Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1998 Dec;18(12):7130–7138. doi: 10.1128/mcb.18.12.7130

Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

RiYun Huang 1, Jian P Lian 2, Dwight Robinson 1, John A Badwey 2,3,*
PMCID: PMC109294  PMID: 9819399

Abstract

Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the α and βγ subunits of complex G proteins.


Neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) exhibit a rapid and transient activation of two p21-activated protein kinases (Paks) with molecular masses of ca. 63 and 69 kDa (1113, 24, 26, 32). Paks are Ser/Thr protein kinases that undergo autophosphorylation and activation upon interacting with the active (GTP-bound) forms of the small GTPase (p21) Rac or Cdc42 (44). Activation of Pak is affected by certain sphingolipids (e.g., d-erythro-sphingosine and C2-ceramide) (5, 28, 39), an inhibitor of heterotrimeric G proteins (pertussis toxin) (12, 26, 32), and antagonists of phosphoinositide 3-kinase (PI 3-K) (14), type 1 and type 2A protein phosphatases (e.g., calyculin A) (1113), and tyrosine kinases (e.g., genistein) (7). Thus, Paks may be capable of integrating messengers from a number of signal transduction pathways.

A variety of studies suggests that Paks can participate in a broad range of cellular events that include rapid cytoskeletal responses as well as certain long-term transcriptional events (for a review, see reference 43). For example, Paks can catalyze the phosphorylation of a conserved serine residue in the heavy chains of myosins 1 and VI (8, 64) and multiple serine residues in p47-phox (the 47-kDa protein component of the phagocyte oxidase) (12, 32); these reactions play an important role in the actin-based cortical processes required for cell migration (52) and superoxide (O2) production (12), respectively. Paks may also be involved in the activation or regulation of several distinct mitogen-activated protein (MAP) kinase cascades which mediate cellular responses to stimuli that range from cytokines, chemoattractants, and various stresses. These MAP kinase cascades include the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and the p38-MAP kinase (43) pathways. Transfection of constitutively active Pak or overexpression of wild-type Pak into certain cells is sufficient to activate JNK/SAPK and to a lesser extent p38-MAP kinase (4, 18, 19, 66). Moreover, activated Pak can potentiate the ability of wild-type Raf-1 or growth factors to stimulate ERKs and MEKs in numerous cell types (18). Substrates for these MAP kinases include transcription factors (e.g., c-Jun), cytoskeletal proteins, and various enzymes (phospholipase A2) (43). Selective antagonists of MEK and p38-MAPK inhibit chemotaxis, degranulation, and O2 production by neutrophils (2, 15, 35, 69).

In this paper, we report that a variety of agonists which bind to serpentine receptors on neutrophils that couple to complex G proteins all stimulate rapid activation of the 63- and 69-kDa Paks. These agonists include sulfatide, which binds to the L-selectin receptor that mediates the initial interactions between neutrophils and the endothelium during an inflammatory event (9, 27, 36, 62), the allergic mediators leukotriene B4 (LTB4) and platelet-activating factor (PAF), and the anaphylatoxin C5a. While fMLP and C5a are thought to activate the same or very similar populations of complex G proteins (22, 63), we demonstrate that these agonists stimulate strikingly different responses in neutrophils with respect to the activation of Pak, JNK, p38-MAPK, and O2 production. Activation of Paks can be transient or chronic, depending on the nature of the stimulus. Moreover, we demonstrate that distinct and separate signals are required for activation and inactivation of the 63- and 69-kDa Paks. Relationships between Pak activation and certain MAP kinase cascades and O2 production are also investigated. These data are discussed in terms of the roles of Paks in the functional responses of neutrophils.

MATERIALS AND METHODS

Materials.

PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), propionyl-PAF (1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine), 2-thioace- tyl–PAF (1-O-hexadecyl-2-thioacetyl-2-deoxy-sn-glycero-3-phosphocholine), lyso-PAF (1-O-hexadecyl-sn-glycero-3-phosphocholine), the PAF antagonist (PAF-A) hexanolamine-PAF [1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho-(N,N,N-trimethyl)-hexanolamine], LTB4 [5(S),12(R)-dihydroxy-6-cis-8-trans-10-trans-14-cis-eicosatetraenoic acid], U75302, d-erythro-sphingosine, herbimycin (from Streptomyces sp.), genistein, and erbstatin analog were purchased from Calbiochem, La Jolla, Calif. 20-Carboxy-LTB4, 20-OH-LTB4, and 14,15-dehydro-LB4 were obtained from BIOMOL Research Laboratories, Plymouth Meeting, Pa. Recombinant human C5a (C5a), sulfatide (cerebroside sulfate), type I and type II galactocerebrosides, and lipopolysaccharide (from Escherichia coli serotype O55:B5) were obtained from Sigma, St. Louis, Mo. Recombinant human interleukin-8 (IL-8) and recombinant human RANTES (acronym for “regulated upon activation, normal T-cell expressed and presumably secreted”) were purchased from R&D Systems Inc., Minneapolis, Minn. An affinity-purified rabbit polyclonal antibody (Ab) raised against a peptide corresponding to amino acid residues 525 to 544 of rat Pak1 [α Pak(C-19) Ab] along with Abs to Pak2 [γ Pak(V-19) Ab] and Pak3 [β Pak(L-18) Ab] were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Affinity-purified rabbit polyclonal Abs that recognize the active (doubly phosphorylated) forms of JNK and p38-MAPK were obtained from Promega Corporation, Madison, Wis. Goat anti-rabbit immunoglobulin G labeled with horseradish peroxidase, a SuperSignal 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, Ill. Sources of all other materials are described elsewhere (1113).

Preparation of neutrophils.

Guinea pig peritoneal neutrophils were prepared as described previously (3). These preparations contained >90% neutrophils with viabilities always >90%.

Detection of renaturable protein kinases in polyacrylamide gels.

Paks and certain other protein kinases were detected directly in gels by the ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed in a gel that corresponds to amino acid residues 297 to 331 of p47-phox. This technique was performed as described elsewhere (11, 12) except that the amount of cells was reduced to 3 × 106/ml.

Immunoblotting.

Neutrophils (7.5 × 106/ml) were stimulated and lysed as described in reference 11. Aliquots of these samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (35 μg/lane) on 9.0% (wt/vol) polyacrylamide slab gels and transferred electrophoretically to Immobilon-P membranes as described in reference 11. Membranes were blocked for 1 h at room temperature with 3.0% (wt/vol) bovine serum albumin (BSA) in 20 mM HEPES (pH 7.4) containing 250 mM NaCl. The blocking buffer was removed, and the membranes were incubated with the primary Ab against active JNK (1:5,000 dilution) or active p38-MAPK (1:2,000 dilution) (55) for 1 h at room temperature in 20 mM Tris (pH 7.4) containing 250 mM NaCl and 1.0% (wt/vol) BSA. The membranes were subsequently washed three times (10 min/wash) with TBST (20 mM Tris-HCl [pH 7.4] containing 150 mM NaCl and 0.01% [vol/vol] Tween 20) and then incubated with the secondary Ab (goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate; 1:10,000 dilution) in TBST for 1 h at room temperature. Membranes were washed four times in TBST (10 min/wash) and once in TBST without Tween 20 (55). The activity of horseradish peroxidase was visualized by incubating the membranes for 20 min at room temperature in a luminol-enhanced chemiluminescence detection system (Pierce) followed by autoradiography for 10 to 30 s (54).

In certain experiments, the immunodetection system was removed from the blot by incubating the membranes with ImmunoPure elution buffer (Pierce) for 30 to 60 min at room temperature followed by two washes with TBST. These blots could then be reprobed with a different primary Ab as described above.

Miscellaneous procedures.

Procedures for immunoprecipitating Paks from neutrophil lysates with the Pak(C-19) Ab and methods for detecting these kinases in immune complexes by the renaturation assay with the p47-phox peptide are described in references 13 and 38. Superoxide release from neutrophils was measured as described previously (12). Unstimulated cells were treated with dimethyl sulfoxide (Me2SO4) or phosphate-buffered saline (PBS), as indicated in the figure legends.

