimulated neutrophils. Neutrophils were stimulated with 50 nM PMA for 3 min. Cells were treated for 30 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (unstimulated cells) (lane a), 0.25% DMSO followed by PMA (lane b), 50 µM PD 98059 followed by PMA (lane c), 50 µM SB 203580 followed by PMA (lane d), and 50 µM SB 202474 followed by PMA (lane e). Stimulation of the cells and Western blotting were performed as described under Materials and Methods. p42ERK, p44ERK, p38MAPK, and MEK are designated by a solid arrow, an open arrowhead, a broken arrow, and a closed arrowhead, respectively. PD, PD 98059; SB, SB 203580; and SB-IA, SB 202474.

 
Miscellaneous procedures

Procedures for immunoprecipitating p90RSK2 from neutrophil lysates and methods for detecting this kinase (and possible substrates) in immune complexes by either autophosphorylation/phosphorylation or by the renaturation assay with the p47phox peptide were identical to assays previously utilized for Pak (4, 33). MAPKAP-K2 was assayed in immune complexes with HSP 27 as the exogenous substrate (13). The resulting 32P-labeled HSP 27 was isolated by SDS-PAGE (10.0% (w/v) acrylamide), cut from the gels, and quantified by liquid scintillation counting. Conditions for immunoprecipitating and assaying MAPKAP-K2 are provided in Ref. 13 .

Analysis of data

Unless otherwise noted, all of the autoradiographic observations were confirmed in at least three separate experiments performed on different preparations of cells. The number of observations (n) is based on different cell preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of compounds PD 98059 and SB 203580 on certain renaturable protein kinases in neutrophils

Neutrophils stimulated with FMLP exhibited activation of several renaturable protein kinases that include enzymes with molecular masses of ~90, 69, and 63 kDa (1, 2, 3) (Fig. 1Go). These kinases can be detected directly in gels by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed within a gel. The positions of the kinases are visualized by autoradiography after exposure of the gel to [{gamma}-32P]ATP (2). The peptide utilized corresponds to amino acid residues 297–331 of p47phox which contains several of the phosphorylation sites of this protein (23). Maximal activation of the 69- and 63-kDa enzymes occurred within 15 s of cell stimulation, whereas optimal activation of the 90-kDa kinase was observed at ~1–3 min (1, 2, 3) (Fig. 1Go, AI or AII). The increased content of 32P in the 90-kDa kinase after stimulating the cells with FMLP for 3 min was estimated by densitometry by comparing the height of the band in lane d with that in lane a of Fig. 1GoAI. The increase was 11 ± 3-fold (SD, n = 4). The 63- and 69-kDa enzymes have been identified as Paks (4).



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  		FIGURE 1. Effects of compounds PD 98059, SB 203580, and certain pyridinyl imidazoles on the activation of the renaturable 90-, 69-, and 63-kDa kinases in stimulated neutrophils. Activation of the 63- and 69-kDa Paks and a 90-kDa kinase were monitored by their ability to undergo renaturation and catalyze the phosphorylation of the p47phox peptide fixed within a gel as referenced under Materials and Methods. Neutrophils were incubated with 0.25% (v/v) DMSO (AI and AII, control cells), 50 µM PD 98059 (B), or 50 µM SB 203580 (C) for 30 min at 37°C and then stimulated with 1.0 µM FMLP. The samples shown are for cells treated with 0.25% (v/v) DMSO for 15 s (unstimulated cells) (lane a), FMLP for 15 s (lane b), FMLP for 30 s (lane c), FMLP for 3 min (lane d), and FMLP for 5 min (lane e). Data shown in AI and AII are the control samples for B and C, respectively. Control samples were derived from the same preparation of cells as those tested with the drug and were run on the same gel as the test samples. Autoradiographs for the control and test samples were developed of the same period of time (i.e., control and test samples were in a single autoradiograph). D, The effects of various pyridinyl imidazoles on the activation of the 90-kDa kinase in neutrophils stimulated with 1.0 µM FMLP for 3 min. Cells were treated for 30 min at 37°C with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (unstimulated cells) (lane a), 0.25% (v/v) DMSO followed by FMLP (lane b), 50 µM SB 203580 followed by FMLP (lane c), 50 µM SB 202190 followed by FMLP (lane d), and 50 µM SB 202474 followed by FMLP (lane e). The renaturable 90-, 69-, and 63-kDa kinases are designated by an arrow, an open arrowhead, and a closed arrowhead, respectively.