Analysis of data.

Unless otherwise noted, all of the autoradiographic observations were confirmed in at least three separate experiments performed on different cell preparations. The numbers of observations are also based on different cell preparations.

RESULTS

Stimulation of neutrophils with a variety of agonists triggers rapid activation of Paks.

Neutrophils stimulated with the chemoattractant fMLP are known to exhibit a rapid and transient activation of two Paks with molecular masses of ca. 63 and 69 kDa (Fig. 1A; references 12 and 24). These kinases can be detected directly in gels by the ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed in a gel. Positions of the protein kinases are visualized by autoradiography after exposure of the gel to [γ-32P]ATP (12, 24). The peptide used corresponds to amino acid residues 297 to 331 of p47-phox and contains several of the phosphorylation sites of this protein (16). Myelin basic protein and histone H4 can also serve as substrates for these kinases in this in-gel assay (11, 14).

FIG. 1.

FIG. 1

Effects of various agonists on the activation of the 63- and 69-kDa Paks in neutrophils. Autoradiographs demonstrate the ability of 1.0 μM fMLP (A), 1.0 μM PAF (B), 20 nM LTB4 (C), 12.5 nM IL-8 (D), 65 nM RANTES (E) and 100 μg of sulfatide per ml (F) to trigger activation of the 63- and 69-kDa Paks in neutrophils. Paks were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel. Cells were treated with solvent/vehicle for 15 s (i.e., unstimulated cells) (lane a), agonist for 15 s (lane b), agonist for 30 s (lane c), agonist for 1.0 min (lane d), agonist for 3.0 min (lane e), and solvent/vehicle for 3.0 min (lane f). Positions of the 63- and 69-kDa Paks are designed by arrows and arrowheads, respectively.

Rapid activation of the 63- and 69-kDa Paks was also observed in neutrophils treated with the chemoattractant PAF (1.0 μM), LTB4 (20 nM), or IL-8 (12.5 nM) (Fig. 1B to D), the CC chemokine RANTES (65 nM) (Fig. 1E), and sulfatide (100 μg/ml), a ligand for L-selectin (27, 62) (Fig. 1F). With each of these agonists, the 63- and 69-kDa Paks exhibited maximal activation within 15 s of cell stimulation followed by significant inactivation at 3 min (Fig. 1).

PAF, LTB4, IL-8, RANTES, and sulfatide all triggered activation of the 63- and 69-kDa Paks in neutrophils in a dose-dependent manner (Fig. 2). LTB4, IL-8, and fMLP stimulated maximal activation of these kinases at concentrations of ≥1.0 nM (Fig. 2B and C; reference 12), whereas PAF and RANTES triggered optimal activation at concentrations of ≥50 nM and ca. 65 nM, respectively (Fig. 2A and D). Sulfatide triggered maximal activation of Paks at concentrations of ≥50 μg/ml, with partial activation occurring at 10 μg/ml (Fig. 2E). The activities of the 63- and 69-kDa Paks in neutrophils stimulated with optimal concentrations of PAF, LTB4, IL-8, RANTES, and sulfatide were comparable to those observed when 1.0 μM fMLP was the agonist (Fig. 2; compare lanes b and e in all panels).

FIG. 2.

FIG. 2

Activation of the 63- and 69-kDa Paks in neutrophils treated with different concentrations of agonists. Neutrophils were stimulated with different amounts of the indicated agonists for 15 s, and the 63- and 69-kDa Paks were assayed as described in Materials and Methods. In lanes a and b of all panels, cells were also treated for 15 s with 0.25% (vol/vol) Me2SO (i.e., unstimulated cells) and 1.0 μM fMLP for comparative purposes, respectively. (A) The concentrations of PAF in lane a and in lanes c through h were 0.0 μM (0.25% [vol/vol] Me2SO), 5.0 μM, 1.0 μM, 0.10 μM, 50 nM, 10 nM, and 1.0 nM, respectively. (B) The concentrations of LTB4 in lanes c through i were 0.0 μM (0.25% [vol/vol] ethanol), 0.50 μM, 0.10 μM, 20 nM, 5.0 nM, 1.0 nM, and 0.10 nM. (C) The concentrations of IL-8 in lanes c through i were 0.0 μM (0.001% [wt/vol] BSA in PBS), 0.25 μM, 0.125 μM, 12.5 nM, 6.3 nM, 1.3 nM, and 0.13 nM. (D) The concentrations of RANTES in lanes c through i were 0.0 μM (0.001% [wt/vol] BSA in PBS), 0.13 μM, 65 nM, 13 nM, 6.5 nM, 1.3 nM, and 0.65 nM. (E) The concentrations of sulfatide in lanes c through i were 0.0 μM (PBS), 400 μg/ml, 200 μg/ml, 100 μg/ml, 50 μg/ml, 10 μg/ml, and 1.0 μg/ml. Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively.

Specificity of the lipid agonists in triggering activation of the 63- and 69-kDa Paks.

A number of analogs and antagonists of PAF, LTB4, and sulfatide were tested to determine the specificity of these agonists in triggering activation of the 63- and 69-kDa Paks. Both propionyl-PAF and 2-thioacetyl-PAF, two biologically active analogs of PAF (25), triggered a rapid and transient activation of the 63- and 69-kDa Paks in neutrophils at concentrations of ≥50 nM. In contrast, lyso-PAF, an inactive metabolite of PAF (25), was ineffective in triggering activation of these kinases at concentrations of 50 nM to 1.0 μM (data not shown). The PAF analog hexanolamine-PAF can function as an antagonist of certain PAF receptors (23). Effects of this antagonist on activation of the 63- and 69-kDa Paks are shown in Fig. 3. This drug (0.10 to 5.0 μM) did not trigger activation of the 63- and 69-kDa Paks when incubated with neutrophils alone for 15 s to 5.0 min (data not shown and Fig. 3, lane b). Hexanolamine-PAF (5.0 μM) completely blocked the activation of the 63- and 69-kDa Paks in neutrophils stimulated with 0.10 μM PAF (Fig. 3, lane d). Inhibition also occurred at a hexanolamine-PAF concentration of 0.50 μM but not 0.10 μM (Fig. 3, lanes g and h). In contrast, hexanolamine-PAF (5.0 μM) did not block activation of these kinases when fMLP (0.10 μM) was the stimulus (Fig. 3, lanes e and f). Thus, hexanolamine-PAF can inhibit activation of the 63- and 69-kDa Paks in neutrophils in a stimulus-specific and dose-dependent manner.

FIG. 3.

FIG. 3

Effects of hexanolamine-PAF (PAF-A) on the activation of the 63- and 69-kDa Paks. Autoradiograms shown were from neutrophils treated with 0.25% (vol/vol) Me2SO for 3.0 min followed by 0.25% (vol/vol) Me2SO for 15 s (i.e., unstimulated cells) (lane a), 5.0 μM PAF-A for 3 min followed by 0.25% (vol/vol) Me2SO for 15 s (lane b), 0.25% (vol/vol) Me2SO for 3 min followed by 0.10 μM PAF for 15 s (lane c), 5.0 μM PAF-A for 3 min followed by 0.10 μM PAF for 15 s (lane d), 0.25% (vol/vol) Me2SO for 3.0 min followed by 0.10 μM fMLP for 15 s (lane e), 5.0 μM PAF-A for 3.0 min followed by 1.0 μM fMLP for 15 s (lane f), 0.50 μM PAF-A for 3.0 min followed by 0.10 μM PAF for 15 s (lane g), and 0.10 μM PAF-A for 3.0 min followed by 0.10 μM PAF for 15 s (lane h). Paks were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel. Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively.

20-OH-LTB4 has an affinity for the LTB4 receptor that is comparable to that of LTB4 and is a potent chemoattractant for neutrophils, whereas 20-carboxy-LTB4 lacks these activities (65). We observed that 20-OH-LTB4 stimulated a rapid and transient activation of the 63- and 69-kDa Paks in neutrophils at concentrations of ≥1.0 nM, whereas 20-carboxy-LTB4 was inactive at all concentrations tested (0.10 nM to 1.0 μM) (data not shown). The progress curves for activation of the 63- and 69-kDa Paks in neutrophils stimulated with 20-OH-LTB4 were virtually identical to those observed for LTB4. U75302, a pyridine analog of LTB4, and 14,15-dehydro-LTB4 are frequently used as specific antagonists of the LTB4 receptor (65). However, incubation of neutrophils with either U75302 (0.10 to 5.0 μM) or 14,15-dehydro-LTB4 (0.50 to 1.5 μM) alone for 15 s resulted in a partial activation of the 63- and 69-kDa Paks (data not shown). Nevertheless, the ability of LTB4 and 20-OH-LTB4 to trigger activation of the 63- and 69-kDa Paks at concentrations of ca. 1.0 nM strongly suggests that this response is a receptor-mediated event.

Selectins are a family of surface receptors and adhesion molecules that mediate the initial interactions between leukocytes and the vascular endothelium (9, 36). Recent studies have established that sulfatide, but not cerebrosides, is a ligand for L-selectin (27, 62). Cerebrosides have the same glycolipid structure as sulfatide but lack the sulfate group on the hexose moiety. Neutrophils treated with sulfatide (50 to 200 μg/ml) (Fig. 2E and Fig. 4) exhibited a marked activation of the 63- and 69-kDa Paks, whereas cells treated with type I or II cerebrosides (200 μg/ml) did not (Fig. 4). Type I cerebrosides differ from type II cerebrosides in that they contain ca. 98% α-hydroxy fatty acids.

FIG. 4.

FIG. 4

Effects of sulfatide and cerebrosides on activation of the 63- and 69-kDa Paks in neutrophils. The autoradiograms shown are from neutrophils treated for 15 s with PBS (i.e., unstimulated cells) (lane a), 200 μg of sulfatide per ml (lane b), and 200 μg each of type I (lane c) and type II (lane d) galactocerebrosides per ml. Paks were assayed by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel as described in Materials and Methods. Positions of 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively.

Effects C5a on activation of Paks in neutrophils.

In marked contrast to other stimuli, neutrophils treated with recombinant C5a exhibited a prolonged activation of the 63- and 69-kDa Paks (Fig. 5A). As with other stimuli, C5a triggered maximal activation of these enzymes within 15 s. However, C5a-treated cells were unusual in that the Paks remained active even at time points of 10 to 15 min after stimulation (Fig. 5A). Data (means ± standard deviations [SD]) presented in Fig. 6 summarize this effect for several different preparations of cells; additional examples are shown in Fig. 7 and 9; Figure 7 also compares the activities of the 63- and 69-kDa Paks in the same preparation of cells after stimulation with either fMLP or C5a and analyzed on the same renaturation gel. C5a triggered maximal activation of the 63- and 69-kDa Paks in neutrophils at concentrations of ≥10 nM (Fig. 5B).

FIG. 5.

FIG. 5

Effects of C5a on activation of the 63- and 69-kDa Paks in neutrophils. (A and B) Time course (A) and dose-response curves (B) for activation of the 63- and 69-kDa Paks in neutrophils. Paks were assayed in neutrophil lysates by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed within a gel as described in Materials and Methods. (A) Autoradiograms from neutrophils treated with 0.0025% (wt/vol) BSA for 15 s (i.e., unstimulated cells) (lane a), 50 nM C5a for 15 s (lane b), 50 nM C5a for 1.0 min (lane c), 50 nM C5a for 3.0 min (lane d), 50 nM C5a for 5.0 min (lane e), 50 nM C5a for 10 min (lane f), 50 nM C5a for 15 min (lane g), and 0.0025% (vol/vol) BSA for 15 min (lane h). (B) Autoradiograms from neutrophils treated for 15 s with 0.25% (vol/vol) Me2SO (i.e., unstimulated cells) (lane a), 1.0 μM fMLP (lane b), 0.0025% (wt/vol) BSA (lane c), 0.10 μM C5a (lane d), 50 nM C5a (lane e), 10 nM C5a (lane f), 1.0 nM C5a (lane g), 0.10 nM C5a (lane h), and 0.0025% (wt/vol) BSA (lane i). (C) Immunoprecipitation of Paks from lysates of fMLP- and C5a-stimulated neutrophils. Paks were immunoprecipitated from lysates of neutrophils with the Pak(C-19) Ab, separated by SDS-PAGE, and assayed by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel as described in Materials and Methods. Autoradiograms shown are from immunoprecipitates derived from neutrophils treated with 0.25% (vol/vol) Me2SO for 15 s (lane a), 1.0 μM fMLP for 15 s (lane b), 1.0 μM fMLP for 3.0 min (lane c), 0.0025% (wt/vol) BSA for 15 s (lane d), 25 nM C5a for 15 s (lane e), 25 nM C5a for 30 s (lane f), 25 nM C5a for 1.0 min (lane g), 25 nM C5a for 3.0 min (lane h), 25 nM C5a for 5.0 min (lane i), 25 nM C5a for 10 min (lane j), 25 nM C5a for 15 min (lane k), and 0.0025% (vol/vol) BSA for 15 min (lane l). Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively.

FIG. 6.

FIG. 6

Progress curves for activation and inactivation of the 63- and 69-kDa Paks in neutrophils stimulated with C5a or fMLP. Activities of the 63-kDa (A) and 69-kDa (B) Paks were compared in neutrophils stimulated with either 25 nM C5a (○ and ▵) or 1.0 μM fMLP (• and ▴) for different periods of time. These kinases were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel as described in Materials and Methods. Activities were estimated by densitometry, with the 100% value representing that exhibited by the kinases at 15 s after stimulation with the relevant agonist. Data points represent means ± SD from 7 to 11 separate experiments.

FIG. 7.

FIG. 7

Alterations in activities of the 63- and 69-kDa Paks when fMLP is added to neutrophils after stimulation with C5a. (A) Paks were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel as described in Materials and Methods. Autoradiograms shown were from neutrophils treated with 0.25% (vol/vol) Me2SO for 15 s (lane a), 1.0 μM fMLP for 15 s (lane b), 1.0 μM fMLP for 3.0 min (lane c), 0.0025% (vol/vol) BSA for 15 s (lane d), 25 nM C5a for 15 s (lane e), 25 nM C5a for 3.0 min (lane f), 25 nM C5a for 7.0 min (lane g), 25 nM C5a for 3.0 min with 1.0 μM fMLP added 15 s after C5a (lane h), and 25 nM C5a for 30 s with 1.0 μM fMLP added 15 s after C5a (lane i). Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively. (B and C) Bar graphs summarizing the activities of the 63-kDa (B) and 69-kDa (C) Paks under the conditions described above. Activities were estimated by densitometry. The 100% values are those observed for these kinases at 15 s after cell stimulation with 1.0 μM fMLP (bars a to c) and at 15 s after cell stimulation with 25 nM C5a (bars d to f). The bars represent activities of Paks in neutrophils treated as follows: a, 0.25% (vol/vol) Me2SO for 15 s; b, 1.0 μM fMLP for 15 s; c, 1.0 μM fMLP for 3.0 min; d, 25 nM C5a for 15 s; e, 25 nM C5a for 3.0 min; f, 25 nM C5a for 3.0 min with 1.0 μM fMLP added 15 s after C5a. Data represent means ± SD for three to four separate experiments.

FIG. 9.

FIG. 9

Effects of antagonists of protein tyrosine kinases on activation of the 63- and 69-kDa Paks in neutrophils. Activities of the 63- and 69-kDa Paks were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel as described in Materials and Methods. Neutrophils were incubated with 0.25% (vol/vol) Me2SO (I and II, control cells), 100 μM genistein (III and IV), or 35 μM herbimycin A (V) for 30 min at 37°C prior to stimulation with 1.0 μM fMLP or 25 nM C5a. For panels I and III, the cells were treated with 0.25% (vol/vol) Me2SO for 15 s (lane a), fMLP for 15 s (lane b), fMLP for 30 s (lane c), fMLP for 3.0 min (lane d), and fMLP for 5.0 min (lane e). For panels II, IV, and V, the cells were treated with 0.0025% (wt/vol) BSA for 15 s (lane a), C5a for 15 s (lane b), C5a for 30 s (lane c), C5a for 3.0 min (lane d), and C5a for 5.0 min (lane e). Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively.