 
The effects of compounds PD 98059 and SB 203580 on activation of these renaturable kinases were examined (Fig. 1Go). Compound PD 98059 blocks activation of MEK and its downstream targets (p44ERK and p42ERK) by disrupting the interaction(s) between MEK and its "upstream" activating kinases (15). In contrast, SB 203580 inhibits the activity of p38MAPK by competing with ATP for its substrate binding site (16, 34, 35). Treatment of neutrophils with 50 µM PD 98059 or 50 µM SB 203580 for 30 min at 37°C did not markedly affect the activation of the 69- and 63-kDa Paks at 15 s and 30 s after cell stimulation. However, some inhibition of Pak activation was observed at time points >=3 min (Fig. 1Go, B and C, lanes d and e). In contrast, compounds PD 98059 and SB 203580 dramatically blocked activation of the 90-kDa kinase (Fig. 1Go). The decrease in activity was estimated by densitometry by comparing the height of the 90-kDa band in lane e in Fig. 1Go, B and C, with that in Fig. 1Go, AI and AII. Treatment of neutrophils with PD 98059 (50 µM) or SB 203580 (50 µM) for 30 min at 37o reduced the amount of 32P in the 90-kDa band in cells stimulated with FMLP for 3 min by 90 ± 2% (n = 4) and 89 ± 5% (n = 5, mean ± SD), respectively. The concentration (50 µM) of PD 98059 and SB 203580 utilized in these studies did not affect cell viability, as measured by the exclusion of trypan blue or the release of lactate dehydrogenase.

Fig. 1GoD compares the abilities of different analogues of SB 203580 to block activation of the 90-kDa kinase in FMLP-stimulated neutrophils. Compound SB 202190 is a more potent inhibitor of p38-MAPK than SB 203580 (IC50 = 350 nM vs 650 nM), whereas SB 202474 is an inactive analogue (16). SB 202190 (50 µM) was highly effective in blocking activation of the 90-kDa kinase, whereas SB 202474 was inactive.

Identification of the 90-kDa kinase as p90RSK2

Recent studies have established that ERKs can participate in activation of the protein kinase p90RSK2 (also referred to as MAPKAP-K1 or p90rsk) (36, 37, 38). p90RSK catalyzes the phosphorylation of serine residues in peptides that conform to the consensus sequence Arg-X-Arg-X-X-Ser or Arg-Arg-X-Ser (39). Because the p47phox peptide substrate utilized in the renaturation assay contains two serine residues that conform to this consensus sequence (i.e., Ser304 and Ser328), immunological studies were undertaken to determine whether the renaturable 90-kDa kinase was a member of the RSK family (Fig. 2Go).



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  		FIGURE 2. Immunoprecipitation of the renaturable 90-kDa protein kinase from lysates of FMLP-stimulated neutrophils with an Ab to p90RSK2. A, Autoradiogram compares the renaturable protein kinases in neutrophil lysates (lanes a and b) and in precipitated immune complexes derived from neutrophils with an Ab to p90RSK2 (ips, lanes c–f). The kinases were assayed by their ability to undergo renaturation and catalyze the phosphorylation of the p47phox peptide fixed within a gel as referenced under Materials and Methods. The lysates (lanes a and b) were from neutrophils treated for 5 min with either 0.25% (v/v) DMSO (unstimulated cells) (lane a) or 1.0 µM FMLP (lane b). The immunoprecipitates (lanes c–f) were derived from neutrophils treated for 15 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 5 min (unstimulated cells) (lane c), 0.25% (v/v) DMSO followed by 1.0 µM FMLP for 5 min (lane d), 50 µM PD 98059 followed by 1.0 µM FMLP for 5 min (lane e), and 50 µM SB 203580 followed by 1.0 µM FMLP for 5 min (lane f). The 90-kDa kinase was immunoprecipitated from lysates of neutrophils with the p90RSK2 Ab as described under Materials and Methods. B, Western blot demonstrating the presence of p90RSK2 in the immune complexes. P90RSK2 was immunoprecipitated from lysates of unstimulated (FMLP -) and stimulated (FMLP +) cells as described above and blotted with the p90RSK2 (711–724) Ab. Std., partially purified p90RSK2 from rabbit skeletal muscle; ip, immunoprecipitated immune complexes; and ip. Sup., supernatant remaining after removal of the immune complexes from the lysates. The 90-kDa kinase is designated by an arrow. The 69- and 63-kDa Paks are designated by open and closed arrowheads, respectively. PD, PD 98059; and SB, SB 203580.

 
Treatment of lysed FMLP-stimulated neutrophils with an Ab raised to a peptide corresponding to amino acid residues 711–724 of human RSK2 (Upstate Biotechnology) resulted in immunoprecipitation of the 90-kDa kinase (Fig. 2Go, lane d) and removal of this enzyme from the whole cell lysate (data not shown). The 90-kDa kinase was detected by the "in gel" renaturation assay after separation of the immunoprecipitated proteins by SDS-PAGE (see Materials and Methods). No other renaturable kinases were observed in the immunoprecipitates when assayed by this method (Fig. 2Go, lane d). The kinase immunoprecipitated by the RSK 2 Ab exhibited the same molecular mass on the renaturation gel as the 90-kDa kinase in cell lysates (Fig. 2Go, lanes b and d). Western blotting experiments confirmed that the Ab employed in these studies immunoprecipitated p90RSK2 from neutrophils and that this protein exhibited the same mass as partially purified p90RSK2 from rabbit skeletal muscle (Fig. 2GoB). Little or no activity was observed for the 90-kDa kinase when the immunoprecipitates were obtained from unstimulated neutrophils (Fig. 2Go, lane c) and the amounts of activity were markedly reduced or eliminated if the cells were treated with compounds PD 98059 (50 µM) or SB 203580 (50 µM) before stimulation (Fig. 2Go, lanes e and f). Similar results were obtained with an Ab raised to a peptide that corresponds to residues 724–740 of human RSK2 (Santa Cruz Biotechnology). In contrast, the 90-kDa kinase was not immunoprecipitated with nonimmune serum or with Abs to RSK1 or RSK3 (data not shown).

The specificity of the RSK2 (711–224) Ab employed in the experiments summarized in Fig. 2Go was examined by Western blotting (Fig. 3GoA). Only a single immunoreactive band with a mass of ~90 kDa was observed when lysates of guinea pig neutrophils were examined at several different concentrations of protein (Fig. 3GoA). Interestingly, a number of different 32P-labeled proteins were observed when immunoprecipitates prepared with this Ab were utilized for in vitro autophosphorylation/phosphorylation studies with [{gamma}-32P]ATP (Fig. 3GoB). These phosphorylation reactions were markedly increased in immunoprecipitates obtained from stimulated neutrophils (Fig. 3GoB, lane b) and were diminished in immunoprecipitates obtained from cells treated with SB 203580 (50 µM) before stimulation (Fig. 3GoB, lane c). This pattern was not observed when nonimmune serum was used in place of the p90RSK2 (711–724) Ab (data not shown). These data strongly suggest that the RSK2 (711–724) Ab is highly specific and recognizes only a single protein in neutrophils. The presence of several phosphoprotein bands in Fig. 3GoB suggests that p90RSK2 may form complexes with a variety of proteins/substrates in these cells.



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  		FIGURE 3. Phosphorylation of p90RSK2 and associated proteins in immune complexes derived from neutrophils. A, Western blot shows the presence of a single protein in guinea pig neutrophils that reacted with an Ab to p90RSK2. The concentrations of protein applied to the gel were 6 µg (lane a), 12 µg (lane b), 18 µg (lane c), and 24 µg (lane d). B, Autoradiogram demonstrating the in vitro autophosphorylation/phosphorylation profile of proteins immunoprecipitated from neutrophils with the p90RSK2 Ab. The immunoprecipitates were obtained from neutrophils treated for 30 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 5 min (lane a), 0.25% (v/v) DMSO followed by 1.0 µM FMLP for 5 min (lane b), and 50 µM SB 203580 followed by 1.0 µM FMLP for 5 min (lane c). Western blotting, immunoprecipitation, and the in vitro phosphorylation experiments were performed as referenced under Materials and Methods. The position of p90RSK2 is designated by an arrow.

 
p90RSK2 can form a complex with ERK under certain circumstances (40, 41). A very light signal for ERK was observed in two of four experiments when Western blots similar to that shown in Fig. 3GoA were reprobed with an Ab that recognized both the active and inactive forms of ERK. The vast majority of ERK remained in the supernatant fraction after immunoprecipitation of p90RSK from neutrophil lysates with the RSK2 (711–724) Ab. Thus, it is uncertain whether the 32P-labeled phosphoprotein bands observed in the 40-kDa range in Fig. 3GoB are ERKs.

The time course for activation of p90RSK2 in FMLP-stimulated neutrophils was very similar when this kinase was monitored in either the RSK2 immune complexes (Fig. 4GoA) or in whole cell lysates (Fig. 4GoB). Maximal activation was observed at 1–3 min after cell stimulation with significant diminution of activity by 10 min.