The possibility existed that the 63- and 69-kDa renaturable kinases that underwent chronic activation in C5a-treated neutrophils were not Paks. However, treatment of lysed C5a-stimulated neutrophils with an antipeptide antibody generated to Pak1 resulted in the immunoprecipitation of an active 63-kDa Pak and a very small amount of a 69-kDa kinase (Fig. 5C). The activities of these kinases in the immunoprecipitates were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel (Fig. 5C). The immunoprecipitated 63-kDa Pak exhibited maximal activation within 15 s and continued to exhibit substantial activity throughout the duration of the experiment (i.e., 15 min [Fig. 5C, lane k]). In contrast, the 63-kDa Pak immunoprecipitated from lysates of fMLP-stimulated cells with the same Ab exhibited a dramatic diminution in activity by 5 min (Fig. 5C, lane c). Paks immunoprecipitated with the Pak1 Ab [α Pak(C-19) Ab] from lysates of LTB4 (20 nM)- or PAF (1.0 μM)-stimulated neutrophils behaved similarly to those from fMLP-treated cells with respect to the kinetics of activation (n = 2; data not shown). The low activity of the 69-kDa Pak in immunoprecipitates derived from lysates of both fMLP or C5a-treated cells may have resulted from a selective dephosphorylation and inactivation of this enzyme during the 2-h immunoprecipitation reaction. The antipeptide Ab to Pak1 [α Pak(C-19) Ab] used for Fig. 5 is known to be partially cross-reactive with Pak2 and Pak3. However, compared with this Pak1 Ab, an antipeptide Ab to Pak2 [γ Pak(V-19) Ab] immunoprecipitated only a fraction of the Pak activity from lysates of fMLP- or C5a-treated neutrophils, and a Pak3 Ab [β Pak(L-18) Ab] was inactive (n = 3; data not shown). Similar results were reported previously with Abs generated to glutathione S-transferase fusion proteins containing amino acid residues 175 to 306 of rat Pak1 and amino acid residues 1 to 252 of rat Pak2 (13).

Chronic activation of Paks in C5a-treated neutrophils might have resulted from a bacterial product or contaminant remaining from the expression system used to generate recombinant C5a and/or the use of the carrier protein albumin. (Four different lots of C5a were used in these studies.) While the former possibility cannot be excluded, it is noteworthy that recombinant IL-8 and RANTES triggered a transient activation of Paks and also utilized albumin as the carrier (Fig. 1D and E). Moreover, incubation of neutrophils for 5 min with either albumin (0.0025 to 0.010% [wt/vol]) or lipopolysaccharide from E. coli (10 μg/ml) did not alter the transient nature of the progress curves for activation of the 63- and 69-kDa Paks when fMLP was the stimulus (n = 2; data not shown).

Separate signals regulate activation and inactivation of the Paks in neutrophils.

Mixing experiments were undertaken to investigate the basis for the chronic activation of the 63- and 69-kDa Paks in C5a-stimulated cells (Fig. 7). As noted above, activities of the 63- and 69-kDa Paks were maximal within 15 s of exposure of cells to fMLP and returned to near the basal level by 3 to 5 min (Fig. 1A; Fig. 7A, lanes a to c). In contrast, the Paks in cells stimulated with C5a exhibited substantial activity at time points of even 10 to 15 min (Fig. 5A, lanes f and g). Interestingly, neutrophils stimulated with C5a for 3.0 min with fMLP added 15 s after C5a exhibited substantially less activity of the 63- and 69-kDa Paks than cells stimulated with C5a alone for 3.0 min (Fig. 7A; compare lanes f and h). Since the 63- and 69-kDa Paks were at maximal activation at the time fMLP was added (lane e), these data are consistent with fMLP providing a signal that results in the inactivation of Paks under these circumstances. Figure 7B and C summarize data from several different experiments examining this effect. The differences between bars e and f in Fig. 7B and C are highly significant (P < 0.001).

Experiments were also undertaken to determine if C5a utilizes the same or a similar signal transduction pathway as fMLP to trigger activation of the 63- and 69-kDa Paks. A variety of structurally distinct antagonists of PI 3-K (e.g., wortmannin) (14), type 1 and/or 2A protein phosphatases (e.g., calyculin A) (12), and certain sphingoid bases (e.g., d-erythro-sphingosine) (39) are known to prevent activation of these kinases in fMLP-stimulated neutrophils. Wortmannin (200 nM), calyculin A (20 nM), and d-erythro-sphingosine (10 μM) also blocked activation of these Paks when C5a was the agonist (Fig. 8). As reported previously (1113), neutrophils treated with calyculin A also exhibited activation of several uncharacterized protein kinases (Fig. 8, lane d, open arrowheads).

FIG. 8.

FIG. 8

Effects of various antagonists on activation of the 63- and 69-kDa Paks in neutrophils stimulated with C5a. Autoradiograms demonstrate the effects of wortmannin, calyculin A, and d-erythro-sphingosine on activation of the 63- and 69-kDa Paks. Paks were monitored by the ability to undergo renaturation and catalyze phosphorylation of the p47-phox peptide fixed in a gel. Autoradiograms shown are for cells treated at 37°C with 0.25% (vol/vol) Me2SO for 10 min followed by 0.0025% (wt/vol) BSA for 1.0 min (i.e., unstimulated cells) (lane a), 0.25% (vol/vol) Me2SO for 10 min followed by 25 nM C5a for 1.0 min (lane b), 200 nM wortmannin for 10 min followed by 25 nM C5a for 1.0 min (lane c), 20 nM calyculin A for 10 min followed by 25 nM C5a for 1.0 min (lane d), 15 μM d-erythro-sphingosine for 10 min followed by 25 nM C5a for 1.0 min (lane e), and 25 nM C5a for 1.0 min (lane f). Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively. Uncharacterized protein kinases that undergo marked activation in cells treated with calyculin A (lane d) are designated with open arrowheads.

Antagonists of protein tyrosine kinases (e.g., genistein and erbstatin) also block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils (7). Genistein is an isoflavone compound from Pseudomonas that competes for the ATP binding site in a variety of tyrosine kinases (7). This drug (100 μM) blocked activation of the 63- and 69-kDa Paks, with optimal inhibition occurring at time periods of ≥3.0 min after stimulation of the cells with either fMLP or C5a (Fig. 9I to IV). The decreases in Pak activity were estimated by densitometry by comparing the heights of the bands in Fig. 9IV with those in Fig. 9II. Treatment of neutrophils with 100 μM genistein for 30 min at 37°C reduced the amounts of 32P in the 63- and 69-kDa bands in cells stimulated with C5a for 15 s, 30 s, 3.0 min, and 5.0 min by 7% ± 7% and 37% ± 14%, by 38% ± 23% and 66% ± 15%, by 61% ± 12% and 86% ± 5%, and by 75% ± 7% and 82% ± 2% (means ± SD; n = 4), respectively. Herbimycin A is a benzoquinoid ansamysin antibiotic that is an irreversible inhibitor of various tyrosine kinases and structurally distinct from genistein (60). Herbimycin A (35 μM) blocked activation of the 63- and 69-kDa Paks in C5a-stimulated neutrophils in a manner similar to that observed with genistein (Fig. 9II and V). Treatment of neutrophils with 35 μM herbimycin A for 30 min at 37°C reduced the amounts of 32P in the 63- and 69-kDa bands in cells stimulated with C5a for 15 s, 30 s, 3 min, and 5.0 min by 20% ± 14% and 23% ± 23%, by 32% ± 11% and 44% ± 12%, by 86% ± 10% and 89% ± 6%, and by 92% ± 9% and 94% ± 6% (mean ± range, n = 2), respectively.