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  		FIGURE 4. Time course for the activation of p90RSK2 in neutrophils stimulated with FMLP. p90RSK2 was assayed in immune complexes derived from neutrophils (A) and in lysates of FMLP-stimulated neutrophils (B). The kinase was assayed by its ability to undergo renaturation and catalyze the phosphorylation of the p47phox peptide fixed within a gel as described under Materials and Methods. The amount of FMLP utilized to stimulate the cells was 1.0 µM. A, Cells were treated with 0.25% (v/v) DMSO for 15 s (unstimulated cells) (lane a), FMLP for 15 s (lane b), FMLP for 30 s (lane c), FMLP for 1 min (lane d), FMLP for 3 min (lane e), FMLP for 5 min (lane f), and FMLP for 10 min (lane g). B, Cells were treated as described in A but with the following changes: FMLP for 7 min (lane g), FMLP for 10 min (lane h), and 0.25% (v/v) DMSO for 10 min (lane i). p90RSK2 is designated by an arrow. The 69- and 63-kDa Paks are designated by open and closed arrowheads, respectively.

 
Effects of compounds PD 98059 and SB 203580 on activation of the ERKs and p38MAPK in neutrophils

Studies were undertaken to determine the effects of compounds PD 98059 and SB 203580 on the MAPK cascades in neutrophils as a means of understanding the regulation of p90RSK2 in these cells. Activation of p42ERK and p38MAPK was monitored with Abs that recognized only the activated (doubly phosphorylated) (30) forms of these kinases (Fig. 5Go). Maximal activation of p42ERK and p38MAPK occurred at about 3 min after stimulation of the cells with FMLP (Fig. 5Go, A and B). Activation of a small amount of p44ERK was also apparent. The increases in activity for p42ERK and p38MAPK in neutrophils stimulated with FMLP for 3 min were estimated densitometrically by comparing the heights of the bands in lane e with those in lane a of Fig. 5Go, A and B. The increases for p42ERK and p38MAPK were 8 ± 3-fold (n = 5) and 7.6 ± 4-fold (n = 4), respectively (mean ± SD). Restaining the same blot utilized for monitoring activation of ERK (Fig. 5GoA, arrow and open arrowhead) with an Ab to activated p38MAPK clearly established that these Abs recognized distinct proteins in neutrophils (Fig. 5GoC, broken arrow).

The time courses for activation of ERK and p38MAPK were very similar to that observed for activation of p90RSK2 (Fig. 4Go). These data are consistent with one or both of these kinases functioning as an upstream component in the activation of p90RSK2. Interestingly, both PD 98059 (50 µM) and SB 203580 (50 µM) blocked activation of p42ERK but not p38MAPK in these cells (Fig. 5Go, D and E). As noted above, SB 203580 is frequently utilized as a highly specific/selective inhibitor of the activity of p38MAPK (42). To our knowledge, the ability of this drug to block activation of ERK in cells has not been described previously. Treatment of neutrophils with PD 98059 (50 µM) for 30 min at 37°C reduced the amount of phosphate in the p42ERK and p38MAPK bands in cells stimulated with FMLP for 3 min by 91 ± 6% (n = 8) and 9 ± 18% (n = 4), respectively (mean ± SD). The corresponding values for cells treated with SB 203580 were 85 ± 11% (n = 6) and 24 ± 8% (n = 3), respectively. Identical results with PD 98059 and SB 203580 were observed when assaying activation of these kinases by phosphorylation on only tyrosine residues (data not shown). Data presented in Fig. 5Go, D and E, are for the same Western blot that was first stained for activated p42ERK (arrow) and then restained for activated p38MAPK (broken arrow). At the end of these experiments, the blots were stripped and stained with an Ab that reacted with both the active and inactive forms of ERK (New England Biolabs) to confirm that equal amounts of protein were present in each lane (data not shown).

Compound SB 203580 blocked activation of p42ERK and p90RSK2 in FMLP-stimulated neutrophils in a dose-dependent manner (Fig. 6Go). Activation of both of these kinases was significantly reduced by concentrations of SB 203580 in the range of 10–100 µM (Fig. 6Go). Fig. 6GoC summarizes data from several different experiments examining these inhibitory effects.