Erbstatin A, a stable analog of erbstatin, competes for the peptide/protein-binding site in certain tyrosine kinases (61). Treatment of neutrophils with erbstatin A (100 μg/ml) for 30 min at 37°C blocked activation of the 63- and 69-kDa Paks at all time points examined (15 s, 30 s, 3 min, and 5 min) in cells stimulated with either fMLP or C5a (data not shown). However, these effects with erbstatin A may be nonspecific since a diminution in the activities of two renaturable kinases in the 50- to 60-kDa range and a pronounced activation of a 60-kDa kinase were also observed in these experiments (data not shown). These 50- to 60-kDa kinases were not altered with genistein or herbimycin A (data not shown).

Effects of various chemoattractants on the activation of JNK and p38-MAPK in neutrophils.

p38-MAPK is known to undergo activation in neutrophils stimulated with fMLP (15, 34, 49, 50, 69). Transfection of constitutively activated Pak mutants into a variety of cells results in the activation of JNK and to a lesser extent p38-MAPK (4, 18, 19, 66). The ability of various chemoattractants to trigger activation of JNK and p38-MAPK in neutrophils was therefore investigated by using antibodies that recognized only the activated (doubly phosphorylated) forms of these kinases (55). Neutrophils stimulated with 1.0 μM fMLP exhibited a time-dependent activation of both JNK and p38-MAPK, with maximum activation of each kinase occurring about 3.0 min after cell stimulation (Fig. 10, lanes a to e). In contrast, very little activation of these kinases was observed in cells stimulated with 25 nM C5a (lanes g to k), even though this agonist triggered a pronounced and prolonged activation of the 63- and 69-kDa Paks (Fig. 5). The antibody to JNK used in these experiments can recognize both JNK1 and JNK2 (55). The molecular mass of activated JNK observed in Fig. 10 (<50 kDa) suggests that JNK1 is responsive to neutrophil stimulation with fMLP (55). Stimulation of neutrophils for 3.0 min with optimal amounts of PAF, LTB4, RANTES, and IL-8 also failed to trigger activation of JNK and the p38-MAPK to a level similar to that observed with fMLP (Fig. 11). A recent study has shown that IL-8 triggers a modest (about twofold) increase in p38-MAPK but not JNK in human neutrophils (31). In contrast, each of the agonists listed above triggered at least some activation of ERK1 in these cells when the kinase was assayed with a specific antibody to the activated form of this enzyme (data not shown). Differential activation of ERKs in human neutrophils stimulated with fMLP, C5a, LTB4, PAF, and IL-8 has been reported previously (58).

FIG. 10.

FIG. 10

Effects of fMLP and C5a on activities of JNK and p38-MAPK in neutrophils. Activation of JNK (A) and p38-MAPK (B) in neutrophils stimulated with 1.0 μM fMLP or 25 nM C5a was monitored by Western blotting with antibodies that recognized only the activated (doubly phosphorylated) forms of these kinases. Stimulation of cells and Western blotting were performed as described in Materials and Methods. The blots shown were from cells treated with 0.25% (vol/vol) Me2SO for 30 s (lane a), fMLP for 30 s (lane b), fMLP for 1.0 min (lane c), fMLP for 3.0 min (lane d), fMLP for 5.0 min (lane e), 0.25% (vol/vol) Me2SO for 5 min (lane f), 0.0025% (wt/vol) BSA for 30 s (lane g), C5a for 30 s (lane h), C5a for 1.0 min (lane i), C5a for 3.0 min (lane j), C5a for 5.0 min (lane k), and 0.0025% (wt/vol) BSA for 5.0 min (lane l). Positions of activated JNK and p38-MAPK are designated by arrows and arrowheads, respectively. The broken arrow shows the position of an unknown protein which also reacted with the antibody to p38-MAPK.

FIG. 11.

FIG. 11

Effects of a variety of agonists on the activation of JNK and p38-MAPK in neutrophils. Activation of JNK and p38-MAPK was monitored in neutrophils by Western blotting with antibodies that recognized only the activated (doubly phosphorylated) forms of these kinases. Stimulation of the cells and Western blotting were performed as described in Materials and Methods. Membranes were first blotted with an antibody to activated JNK (arrows) and then reblotted with an antibody to activated p38-MAPK (arrowheads). Neutrophils were treated for 3 min at 37°C with 0.25% (vol/vol) Me2SO (unstimulated cells) (lane a), 1.0 μM fMLP (lane b), 1.0 μM PAF (lane c), 25 nM C5a (lane d), 20 nM LTB4 (lane e), 65 nM RANTES (lane f), 100 μg of sulfatide per ml (lane g), and 12.5 nM IL-8 (lane h). The broken arrow shows the position of an unknown protein which also reacted with the antibody to activated p38-MAPK.

Finally, neutrophils stimulated with optimal amounts of fMLP or C5a also displayed striking differences in activation of the NADPH-oxidase complex as measured by the amounts of O2 released into the medium. Cells treated with fMLP (1.0 μM) or C5a (50 to 200 nM) exhibited rates of O2 release of 46 ± 9 (12) and 5.3 ± 2.5 (SD, n = 3) nmol of O2/min/107 cells, respectively. Moreover, O2 release from neutrophils stimulated with fMLP lasted for 3 to 5 min (12), whereas that triggered by C5a persisted for ≤1.0 min (data not shown). Treatment of neutrophils with 200 nM or 1.0 μM wortmannin for 30 min at 37°C did not significantly (<20%) block the activation of JNK or p38-MAPK in cells stimulated with fMLP for 3 min (n = 3; data not shown). However, wortmannin (100 nM) is known to block the stimulation of O2 production and activation of the 63- and 69-Paks in fMLP-stimulated neutrophils (14). These data indicate that JNK and p38-MAPK are not likely to be downstream targets of Pak in these cells and that the large quantities of O2 (and hence H2O2 by dismutation) stimulated by fMLP were not responsible for the pronounced activation of JNK and p38-MAPK that was observed with this stimulus.

DISCUSSION

In this paper, we report that occupation of a variety of diverse receptors on neutrophils that couple to heterotrimeric G proteins (10, 21, 30) triggers rapid activation of the 63- and 69-kDa Paks. Activation of these kinases is usually transient but may also be chronic, depending on the nature of the stimulus. Evidence is presented that separate signals are required for activation and inactivation of Paks. In addition, we demonstrate that activation of Pak alone does not trigger activation of certain MAP kinases or O2 production in these cells. The significance of these and other novel observations to the function(s) of Pak in neutrophils is discussed below.

With most agonists, the 63- and 69-kDa Paks exhibited maximal activation within 15 s, followed by a marked inactivation at 3 min (Fig. 1). In contrast, neutrophils stimulated with C5a exhibited a prolonged activation of these kinases, with ca. 50% of the activity remaining even after 10 to 15 min (Fig. 5 and 6). However, addition of fMLP to neutrophils already stimulated with C5a resulted in a pronounced inactivation of the 63- and 69-kDa Paks (Fig. 7). The most straightforward explanation for these results is that separate and distinct signals are required for activation and inactivation of these kinases, with the latter signal being diminished in C5a-stimulated cells (see below).

Paks are subjected to a complex array of regulatory phenomena. These kinases must clearly undergo covalent modification (phosphorylation) (24) during neutrophil stimulation with each of the agonists tested since the enhanced activity persists even after SDS-PAGE and the denaturation/renaturation procedure. Thus, the inactivation of the 63- and 69-kDa Paks observed in Fig. 1 and 7 likely involves dephosphorylation of these kinases. As noted above, Paks undergo activation upon interacting with the GTP-bound form of Cdc42 or Rac (44). Interestingly, Pak can form a tight complex with PIX, a guanine nucleotide exchange factor for Rac (45). Paks can also undergo a Rac/Cdc42-independent activation upon associating with membrane or certain lipids (5, 40). The small adapter protein Nck and the β subunit of a complex G protein bind specifically to Pak and may mediate this association with the membrane (6, 20, 37). Both the GTPase-dependent and GTPase-independent modes of Pak activation require (auto)phosphorylation of the kinase, and these reactions take from several minutes to 1 h in vitro (5, 41, 46). In contrast, optimal activation of the 63- and 69-kDa Paks occurred within 15 s in stimulated neutrophils (Fig. 1).