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  		FIGURE 6. Effects of different concentrations of SB 203580 on the activation of p90RSK2 and p42ERK in stimulated neutrophils. p90RSK2 (A) and p42ERK (B) were monitored in neutrophils treated with various amounts of compound SB 203580 for 30 min and then stimulated with 1.0 µM FMLP. A, Activation of p90RSK2 was assayed by its ability to undergo renaturation and catalyze the phosphorylation of the p47phox peptide fixed within a gel. B, Activation of ERKS was monitored by Western blotting with an Ab that recognized only the activated (doubly phosphorylated) forms of these kinases. Unless otherwise indicated, cells were treated with 1.0 µM FMLP for 3 min. In A and B, cells were treated for 30 min at 37°C with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (unstimulated cells) (lane a), 0.25% (v/v) DMSO followed by FMLP for 15 s (lane b1), 0.25% (v/v) DMSO followed by FMLP for 3 min (lane b), 100 µM SB 203580 followed by FMLP (lane c), 50 µM SB 203580 followed by FMLP (lane d), 25 µM SB 203580 followed by FMLP (lane e), 10 µM SB 203580 followed by FMLP (lane f), 5.0 µM SB 203580 followed by FMLP (lane g), 1.0 µM SB 20358 followed by FMLP (lane h), and 0.10 µM SB 203580 followed by FMLP (lane i). p90RSK2, p42ERK, and p44ERK are designated by a solid arrow, a broken arrow, and an asterisk, respectively. The 69- and 63-kDa Paks are designated by open and closed arrowheads, respectively. C, Bar graph summarizing the effects of SB 203580 on the activation of p42ERK and p90RSK2 in neutrophils. Cells were incubated with different amounts of SB 203580 for 30 min before stimulation with 1.0 µM FMLP for 3 min. Activities were estimated by densitometry. The 100% values are the activities of p42ERK and p90RSK2 in neutrophils stimulated with FMLP (1.0 µM) for 3 min in the absence of inhibitors. Data represent mean values ± SD for three to four separate experiments performed on different preparations of cells.

 
A recent study has demonstrated that compounds SB 203580 and PD 98059 directly inhibit purified cyclooxygenase in vitro at concentrations of 0.01–10 µM (43). However, treatment of neutrophils with the cyclooxygenase antagonist acetylsalicylic acid (10 and 100 µM) or indomethacin (100 µM) for 30 min before stimulation with 1.0 µM FMLP for 5 min did not block activation of the ERKs or p90RSK2 in these cells (n = 3; data not shown). These data strongly suggest that the inhibitory effects of SB 203580 or PD 98059 on the activation of ERKs and p90RSK2 in neutrophils were not mediated by cyclooxygenase.

Targets of SB 203580 in neutrophils

Could p38MAPK mediate activation of p42ERK in neutrophils? Such a situation would account for the sensitivity of p42ERK activation to SB 203580. To test this possibility, we examined the ability of SB 203580 to block activation of MAPKAP-K2 in these cells. Previous studies have established that p38MAPK catalyzes the phosphorylation/activation of MAPKAP-K2 in human neutrophils and that low concentrations of SB 203580 block this reaction (13, 25). Thus, MAPKAP-K2 can serve as a substrate to measure the activity of p38MAPK in vivo. MAPKAP-K2 was immunoprecipitated from neutrophil lysates and assayed in the immune complexes with HSP 27 as the substrate (Fig. 7GoB). As expected, a pronounced increase in the activity of this enzyme was observed in cells stimulated with FMLP for 3 min (13, 25). Importantly, treatment of neutrophils with only 5.0 µM SB 203580 for 30 min before stimulation of the cells with FMLP blocked activation of MAPKAP-K2 by 90% (Fig. 7Go, B and D). Similar results were observed if MAPKAP-K2 was monitored by autophosphorylation (Fig. 7GoA). In contrast, this concentration of SB 203580 blocked activation p42ERK by <=25% (Fig. 6Go). These data indicate that p42ERK does not lie downstream of p38MAPK in neutrophils.



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  		FIGURE 7. Effects of SB 203580 on the activation of MAPKAP-K2 in stimulated neutrophils. A and B, MAPKAP-K2 was immunoprecipitated from neutrophil lysates with the MAPKAP-K2 (310–325) Ab and monitored by it’s ability to undergo autophosphorylation (A) or to catalyze the phosphorylation of HSP 27 (B) as referenced under Materials and Methods. Neutrophils were stimulated with 1.0 µM FMLP for 3 min. The autoradiograms shown were derived from cells treated for 30 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (unstimulated cells) (lane a), 0.25% (v/v) DMSO followed by FMLP (lane b), 5.0 µM SB 203580 followed by 0.25% (v/v) DMSO for 3 min (lane c), and 5.0 µM SB 203580 followed by FMLP (lane d). Autoradiograms shown in A and B were developed for 7 days and 24 h, respectively. C, Aliquots of the immunoprecipitated samples of B were subjected to Western blotting with the MAPKAP-K2 (C-18) Ab which recognizes both the active and inactive forms of this enzyme. D, Bar graph summarizing data from three separate experiments identical to that described in B. The HSP 27 band was cut from the gel and 32P quantified by scintillation counting.