The exact events which trigger the rapid activation and inactivation of Paks in neutrophils are not known. As noted above, it is likely that Paks undergo both phosphorylation and dephosphorylation in stimulated neutrophils, with the phosphorylation reaction predominating in the early time points after cell stimulation (15 s to 3.0 min). Under these circumstances, the missing inactivation signal that fMLP provides to C5a-treated neutrophils may involve any one of a number of different possibilities, including stimulation of a GTPase-activating protein for Rac/Cdc42, inactivation of a putative upstream kinase that catalyzes the rapid phosphorylation/activation of Pak (see below), stimulation of a phosphatase that recognizes Pak, and/or the dissolution of a complex that shields Paks from phosphatases.

The overall mechanism(s) responsible for the rapid activation of the 63- and 69-kDa Paks is likely to be the same for neutrophils stimulated with C5a or fMLP. Activation of Paks in neutrophils stimulated with either fMLP (7, 12, 14) or C5a (Fig. 8 and 9) is sensitive to antagonists of PI 3-K, type 1 and/or 2A protein phosphatases, and tyrosine kinases. Interestingly, neutrophils contain an isoform of PI 3-K that is synergistically activated by the βγ subunits of complex G proteins and tyrosine-phosphorylated proteins (53). Location of this isoform of PI 3-K upstream of Pak could account for the sensitivity of the Pak stimulatory pathway to pertussis toxin (12, 24), wortmannin (reference 14 and Fig. 8), and herbimycin and genistein (reference 7 and Fig. 9). 3-Phosphorylated inositides may function in the activation of Pak by stimulating a guanine nucleotide exchange factor and/or by activating a protein kinase that catalyzes the phosphorylation/activation of Pak (59).

The C5a and fMLP receptors appear to trigger activation of the same or a very similar population of complex G proteins (22, 63). Why, then, should the kinetics of Pak activation and certain other cellular responses (i.e., activation of JNK/p38-MAPK and O2 production) differ for these stimuli? While the answer to this question is speculative, recent studies have demonstrated that some serpentine receptors can form signaling complexes with proteins in addition to the complex G protein (e.g., Rho and JAK) (47, 48). Perhaps unique regions in the fMLP receptor can form such complexes and trigger at least some of the cellular responses listed above. The possibility also exists that the fMLP or C5a receptors selectively activate different low-abundance G proteins in neutrophils that may also be involved in these responses (1).

Transfection of constitutively activated Pak mutants or overexpression of wild-type Pak into certain cells is sufficient to activate JNK and to a lesser extent p38-MAPK (4, 18, 19, 43, 66). However, activation of Pak alone is not sufficient to trigger stimulation of these MAP kinases in neutrophils. In particular, C5a triggers a pronounced and prolonged activation of the 63- and 69-kDa Paks but stimulates little or no activation of JNK or p38-MAPK compared to fMLP (Fig. 10 and 11). Interestingly, a recent study has also concluded that Pak1 does not mediate JNK activation by Rac in Cos-1 cells (57). The possibility also existed that JNK or p38-MAPK provided a signal that promotes the inactivation of Pak. Thus, the failure of these MAP kinases to undergo activation in C5a-treated neutrophils could account for the chronic stimulation of Pak. However, neutrophils stimulated with PAF, LTB4, RANTES, or IL-8 exhibited a transient activation of PAK with little or no activation of JNK or p38-MAPK (Fig. 11). Previous studies have shown that human neutrophils stimulated with IL-8 or PAF exhibit increases in p38-MAPK activity of ca. 200 and 500%, respectively (31, 49). Activation of p38-MAPK in fMLP-stimulated human neutrophils as measured by the content of phosphotyrosine was also partially inhibited by wortmannin (34). Whether these differences from our results reflect the different species used and/or differences between blood and elicited peritoneal neutrophils is not known.

What is the function(s) of the Paks in neutrophils? As noted above, activation of these kinases (and their upstream signals) triggers neither activation of JNK or p38-MAPK nor optimal O2 production. Thus, if Paks are involved in these responses, additional messengers or signals are required. We have previously reported that optimal activation of Paks can occur at concentrations of fMLP that trigger only shape changes and chemotaxis in neutrophils (12). The same situation may also exist for the chemoattractants C5a, LTB4, IL-8, and PAF (Fig. 2; references 17 and 30). Neutrophils undergoing chemotaxis exhibit a polarized morphology and marked increases in total F-actin and cytoskeletal actin (33, 51, 68). As noted above, wortmannin and calyculin A block activation of the 63- and 69-kDa Paks (Fig. 8; references 12 and 14) and inhibit the increase in cytoskeletal actin in fMLP-stimulated cells (33, 51). Wortmannin also blocks cell polarization and chemotaxis in neutrophils stimulated with fMLP (51). Thus, there appears to be a correlation between the activity of Pak and the association and organization of actin with the cytoskeleton. Occupation of the L-selectin receptor by sulfatide also triggers activation of the 63- and 69-kDa Paks (Fig. 1, 2, and 4). This receptor mediates “rolling” of neutrophils along endothelial cells (9, 36), a phenomena that is also dependent on the cytoskeleton (29). Transfection studies have shown that a variety of Pak mutants can promote cytoskeletal changes in fibroblasts (42, 45, 56, 67). Identification of the substrates of Pak will enhance our knowledge of these critical cellular events.

ACKNOWLEDGMENTS

This study was supported by National Institutes of Health grants DK50015, AI23323 (to J.A.B.), and AR43518 (to D.R.R.).