 
It was also possible that compound PD 98059 and/or SB 203580 blocked activation of ERKs simply by interfering with the binding of FMLP to its receptor. The effects of these antagonists were therefore evaluated in neutrophils stimulated with PMA (Fig. 8Go). PMA bypasses the surface receptors on neutrophils and directly activates protein kinase C (PKC) (44). Activated PKC triggers the activation of ERKs in cells through a pathway that requires Ras activation and the formation of Ras-GTP-Raf complexes (45). Data presented in Fig. 8Go show the activation of ERKs, p38MAPK, and MEK in neutrophils stimulated with PMA for various periods of time. Optimal activation of each of these kinases occurred at ~3 min after stimulation of the cells with PMA. A single blot was first stained for activated ERKs (Fig. 8GoA), restained for activated p38MAPK (Fig. 8GoB, broken arrow), and then stained again for activated MEK (Fig. 8GoC, closed arrowhead). The small differences in these time courses may reflect different affinities of the Abs for their Ags. These data clearly demonstrate that the Abs utilized recognize specific proteins in neutrophils. Most importantly, compounds PD 98059 (50 µM) and SB 203580 (50 µM) blocked activation of p44ERK, p42ERK, and MEK (see Discussion) but not p38MAPK (Fig. 8GoD). Thus, the inhibitory effects observed in Fig. 5Go were not simply the result of compounds PD 98059 or SB 203580 interfering with the binding of FMLP to its receptor. As in Fig. 5Go, these blots were stripped and stained with an Ab that reacted with both the active and inactive forms of ERK to confirm that equal amounts of protein were present in each lane (data not shown).

Phosphorylation of CREB in neutrophils

Purified p90RSK catalyzes phosphorylation of the transcription factor CREB on Ser133 (28) and the transcription factor inhibitor I{kappa}B{alpha} on Ser32 (46). Experiments were undertaken to determine whether these reactions occurred in neutrophils utilizing phospho-specific Abs that recognized only the phosphorylated forms of these proteins. Neutrophils stimulated with FMLP (1.0 µM) exhibited a time-dependent phosphorylation of CREB on Ser133 with maximal phosphorylation occurring at ~3 min (Fig. 9GoA; arrow). The Ab utilized in these studies was highly selective and recognized only two major proteins in these cells. The low m.w. protein that reacted with the phospho-specific Ab (Fig. 9GoA, broken arrow) may be the transcription factor ATF-1 (activating transcription factor-1) (47, 48). The location of CREB on the blot was verified with a second Ab to this protein. Interestingly, while PD 98059 (50 µM) substantially blocked activation of p90RSK2 in FMLP-stimulated neutrophils (Fig. 1Go), it only partially inhibited the phosphorylation of CREB (Fig. 9Go, B and C). In contrast, SB 203580 substantially inhibited the phosphorylation of CREB at 50 µM and partially blocked this reaction at 5.0 µM (Fig. 9GoC). These data are consistent with protein kinases downstream of ERK (i.e., p90RSK2) and p38MAPK (e.g., MAPKAP-K2) (47) utilizing CREB as a substrate (see Discussion).



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  		FIGURE 9. Phosphorylation of CREB in stimulated neutrophils. Effects of compounds PD 98059 and SB 203580. A, Phosphorylation of CREB in neutrophils was monitored by Western blotting with an Ab that recognized CREB only when phosphorylated on Ser133. Stimulation of the cells with 1.0 µM FMLP and Western blotting were performed as described under Materials and Methods. The blot shown in A was from cells treated with 0.25% (v/v) DMSO for 15 s (unstimulated cells) (lane a), FMLP for 15 s (lane b), FMLP for 30 s (lane c), FMLP for 1 min (lane d), FMLP for 3 min (lane e), FMLP for 5 min (lane f), FMLP for 10 min (lane g), FMLP for 15 min (lane h), and 0.25% (v/v) DMSO for 15 min (lane i). B shows the effects of compounds PD 98059, SB 203580, and SB 202474 on the phosphorylation of CREB in stimulated neutrophils. Neutrophils were stimulated with 1.0 µM FMLP for 3 min. Cells were treated for 30 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (unstimulated cells) (lane a), 0.25% (v/v) DMSO followed by FMLP (lane b), 50 µM SB 203580 followed by FMLP (lane c), 50 µM SB 202474 followed by FMLP (lane d), and 50 µM PD 98059 followed by FMLP (lane e). C, Bar graph summarizing the effects of various antagonists on the phosphorylation of CREB. Neutrophils were stimulated with 1.0 µM FMLP for 3 min. Cells were treated for 30 min with 0.25% (v/v) DMSO followed by an additional 0.25% (v/v) DMSO for 3 min (unstimulated cells) (lane a), 0.25% (v/v) DMSO followed by FMLP (lane b), 50 µM SB 203580 followed by FMLP (lane c), 5.0 µM SB 203580 followed by FMLP (lane d), 50 µM SB 202474 followed by FMLP (lane e), and 50 µM PD 98059 followed by FMLP (lane f). Activities were estimated by densitometry. Data represent mean values ± SD for seven separate experiments. CREB is designated by a solid arrow. The immunoreactive band designated by the broken arrow may be ATF-1 (activating transcription factor-1). SB, SB 203580; SB-IA, SB 202474; and PD, PD 98059.

 
The isoquinoline sulfonamide HA1004 is a cell-permeable, selective inhibitor of the cyclic nucleotide dependent protein kinases (49). Treatment of neutrophils with HA1004 (100 µM) for 30 min did not reduce the basal level of CREB phosphorylation in unstimulated cells nor did it block phosphorylation of CREB after stimulation of the cells with FMLP (n = 2). Moreover, treatment of neutrophils with both HA1004 (100 µM) and SB 203580 (50 µM) for 30 min followed by stimulation of the cells with FMLP did not reduce the residual level of CREB phosphorylation below that observed with SB 203580 (50 µM) alone (n = 2; data not shown). At the end of these experiments, the blots were stripped and stained with an Ab that reacted with both the phosphorylated and nonphosporylated forms of CREB to confirm that equal amounts of protein were present in each lane. In contrast to CREB, we did not consistently observe phosphorylation of I{kappa}B{alpha} on Ser32 in neutrophils after stimulation of the cells with FMLP (1.0 µM) for time periods ranging from 15 s to 20 min (n = 5; data not shown) (50).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several novel observations are presented in this paper. In particular, we identify the 90-kDa renaturable protein kinase that undergoes activation in stimulated neutrophils as p90RSK2 and report that activation of this kinase is sensitive to both compounds PD 98059 and SB 203580. In contrast to the striking specificity of SB 203580 that is observed for isolated p38MAPK in vitro even at a concentration of 100 µM (42), we report that in stimulated neutrophils this drug also reduced activation of ERK at concentrations of 10–50 µM. These studies are relevant to the wide use of SB 203580 to implicate p38MAPK in a variety of cellular activities and for the development of novel anti-inflammatory agents. In addition, we report that CREB, a substrate for p90RSK2 (28), undergoes phosphorylation/activation in stimulated neutrophils. The kinetics of CREB phosphorylation are consistent with this transcription factor being located downstream of both the ERK/p90RSK2 and p38MAPK/MAPKAP-K2 cascades. These observations are developed below.

p90RSK2 is a highly unusual enzyme in that it contains two distinct active sites/protein kinase domains that reside in the N-terminal and C-terminal regions of the protein (51). The N-terminal kinase domain is necessary to catalyze the phosphorylation of exogenous substrates, whereas the C-terminal domain is required for complete activation of the N-terminal kinase domain (38). Reconstitution experiments and transfection studies have demonstrated that ERKs can participate in the phosphorylation/activation of p90RSK2 (36, 37, 38). Our observations on the activation of p90RSK2 in stimulated neutrophils are consistent with those studies. In particular, the progress curves for activation of p42ERK and p90RSK2 are very similar (Figs. 4Go and 5Go) and compound PD 98059, which blocked activation of ERK, also prevented activation of p90RSK2 in these cells (Figs. 5Go and 8Go). Surprisingly, we observed that the drug SB 203580 at concentrations >=10 µM also blocked activation of ERK in stimulated neutrophils (Figs. 5Go, 6Go, and 8Go). The dose-response curves for SB 203580 in blocking activation of ERK and p90RSK2 were very similar with IC50 values of ~10 µM (Fig. 6Go). These data are consistent with the inhibitory effects of SB 203580 on p90RSK2 activation being mediated through ERK (see below).

SB 203580 had only minor effects (<25%) on the activation of ERK and p90RSK2 in neutrophils at concentrations <=5.0 µM (Fig. 6Go). In contrast, SB 203580 substantially blocked activation of MAPKAP-K2, a substrate for p38MAPK, in neutrophils at a concentration of only 5.0 µM (Fig. 7Go). Thus, major inhibitory effects of SB 203580 on neutrophils at concentrations <=5.0 µM should primarily reflect inhibition of p38MAPK activity and not blockade of ERK or p90RSK2 activation. SB 203580 and related compounds abrogate a number of cellular responses at concentrations <=5.0 µM. These responses include cytokine biosynthesis (16, 21), activation of HIV (52, 53), apoptosis (54), expression of the low affinity IgE-receptor (55), and production of NO (56). Several isoforms of p38MAPK are known to exist (e.g., {alpha}, ß, {gamma}, {delta}), and some of these forms are insensitive to SB 203580 ({gamma}, {delta}) (57). The {alpha} and {delta} isoforms of p38MAPK are present in human neutrophils (57).

MEK-1 and -2 undergo activation in stimulated neutrophils and catalyze the phosphorylation/activation of ERKs (17, 58). In addition to ERK and p90RSK2, a high concentration of SB 203580 (50 µM) also blocked the activation of MEK in PMA-stimulated neutrophils (Fig. 8GoD). As noted above, PMA activates ERKs in cells through a pathway that requires Ras activation and the formation of Ras-GTP-Raf complexes (10, 45). Compound PD 98059 binds to the dephosphorylated/inactive form of MEK and prevents its interactions with "upstream" activating kinases (i.e., Raf or MEKK) (15). It is possible that high concentrations of SB 203580 also block activation of MEK (Fig. 8GoD) (and hence ERK and p90RSK2) in neutrophils through a similar mechanism. PD 98059 and SB 203580 have somewhat similar structures in that each of these molecules are heterocylic compounds. As noted above, both SB 203580 and PD 98059 inhibit cyclooxygenase in vitro which indicates that certain binding sites on proteins may accommodate either of these ligands (43).

We have previously reported that wortmannin, an inhibitor of phosphoinositide 3-kinase, blocks activation of a renaturable 40-kDa kinase but not the 90-kDa kinase (p90RSK2) in FMLP-stimulated neutrophils (6). It is unlikely that the renaturable 40-kDa kinase is ERK or p38MAPK. The p47phox peptide that is used as a substrate in the renaturation assay to detect the 40-kDa kinase does not contain the minimal consensus sequence -(S/T)P- recognized by ERKs or p38MAPK (2, 14). Moreover, the renaturable 40-kDa kinase exhibited optimal activation within 15 s of cell stimulation by FMLP followed by significant inactivation at 3 min. In contrast, the ERKs and p38MAPK in neutrophils did not exhibit optimal activation until 3 min after stimulation with little or no activity at 15 s. (Fig. 5Go). Treatment of neutrophils with 200 nM wortmannin for 30 min at 37°C did not significantly (<20%) block activation of p38MAPK (32) or the ERKs (data not shown) upon subsequent stimulation of the cells with FMLP (1.0 µM) for 3 min. Activation of p38MAPK in FMLP-stimulated human neutrophils is partially inhibited by wortmannin (13). Whether this difference from our results reflect the different species used and/or differences between blood and elicited peritoneal neutrophils is not known.

What are the functions of p90RSK2 in neutrophils? Stimulated neutrophils exhibit phosphorylation of CREB on Ser133 (Fig. 9Go). A number of protein kinases can catalyze this reaction (e.g., p90RSK2, MAPKAP-K2, PKC, calmodulin kinases) (28, 47, 48, 59, 60). As noted above, p90RSK2 and MAPKAP-K2 can be activated by ERK and p38MAPK, respectively (13, 38, 47). Phosphorylation of CREB in stimulated neutrophils was only partially inhibited by PD 98059 (50 µM) (Fig. 9Go) at a concentration of this antagonist that substantially blocked (90%) activation of p90RSK2 (Fig. 1GoB). Similarly, a concentration of SB 203580 (5.0 µM) that substantially blocked (90%) activation of MAPKAP-K2 (Fig. 7Go) (but not activation of p90RSK2) also caused a partial inhibition of CREB phosphorylation (Fig. 9Go). However, a concentration of SB 203580 (50 µM) that blocked activation of both ERK and p38MAPK substantially inhibited the phosphorylation of CREB in FMLP-stimulated cells (Fig. 9GoC, lane c). These data suggest that CREB may be a substrate for p90RSK2 and certain other protein kinases in neutrophils (e.g., MAPKAP-K2). Finally, compound PD 98059 also inhibits chemotaxis and phagocytosis by neutrophils (18, 19, 20). Perhaps p90RSK2 as a downstream component of ERK has a role in these responses. Identification of the substrates for p90RSK2 in neutrophils may implicate this kinase in a number of the functional responses of these cells.


   Footnotes
 
1 These studies were supported by National Institutes of Health Grants DK 50015, AI 23323 (to J.A.B.), and AR 43518 (to D.R.). Back

2 Address correspondence and reprint requests to Dr. John A. Badwey, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114. Back

3 Abbreviations used in this paper: MAP, mitogen-activated protein; Pak, p21-activated protein kinase; p90RSK2, 90-kDa ribosomal S6 kinase (also referred to as p90rsk or MAPKAP-K1); ERK, extracellular-regulated kinase; MAPK, MAP kinase, MEK, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase; MAPKAP, mitogen-activated protein kinase-activated protein; MAPKAP-K2, MAPKAP-kinase 2; p47phox, 47-kDa protein component of the phagocyte oxidase; HSP 27, heat shock protein 27; PD 98059, (2-(2'-amino-3'-methoxyphenol)-axanapthalen-4-one); SB 203580, 4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)imidazole; PKC, protein kinase C; CREB, cAMP response element binding protein. Back

Received for publication April 1, 1999. Accepted for publication July 30, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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