REFERENCES

  • 1.Amatruda T T, III, Gerard N P, Gerard C, Simon M I. Specific interactions of chemoattractant factor receptors with G-proteins. J Biol Chem. 1993;268:10139–10144. [PubMed] [Google Scholar]
  • 2.Avdi N J, Winston B W, Russel M, Young S K, Johnson G L, Worthen G S. Activation of MEKK by formyl-methionyl-leucyl-phenylalanine in human neutrophils. Mapping pathways for mitogen-activated protein kinase activation. J Biol Chem. 1996;52:33598–33606. doi: 10.1074/jbc.271.52.33598. [DOI] [PubMed] [Google Scholar]
  • 3.Badwey J A, Karnovsky M L. NADH-oxidase and aldehyde oxidase from polymorphonuclear leukocytes. Methods Enzymol. 1986;132:365–368. doi: 10.1016/s0076-6879(86)32021-4. [DOI] [PubMed] [Google Scholar]
  • 4.Bagrodia S, Derijard B, Davis R J, Cerione R A. Cdc42 and Pak-mediated signaling leads to JUN kinase and p38 MAP kinase activation. J Biol Chem. 1995;270:22731–22737. doi: 10.1074/jbc.270.47.27995. [DOI] [PubMed] [Google Scholar]
  • 5.Bokoch G M, Reilly A M, Daniels R H, King C C, Olivera A, Spiegel S, Knaus U G. A GTPase-independent mechanism of Pak activation: regulation by sphingosine and other biologically active lipids. J Biol Chem. 1998;273:8137–8144. doi: 10.1074/jbc.273.14.8137. [DOI] [PubMed] [Google Scholar]
  • 6.Bokoch G M, Wang Y, Bohl B P, Sells M A, Quilliam L A, Knaus U G. Interaction of the Nck adapter protein with p21-activated kinase (Pak1) J Biol Chem. 1996;271:25746–25749. doi: 10.1074/jbc.271.42.25746. [DOI] [PubMed] [Google Scholar]
  • 7.Brumell J H, Grinstein S. Serine/threonine kinase activation in human neutrophils: relationship to tyrosine phosphorylation. Am J Physiol. 1994;267:C1574–C1581. doi: 10.1152/ajpcell.1994.267.6.C1574. [DOI] [PubMed] [Google Scholar]
  • 8.Brzeska H, Knaus U G, Wang Z-Y, Bokoch G M, Korn E D. p21-Activated kinase has substrate specificity similar to Acanthamoeba myosin-1 heavy chain kinase and activates Acanthamoeba myosin-1. Proc Natl Acad Sci USA. 1997;94:1092–1095. doi: 10.1073/pnas.94.4.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Butcher E C. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991;67:1033–1036. doi: 10.1016/0092-8674(91)90279-8. [DOI] [PubMed] [Google Scholar]
  • 10.Didsbury J R, Uhing R J, Tomhave E, Gerard C, Gerard N, Snyderman R. Receptor class desensitization of leukocyte chemoattractant receptors. Proc Natl Acad Sci USA. 1991;88:11564–11568. doi: 10.1073/pnas.88.24.11564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ding J, Badwey J A. Neutrophils stimulated with a chemotactic peptide or a phorbol ester exhibit different alterations in the activities of a battery of protein kinases. J Biol Chem. 1993;268:5234–5240. [PubMed] [Google Scholar]
  • 12.Ding J, Badwey J A. Stimulation of neutrophils with a chemoattractant activates several novel protein kinases that can catalyze the phosphorylation of peptides derived from p47-phox and MARKS. J Biol Chem. 1993;268:17326–17333. [PubMed] [Google Scholar]
  • 13.Ding J, Knaus U G, Lian J P, Bokoch G M, Badwey J A. The renaturable 69 and 63 kDa protein kinases that undergo rapid activation in chemoattractant-stimulated guinea pig neutrophils are p21-activated kinases (Paks) J Biol Chem. 1996;271:24869–24873. doi: 10.1074/jbc.271.40.24869. [DOI] [PubMed] [Google Scholar]
  • 14.Ding J, Vlahos C J, Liu R, Brown R F, Badwey J A. Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J Biol Chem. 1995;270:11684–11691. doi: 10.1074/jbc.270.19.11684. [DOI] [PubMed] [Google Scholar]
  • 15.Downey G P, Butler J R, Tapper H, Fialkow L, Saltiel A R, Rubin B R, Grinstein S. Importance of MEK in neutrophil microbicidal responsiveness. J Immunol. 1997;160:434–443. [PubMed] [Google Scholar]
  • 16.El Benna J, Faust L P, Babior B M. The phosphorylation of the respiratory burst oxidase component p47-phox during neutrophil activation. J Biol Chem. 1994;269:23431–23436. [PubMed] [Google Scholar]
  • 17.Foxman E F, Campbell J J, Butcher E C. Multistep navigation and the combinatorial control of leukocyte chemotaxis. J Cell Biol. 1997;139:1349–1360. doi: 10.1083/jcb.139.5.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Frost J A, Steen H, Shapiro P, Lewis T, Ahn N, Shaw P E, Cobb M H. Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. EMBO J. 1997;16:6426–6438. doi: 10.1093/emboj/16.21.6426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Frost J A, Xu S C, Hutchison M R, Marcus S, Cobb M H. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members. Mol Cell Biol. 1996;16:3707–3713. doi: 10.1128/mcb.16.7.3707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Galisteo M L, Chernoff J, Su Y-C, Skolnick E, Schlessinger J. The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak 1. J Biol Chem. 1996;271:20997–21000. doi: 10.1074/jbc.271.35.20997. [DOI] [PubMed] [Google Scholar]
  • 21.Gerard C, Gerard N P. C5a anaphylatoxin and its seven transmembrane-segment receptor. Annu Rev Immunol. 1994;12:775–808. doi: 10.1146/annurev.iy.12.040194.004015. [DOI] [PubMed] [Google Scholar]
  • 22.Goldman D W, Chang F-H, Gifford L A, Goetzyl E J, Bourne H R. Pertussis toxin inhibition of chemotactic factor-induced calcium mobilization and function in human polymorphonuclear leukocytes. J Exp Med. 1985;162:145–156. doi: 10.1084/jem.162.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Grigoriadis G, Stewart A G. 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine: an analogue of platelet-activating factor with partial agonist activity. Br J Pharmacol. 1991;104:171–177. doi: 10.1111/j.1476-5381.1991.tb12403.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Grinstein S, Furuya W, Butler J R, Tseng J. Receptor mediated activation of multiple serine/threonine kinases in human neutrophils. J Biol Chem. 1993;268:20223–20231. [PubMed] [Google Scholar]
  • 25.Hanahan D J. Platelet activating factor: a biologically active phosphoglyceride. Annu Rev Biochem. 1986;55:483–509. doi: 10.1146/annurev.bi.55.070186.002411. [DOI] [PubMed] [Google Scholar]
  • 26.Huang C-K, Laramee G F, Yamazaki M, Sha’afi R I. Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils treated with chemotactic factors. Lack of requirements of calcium mobilization and protein kinase C activation. J Cell Biochem. 1990;44:221–228. doi: 10.1002/jcb.240440404. [DOI] [PubMed] [Google Scholar]
  • 27.Imai Y, True D D, Singer M S, Rosen S D. Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor. J Cell Biol. 1990;111:1225–1232. doi: 10.1083/jcb.111.3.1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kaga S, Ragg S, Rogers K A, Ochi A. Activation of p21-Cdc42/Rac-activated kinases by CD28 signalling: p21-activated kinase (PAK) and MEK kinase 1 (MEKK1) may mediate the interplay between CD3 and CD28 signals. J Immunol. 1998;160:4182–4189. [PubMed] [Google Scholar]
  • 29.Kansas G S, Ley K, Munro J M, Tedder T F. Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin. J Exp Med. 1993;177:833–838. doi: 10.1084/jem.177.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kitayama J, Carr M W, Roth S J, Buccola J, Springer T A. Contrasting responses to multiple chemotactic stimuli in transendothelial migration. Heterologous desensitization in neutrophils and augmentation of migration in eosinophils. J Immunol. 1997;158:2340–2349. [PubMed] [Google Scholar]
  • 31.Knall C, Worthen G S, Johnson G L. Interleukin-8 stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen activated protein kinases. Proc Natl Acad Sci USA. 1997;94:3052–3057. doi: 10.1073/pnas.94.7.3052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Knaus U G, Morris S, Dong H-J, Chernoff J, Bokoch G M. Regulation of human leukocyte p21-activated kinases through G-protein coupled receptors. Science. 1995;269:221–223. doi: 10.1126/science.7618083. [DOI] [PubMed] [Google Scholar]
  • 33.Kreienbuhl P, Keller H U, Niggli V. Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils. Blood. 1992;80:2911–2919. [PubMed] [Google Scholar]
  • 34.Krump E, Sanghera J S, Pelech S L, Furuya W, Grinstein S. Chemotactic peptide N-formyl-Met-Leu-Phe activation of p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase-2 in human neutrophils. J Biol Chem. 1997;272:937–944. doi: 10.1074/jbc.272.2.937. [DOI] [PubMed] [Google Scholar]
  • 35.Kuroki M, O’Flaherty J T. Differential effects of a mitogen-activated protein kinase inhibitor on human neutrophil responses to chemotactic factors. Biochem Biophys Res Commun. 1997;232:474–477. doi: 10.1006/bbrc.1997.6296. [DOI] [PubMed] [Google Scholar]
  • 36.Lawrence M B, Springer T A. Leukocytes roll on a selectin at physiologic flow rates: dissociation from a prerequisite for adhesion through integrins. Cell. 1991;65:859–873. doi: 10.1016/0092-8674(91)90393-d. [DOI] [PubMed] [Google Scholar]
  • 37.Leeuw T, Wu C, Shrag J D, Whiteway M, Thomas D Y, Leberer E. Interaction of a G-protein β-subunit with a conserved sequence in Ste20/PAK family protein kinases. Nature. 1998;391:191–198. doi: 10.1038/34448. [DOI] [PubMed] [Google Scholar]
  • 38.Lian J P, Badwey J A. Activation of the p21-activated protein kinases from neutrophils with an antibody that reacts with the N-terminal region of Pak 1. FEBS Lett. 1997;404:211–215. doi: 10.1016/s0014-5793(97)00134-8. [DOI] [PubMed] [Google Scholar]
  • 39.Lian J P, Huang R-Y, Robinson D R, Badwey J A. Products of sphingolipid catabolism block activation of the p21-activated protein kinases in neutrophils. J Immunol. 1998;161:4375–4381. [PubMed] [Google Scholar]
  • 40.Lu W, Katz S, Gupta R, Mayer B J. Activation of Pak by membrane localization mediated by Nck. Curr Biol. 1997;7:85–94. doi: 10.1016/s0960-9822(06)00052-2. [DOI] [PubMed] [Google Scholar]
  • 41.Manser E, Chong C, Zhao Z-S, Leung T, Michael G, Hall C, Lim L. Molecular cloning of a new member of the p21-Cdc42/Rac activated kinase (PAK) family. J Biol Chem. 1995;270:25070–25078. doi: 10.1074/jbc.270.42.25070. [DOI] [PubMed] [Google Scholar]
  • 42.Manser E, Huang H-Y, Loo T-H, Chen X-Q, Dong J-M, Leung T, Lim L. Expression of constitutively active αPak reveals effects of the kinase on actin and focal complexes. Mol Cell Biol. 1997;17:1129–1143. doi: 10.1128/mcb.17.3.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Manser E, Huang H-Y, Loo T-H, Chen X-Q, Dong J-M, Leung T, Lim L. Regulation of phosphorylation pathways by p21-GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur J Biochem. 1997;242:171–185. doi: 10.1111/j.1432-1033.1996.0171r.x. [DOI] [PubMed] [Google Scholar]
  • 44.Manser E, Leung T, Salihuddin H, Zhao Z-S, Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac 1. Nature. 1994;367:40–46. doi: 10.1038/367040a0. [DOI] [PubMed] [Google Scholar]
  • 45.Manser E, Loo T-H, Koh C-G, Zhao Z-S, Chen X-Q, Tan L, Tan I, Leung T, Lim L. Pak kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol Cell. 1998;1:183–192. doi: 10.1016/s1097-2765(00)80019-2. [DOI] [PubMed] [Google Scholar]
  • 46.Martin G A, Bollag G, McCormick F, Abo A. A novel serine kinase activated by Rac1/Cdc42 Hs-dependent autophosphorylation is related to Pak 65 and STE 20. EMBO J. 1995;14:1970–1978. doi: 10.1002/j.1460-2075.1995.tb07189.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.McWhinney C D, Hunt R A, Conrad K M, Dostal D E, Baker K M. The type 1 angiotensin II receptor couples to Stat1 and Stat3 activation through Jak2 kinase in neonatal rat cardiac myocytes. J Mol Cell Cardiol. 1997;29:2513–2524. doi: 10.1006/jmcc.1997.0489. [DOI] [PubMed] [Google Scholar]
  • 48.Mitchell R, McCulloch D, Lutz E, Johnson M, MacKenzie C, Fennell M, Fink G, Zhou W, Sealfon S C. Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature. 1998;392:411–414. doi: 10.1038/32937. [DOI] [PubMed] [Google Scholar]
  • 49.Nahas N, Molski T F P, Fernandez G A, Sha’afi R I. Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists. Biochem J. 1996;318:247–253. doi: 10.1042/bj3180247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Nick J A, Avdi N J, Young S K, Knall C, Gerwins P, Johnson G L, Worthen G S. Common and distinct intracellular signalling pathways in human neutrophils utilized by platelet activating factor and fMLP. J Clin Investig. 1997;99:975–986. doi: 10.1172/JCI119263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Niggli V, Keller H. The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptide-induced locomotion and increases in cytoskeletal actin in human neutrophils. Eur J Pharmacol. 1997;335:43–52. doi: 10.1016/s0014-2999(97)01169-2. [DOI] [PubMed] [Google Scholar]
  • 52.Novak K D, Titus M A. Myosin I overexpression impairs cell migration. J Cell Biol. 1997;136:633–647. doi: 10.1083/jcb.136.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Okada T, Hazeki O, Ui M, Katada T. Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and βγ-subunits of GTP-binding proteins. Biochem J. 1996;317:475–480. doi: 10.1042/bj3170475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Pierce Chemical Co. SuperSignal substrate Western blotting kits. No. 34081-34086. Rockford, Ill: Pierce Chemical Co.; 1996. [Google Scholar]
  • 55.Promega. Promega technical bulletin no. 262. Madison, Wis: Promega; 1998. [Google Scholar]
  • 56.Sells M A, Knaus U G, Bagrodia S, Ambrose D M, Bokoch G M, Chernoff J. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr Biol. 1997;7:202–210. doi: 10.1016/s0960-9822(97)70091-5. [DOI] [PubMed] [Google Scholar]
  • 57.Tapson N, Nagata K-I, Lamarche N, Hall A. A new Rac target POSH is an SH3-containing scaffold protein involved in JNK and NF-κB signalling pathways. EMBO J. 1998;17:1395–1404. doi: 10.1093/emboj/17.5.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Thompson H L, Marshall C J, Saklatvala J. Characterization of two different forms of mitogen-activated protein kinase in polymorphonuclear leukocytes following stimulation by N-formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor. J Biol Chem. 1994;269:9486–9492. [PubMed] [Google Scholar]
  • 59.Toker A, Cantley L C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997;387:673–676. doi: 10.1038/42648. [DOI] [PubMed] [Google Scholar]
  • 60.Uehara Y, Fukazawa H, Murakami Y, Mizuno S. Irreversible inhibition of the v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds. Biochem Biophys Res Commun. 1989;163:803–809. doi: 10.1016/0006-291x(89)92293-6. [DOI] [PubMed] [Google Scholar]
  • 61.Umezawa K, Hori T, Tajima H, Imoto M, Isshiki K, Takeuchi T. Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors. FEBS Lett. 1990;260:198–200. doi: 10.1016/0014-5793(90)80102-o. [DOI] [PubMed] [Google Scholar]
  • 62.Waddell T K, Fialkow L, Chan C K, Kishimoto T K, Downey G P. Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase. J Biol Chem. 1995;270:15403–15411. doi: 10.1074/jbc.270.25.15403. [DOI] [PubMed] [Google Scholar]
  • 63.Wilde M W, Carlson K E, Manning D R, Zigmond S H. Chemoattractant-stimulated GTPase activity is decreased on membranes from polymorphonuclear leukocytes incubated in chemoattractant. J Biol Chem. 1989;264:190–196. [PubMed] [Google Scholar]
  • 64.Wu C, Lee S-F, Furmaniak-Kazmierczak E, Cote G P, Thomas D Y, Leberer E. Activation of myosin-1 by members of the Ste20p protein kinase family. J Biol Chem. 1996;271:31787–31790. doi: 10.1074/jbc.271.50.31787. [DOI] [PubMed] [Google Scholar]
  • 65.Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature. 1997;387:620–624. doi: 10.1038/42506. [DOI] [PubMed] [Google Scholar]
  • 66.Zhang S, Han J, Sells M A, Chernoff J, Knaus U G, Ulevitch R J, Bokoch G M. Rho family GTPases regulate p38 MAP kinase through the downstream mediator Pak 1. J Biol Chem. 1995;270:23934–23936. doi: 10.1074/jbc.270.41.23934. [DOI] [PubMed] [Google Scholar]
  • 67.Zhao Z-S, Manser E, Chen X-Q, Chong C, Leung T, Lim L. A conserved negative regulatory region in αPak: inhibition of Pak kinases reveals their morphological roles downstream of Cdc42 and Rac1. Mol Cell Biol. 1998;18:2153–2163. doi: 10.1128/mcb.18.4.2153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Zigmond S H. Signal transduction and actin filament organization. Curr Opin Cell Biol. 1996;8:66–73. doi: 10.1016/s0955-0674(96)80050-0. [DOI] [PubMed] [Google Scholar]
  • 69.Zu Y-L, Qi J, Gilchrist A, Fernandez G A, Vazquez-Abad D, Kreutzer D L, Huang C-K, Sha’afi R I. p38-mitogen activated protein kinase activation is required for human neutrophil function triggered by TNF-α or fMLP stimulation. J Immunol. 1998;160:1982–1989. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES