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The Journal of Immunology, 2001, 166: 6349-6357.
Copyright © 2001 by The American Association of Immunologists

Phosphorylation of the Activation Loop of {gamma} p21-Activated Kinase ({gamma}-Pak) and Related Kinases (MSTs) in Normal and Stressed Neutrophils1

Jian P. Lian*, Alex Toker{dagger} and John A. Badwey2,*,{ddagger}

* Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA 02115; {dagger} Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02115; and {ddagger} Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutrophils stimulated with a variety of chemoattractants exhibit a rapid activation of two p21-activated kinases (Paks) with molecular masses of ~63 and 69 kDa ({gamma}- and {alpha}-Pak). A number of in vitro studies suggest that modification of Thr402 in the activation loop (AL) of {gamma}-Pak can play a critical role in the regulation of this kinase under certain circumstances. A phosphospecific Ab was generated to this region of Pak (pPak(AL)Ab). This Ab reacted with activated {gamma}- and {alpha}-Pak from fMLP-stimulated neutrophils that contain the sequence KRXT(P)XXGTP in their ALs. The rapid but transient activation of Paks in normal stimulated neutrophils coincided with phosphorylation and dephosphorylation at the ALs of these enzymes. In contrast, stressed cells exhibited a prolonged phosphorylation at Thr402 in both intact {gamma}-Pak and a proteolytic fragment of this kinase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like kinases (MSTs) (members of the Pak family) in osmotically stressed neutrophils and neutrophils treated with certain apoptotic agents (i.e., tumor promoters that inhibit type 1 and 2A protein phosphatases) but not in normal fMLP-stimulated cells. Thus, our results indicate that the AL of {gamma}-Pak undergoes transient phosphorylation during normal neutrophil stimulation and chronic phosphorylation in stressed cells. In addition, we demonstrate that a number of MSTs are present in neutrophils and also undergo phosphorylation during stressful circumstances.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils stimulated with a variety of chemoattractants exhibit a pronounced activation of the {gamma}-isoform of p21-activated kinase (Pak)3 along with a minor activation of {alpha}-Pak (1, 2, 3). Paks are Ser/Thr protein kinases that undergo autophosphorylation/activation upon binding the active (GTP bound) forms of the small GTPases (p21) Rac or Cdc42 (4). Paks can participate in a broad range of cellular events that include rapid cytoskeletal responses, activation/potentiation of several distinct mitogen-activated protein kinase (MAPK) cascades, and apoptosis (for review, see Refs. 5 and 6). Recent studies have implicated Pak in tumor growth (7), the invasiveness of breast cancer cells (8), and the pathogenesis of HIV (9, 10). Paks are also involved in the cross-talk between the CD28 costimulatory signal and the TCR signal (11), activation of NF-{kappa}B in macrophages (12), and activation of NFAT in T cells (13). The TCR linker protein SLP-76, which is required for a functional TCR, may serve as a scaffold to colocalize Pak and the Wiscott-Aldrich syndrome protein that regulates actin polymerization (14). The possibility exists that Pak and Wiscott-Aldrich syndrome protein form a complex that is involved in cytoskeletal regulation (15).

Considerable effort has been made to understand the regulation of Pak at the molecular level. The kinase domain (KD) is located within the C-terminal region of the enzyme (4), whereas the N-terminal region contains the Cdc42/Rac interactive binding motif (CRIB) (16) and an autoinhibitory domain (ID) (17, 18). Binding of activated Rac or Cdc42 to CRIB promotes autophosphorylation of the activation loop (AL) in Pak (4, 19), which converts this kinase to a form that is active in the absence of Rac or Cdc42 (19, 20, 21). X-ray analysis has revealed a complex series of conformational changes in Pak upon binding Cdc42 · GTP (15). {gamma}-Pak also exhibits optimal activity with some basic substrates without autophosphorylation of the AL or activated Cdc42/Rac (22). Thus, phosphorylation of the AL in Pak increases the spectrum of substrates recognized by this kinase.

Paks undergo a rapid but transient activation in neutrophils stimulated with a wide variety of physiological agonists (1, 2, 3). In contrast, both intact {gamma}-Pak and a caspase-3-mediated product of {gamma}-Pak exhibit a prolonged activation during stress or apoptosis in other cell types (23, 24, 25). Transfection of activated Pak into certain cells can trigger aspects of apoptosis (23). Whether a similar situation occurs in stressed neutrophils has not been investigated until now. Resolution of inflammation requires the recognition and phagocytosis of apoptotic neutrophils by macrophages (26).

In this paper, we describe a phosphospecific Ab that recognizes the AL in Pak only when it is phosphorylated. This Ab was used to study the regulation of Pak in normal and stressed neutrophils. In addition, we report that the mammalian sterile twenty-like kinases (MSTs), members of the Pak family (27, 28, 29), are also present in neutrophils and undergo phosphorylation when these cells are subjected to chemical or osmotic stress.


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

The phosphopeptide PEQSKRST(P)MVGTP, which corresponds to residues 395–408 of activated (phosphorylated) {gamma}-Pak (63-kDa Pak), was synthesized and used to generate antisera that contained a phosphospecific Ab to Pak by Research Genetics (Huntsville, AL). Goat polyclonal Abs raised to peptides derived from the carboxyl terminus ({gamma}-Pak(C-19) Ab) and amino terminus of human {gamma}-Pak ({gamma}-Pak(N-19) Ab) and a rabbit polyclonal Ab raised to a peptide derived from the carboxyl terminus of rat {alpha}-Pak ({alpha}-Pak(C-19) Ab) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A phosphospecific Ab that only recognized Akt when this kinase was phosphorylated on Thr308 was obtained from New England Biolabs (Beverly, MA). Mouse mAbs generated to residues 331–483 of human MST-1 and residues 275–393 of human MST-3 were obtained from PharMingen Transduction Laboratories (San Diego, CA). Calyculin A, wortmannin, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) were purchased from Calbiochem (La Jolla, CA). Affi-Gel 10 (N-hydroxysuccinimide active ester agarose) was a product of Bio-Rad (Richmond, CA). Sources of all other materials are provided elsewhere (1, 2, 3, 30).

Methods

Preparation of neutrophils. Guinea pig peritoneal neutrophils were prepared as described previously (31). These preparations contained >90% neutrophils with viabilities always >90%.

Preparation of samples and detection of renaturable protein kinases (Paks) in polyacrylamide gels. Neutrophils (6 x 106/ml) were stimulated in disposable 1-cm plastic cuvettes at 37°C. The standard reaction mixture consisted of a modified Dulbecco’s PBS medium (135 mM NaCl, 2.7 mM KCl, 16.2 mM Na2HPO4, 1.47 mM KH2PO4, 0.90 mM CaCl2, and 0.50 mM MgCl2, pH 7.35) containing 7.5 mM D-glucose. Cells were generally incubated in this reaction medium for 3.0 min at 37°C before stimulation with 1.0 µM fMLP. At the appropriate time, the cells were rapidly lysed by transferring 0.50 ml of the reaction mixture to a microcentrifuge tube containing 0.50 ml of 2x concentrated "solubilization buffer" (SDS-B). The final composition of SDS-B after mixing was 2.3% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), 5.0 mM EDTA, 10.0% (w/v) glycerol, 5.0% (w/v) 2-ME, and 0.002% (w/v) bromophenol blue. 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 elsewhere (1) except that the amount of cells was reduced to 3 x 106/ml.

Immunoblotting of protein kinases. Neutrophils (7.5 x 106/ml) were stimulated and rapidly lysed in SDS-B as described above. Aliquots of these samples were subjected to SDS-PAGE (35 µg/ml) on 9.0% (w/v) polyacrylamide slab gels and transferred electrophoretically to Immobilon-P membranes (Millipore, Bedford, MA) as described previously (30). Membranes were blocked for 1 h at room temperature with 3.0% (w/v) 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 for 1 h at room temperature in 20 mM Tris (pH 7.4) containing 250 mM NaCl and 1.0% (w/v) 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% (v/v) Tween 20) and then incubated with the secondary Ab (goat anti-rabbit Ig G-HRP 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 (3). The activity of HRP was visualized by incubating the membranes for 20 min at room temperature in a luminol-ECL detection system (Pierce, Rockford, IL) followed by autoradiography for 10–30 s (e.g., Ref. 3).

In certain experiments (see Fig. 4GoD), products of the chemiluminescence detection system were removed by washing the membranes two times (10 min/wash) with TBST. These blots were then reprobed with a different Ab as described above so that both Ags could be visualized simultaneously (cf Ref. 32). 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 Akt or extracellular signal-regulated kinase to confirm that equal amounts of protein were present in each lane of the gel.



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  		FIGURE 4. Time course for the activation and phosphorylation of the Paks in stimulated neutrophils. Specificity of the pPak(AL) Ab. A, Activation of {gamma}- and {alpha}-Pak in neutrophils stimulated with 1.0 µM fMLP for different time periods was monitored by the ability of these enzymes to undergo renaturation and catalyze the phosphorylation of the p47phox peptide fixed within a gel as referenced under Materials and Methods. B, Phosphorylation of {gamma}- and {alpha}-Pak was monitored in neutrophils stimulated with 1.0 µM fMLP for different time periods by Western blotting with the pPak(AL) Ab. C, Time course for activation of {gamma}- and {alpha}-Pak in stimulated neutrophils monitored by a mobility shift during Western blotting with the {alpha}-Pak(C-19) Ab. D, The same membrane shown in B that was blotted with the pPak(AL) Ab was reblotted with a phosphospecific Ab that only recognized Thr308 in Akt when this residue was phosphorylated (*). E, Time-course for the phosphorylation of Akt on Thr308 in stimulated neutrophils. Stimulation of the cells and Western blotting were performed as descried under Materials and Methods. {gamma}- and {alpha}-Pak are designated by an arrow and filled arrowhead, respectively. Akt is designated by an asterick. The kinase in A that undergoes activation at time points >1.0 min (broken arrow) is p90-RSK2 (Ref. 32 ).

 
Immunoprecipitation of protein kinases. Neutrophils (3.0–5.0 x 107/ml) were treated with 50 nM calyculin A or stimulated with 1.0 µM fMLP in the modified Dulbecco’s PBS medium as described above. At the appropriate time, the cells were rapidly lysed by transferring 0.50 ml of the reaction mixture to a microcentrifuge tube containing 2x concentrated "immunoprecipitation buffer" (ip-B) and rapidly mixed. The final composition of ip-B was 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5.0 mM MgCl2, 1.0 mM EGTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1.0% (v/v) Nonidet P-40, 2.5% (v/v) glycerol, 1.0 mM Na3VO4, and 1.0 mM PMSF (2, 33). After 15 min at 4°C, the lysates were centrifuged for 10 min at 1000 x g to remove insoluble debris, and the appropriate Ab (10 µg/ml) was added to the soluble fraction, which was then incubated for 2 h at 4°C. A 50% slurry of protein-A Sepharose beads (60 µl) was added to the reaction mixture, which was then incubated for one additional hour at 4°C. The resulting beads containing the bound immune complexes were washed five times with the ip-B buffer (1.0 ml/wash) and then suspended in 0.10 ml of SDS-B. Samples were immersed in a boiling water bath for 3.0 min and then subjected to immunoblotting as described above.

Purification of the phosphospecific Ab to Pak (pPak(AL) Ab). The phosphopeptide Ag and corresponding nonphosphorylated peptide were coupled to Affi-Gel 10 according to the instructions of the manufacturer (Bio-Rad). Anti-phospho Pak serum from rabbits (30 ml) was applied to a small phosphopeptide affinity column and washed extensively with Tris-HCl (pH 8.0) containing 50 mM NaCl. The pPak(AL) Ab was eluted from the column with 0.10 M Tris-glycine buffer (pH 2.8), neutralized with 2.0 M Tris base, and extensively dialyzed against 50 mM Tris (pH 8.0) at 4°C. The dialyzed samples were applied to a nonphosphorylated peptide affinity column, and the "flow-through" fractions containing the pPak(AL) Ab were stored at -20°C.

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 (n) are also based on different cell preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preparation of a phosphospecific Ab to the AL of Pak

Neutrophils stimulated with the chemoattractant fMLP exhibit rapid activation of two Paks with molecular masses of ~63 and 69 kDa ({gamma}- and {alpha}-Pak) (Fig. 1GoA; Refs. 1, 2, 3). The activities of these kinases can be conveniently assayed 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. The positions of the kinases are visualized by autoradiography after exposure of the gel to [{gamma}-32P]ATP (1, 2, 3).



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  		FIGURE 1. Activation and phosphorylation of Paks in neutrophils. A, Activation of the 63- and 69-kDa Paks was monitored by the ability of these kinases to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed within a gel. B, Phosphorylation of the 63- and 69-kDa Paks was assayed by Western blotting with an Ab generated to a phosphopeptide derived from the AL of {gamma}-Pak (pPak(AL) Ab). Conditions for the in gel kinase reaction and Western blotting are referenced in Materials and Methods. Samples shown are for neutrophils treated for 15 s with: (lane a) 0.25% (v/v) DMSO (unstimulated cells) and (lane b) 1.0 µM fMLP (stimulated cells). The 63- and 69-kDa Paks are designated by an arrow and filled arrowhead, respectively.

 
An Ab to a synthetic phosphopeptide (PEQSKRST(P)MVGTPY) corresponding to a portion of the AL of activated Pak (referred to as pPak(AL) Ab) was generated in rabbits (Research Genetics) and purified in our laboratory by affinity chromatography as described in Materials and Methods. There is complete homology between the phosphopeptide Ag and the corresponding regions in the ALs of {gamma}-Pak (residues 395–408) and {alpha}-Pak (residues 415–428) (e.g., Ref. 34) (see Fig. 5Go). Fig. 1GoB presents a Western blot comparing the proteins that reacted with this Ab in unstimulated and stimulated cells. The Ab recognized several proteins in unstimulated cells. Upon stimulation of neutrophils with 1.0 µM fMLP, two additional bands in the 60- to 70-kDa region were also observed, a prominent lower band and a minor upper band (Fig. 1GoB). This pattern was very similar to that of {gamma}- and {alpha}-Pak observed in the renaturation assay (Fig. 1GoA).



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  		FIGURE 5. Amino acid sequences of portions of the AL of {gamma}- and {alpha}-Pak and related Ser/Thr protein kinases. The preferred substrate sequence/consensus sequence recognized by Pak is present in the AL of {gamma}- and {alpha}-Pak and MST-1/2. This sequence is not present in several other protein kinases that undergo rapid activation in stimulated neutrophils.

 
Studies were undertaken to determine whether the pPak(AL) Ab recognized only phosphorylated forms of {gamma}- and {alpha}-Pak. Fig. 2GoA presents a Western Blot comparing lysates from unstimulated and stimulated neutrophils blotted with commercial Abs to {gamma}-Pak ({gamma}-Pak(N-19) Ab) and {alpha}-Pak ({alpha}-Pak(C-19) Ab). These Abs react with both the phosphorylated and nonphosphorylated forms of the kinases. The light, lower band observed with the {alpha}-Pak(C-19) Ab is likely to be {gamma}-Pak because it has the expected mass for this kinase and this Ab exhibits partial cross-reactivity toward {gamma}-Pak. Stimulation of neutrophils with 1.0 µM fMLP for 15 s resulted in a pronounced "mobility shift" in all of these bands (Fig. 2GoA, lanes b and d), which indicated that a large fraction of {gamma}- and {alpha}-Pak underwent rapid modification/phosphorylation in stimulated cells. Importantly, these Abs were highly specific as they did not recognize any other major bands in neutrophils. Thus, they could be used in immunoprecipitation studies to evaluate the specificity of the pPak(AL) Ab.



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  		FIGURE 2. Identification of the 63- and 69-kDa Paks ({gamma}- and {alpha}-Pak) in neutrophils as targets of the pPak(AL) Ab. A, Western blot demonstrates the presence of proteins in neutrophils that react with Abs to {gamma}- and {alpha}-Pak and the modification of these enzymes during cell stimulation measured as a mobility shift during SDS-PAGE. Blots shown are for cells treated for 15 s with: (lanes a and c) 0.25% (v/v) DMSO and (lanes b and d) 1.0 µM fMLP. The {alpha}-Pak(C-19) Ab is known to partially cross-react with {gamma}-Pak (lower light band). B,. The pPak(AL) Ab reacts with Paks only from stimulated neutrophils. Paks were immunoprecipitated from neutrophil lysates with the {gamma}-Pak(N-19) Ab (lanes a and b) or the {alpha}-Pak(C-19) Ab (lanes c and d) and subjected to Western blotting with the pPak(AL) Ab. The immunoprecipitates were derived from neutrophils treated for 15 s with: (lanes a and c) 0.25% (v/v) DMSO and (lanes b and d) 1.0 µM fMLP. Preparation of neutrophil lysates and conditions for immunoprecipitation and Western blotting are described in Materials and Methods. ip, Immunoprecipitated immune complexes. The 63-kDa ({gamma}) and 69-kDa ({alpha}) Paks are designated by an arrow and filled arrowhead, respectively.

 
When {gamma}-Pak was immunoprecipitated from stimulated neutrophils with the {gamma}-Pak(N-19) Ab and blotted with the pPak(AL) Ab, a significant immunoreactive band was observed with the expected mass of 63 kDa (Fig. 2GoB, lane b). Similarly, when {gamma}-Pak and {alpha}-Pak were immunoprecipitated together from stimulated neutrophils with the {alpha}-Pak(C-19) Ab and blotted with the pPak(AL) Ab two immunoreactive bands were observed with masses of ~63 and 69 kDa (Fig. 2GoB, lane d). The similar staining intensities for {gamma}- and {alpha}-Pak in Fig. 2GoB (lane d) probably reflects the fact that while {gamma}-Pak is the major isoform of Pak that undergoes activation in fMLP-stimulated neutrophils (Fig. 1Go) the {alpha}-Pak(C-19) Ab is more effective against {alpha}-Pak (Fig. 2GoA, lane c). Treatment of the immunoprecipitated Paks from stimulated neutrophils with protein phosphatase 2A (PP2A) (5.0 U/ml) for 10 min at 37°C eliminated their ability to react with the pPak(AL) Ab (Fig. 3GoA). This treatment with PP2A did not reduce the amounts of {gamma}- and {alpha}-Pak that were present in the immunoprecipitates as monitored by Western blotting with the {alpha}-Pak(C-19) Ab (Fig. 3GoB). In addition, the {gamma}- and {alpha}-Pak bands observed with the pPak(AL) Ab were blocked by the phosphopeptide Ag (2.0 µM) but not by the corresponding nonphosphorylated peptide (2.0 µM) (data not shown). Data presented in Figs. 2Go and 3Go strongly suggest that the pPak(AL) Ab recognizes {gamma}- and {alpha}-Pak only when they are phosphorylated.



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  		FIGURE 3. The pPak(AL) Ab recognizes only a phosphorylated form of Pak in stimulated neutrophils. A, The 63- and 69-kDa Paks were immunoprecipitated from neutrophil lysates with the {alpha}-Pak(C-19) Ab. The resulting immune complexes were washed, resuspended in buffer b (40 mM HEPES (pH 7.4), 1.0 mM EDTA, 150 mM NaCl, and 10 mM MgCl2) and incubated in the absence (lanes a and b) or presence of PP2A (5.0 U/ml) (lane c) for 10 min at 37°C. The immune complexes were then subjected to SDS-PAGE and Western blotting with the pPak(AL) Ab. The samples shown are from: (lane a) unstimulated cells; (lane b) stimulated cells, and (lane c) stimulated cells treated with PP2A. Neutrophils were stimulated with 1.0 µM fMLP for 15 s. Preparation of neutrophil lysates and conditions for immunoprecipitation/Western blotting are described under Materials and Methods. B, The same samples described above but blotted with the {alpha}-Pak(C-19) Ab are shown. The 63-kDa ({gamma}) and 69-kDa ({alpha}) Paks are designated by an arrow and filled arrowhead, respectively.

 
Time-course and specificity of the pPak(AL) Ab

{gamma}- and {alpha}-Pak exhibited maximal activation within 15 s of neutrophil stimulation followed by significant inactivation at 5.0 min when assayed with the "in gel" renaturation assay (Refs. 1 and 3 ; Fig. 4GoA). Very similar time courses were observed when phosphorylation of these enzymes was monitored with the pPak(AL) Ab (Fig. 4GoB) or as a "mobility shift" with the {alpha}-Pak(C-19)Ab (Fig. 4GoC). The Western blot in Fig. 4CGo indicated that there was little loss in the cellular content (proteolysis) of these kinases at 3–5 min after cell stimulation. These data strongly suggest that the loss of reactivity of {gamma}- and {alpha}-Pak toward the pPak(AL) Ab in stimulated neutrophils at time points >= 30 s (Fig. 4GoB) was due to a dephosphorylation reaction rather than proteolysis of these kinases.

As noted above, the pPak(AL) Ab was generated to a phosphopeptide that corresponded to the Thr402 phosphorylation site in {gamma}-Pak (Thr422 in {alpha}-Pak) (34). Interestingly, Thr402 resides in the optimal consensus sequence for substrate phosphorylation by {gamma}-Pak ((K/R)-R-X-(S/T)) (35). A number of protein kinases including protein kinase C (PKC), protein kinase B (PKB)/Akt, and p90-RSK undergo rapid activation (<=1.0 min) in stimulated neutrophils (e.g., see p90-RSK in Fig. 4AGo, broken arrow) (e.g., Ref. 32). Several of these kinases contain phosphorylation sites in their ALs that exhibit some homology to the C-terminal flanking region of Thr402 in {gamma}-Pak (i.e., TXXGTP) (Fig. 5Go). However, none of these sites contain the N-terminal flanking sequence KRXT found in Pak and only {gamma}- and {alpha}-Pak were recognized by the pPak(AL) Ab in blots of stimulated neutrophils (Fig. 1GoB and 4B). For example, Fig. 4Go shows a Western blot that was first probed with the pPak(AL) Ab (Fig. 4GoB, arrow and arrowhead) and then reblotted with a phosphospecific Ab that only recognized PKB/Akt when phosphorylated on its AL (Fig. 4GoD, asterisk). Phosphorylation of Akt was clearly evident in this blot yet the pPak(AL) Ab did not react with this kinase. In contrast, data provided below (see Fig. 7Go) show that the pPak(AL) Ab recognized the activated forms of MST-1 and MST-3, which do contain the sequence KRXTXXGTP in their ALs (Refs. 27, 28, 29 ; see Fig. 5Go). These data suggest that Thr402 may be the only residue in {gamma}-Pak that is recognized by the pPak(AL) Ab. Additional data on the specificity of the pPak(AL)Ab are provided below.



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  		FIGURE 7. Identification of the 50- to 60-kDa proteins that react with the pPak(AL) Ab in calyculin A-treated neutrophils as MST kinases. A–C, Western blots demonstrate that MST-1 and -3 are present in neutrophils and exhibit the same mass/mobility on SDS-PAGE as the 50- to 60-kDa proteins in calyculin A-treated cells that react with the pPak(AL) Ab. Lysates from neutrophils treated for 3.0 min with 50 nM calyculin A (Caly A) were subjected to SDS-PAGE and then transferred to an Immobilon-P membrane. The membrane was cut into three sections (A, B, and C) and used for Western blotting with different Abs. A, B, and C were immunoblotted with the pPak(AL) Ab, an Ab to MST-1, and an Ab to MST-3, respectively. Lanes shown are for cells treated with: (lane a) 0.25% (v/v) DMSO for 15 s; (lane b) 1.0 µM fMLP for 15 s; (lane c) 0.25% (v/v) DMSO for 3.0 min; and (lane d) 50 nM calyculin A for 3.0 min. D, Immunoprecipitation of two of the 50- to 60-kDa proteins that react with the pPak(AL) Ab from calyculin A-treated neutrophils with Abs to MST-1 and MST-3. Western blot compares the proteins that react with the pPak(AL) Ab in total neutrophil lysates with those in immune complexes (ips) derived from lysates treated with Abs to MST-1 and MST-3. MST-1 and MST-3 were immunoprecipitated from lysates of untreated cells (Caly A-) and cells treated with calyculin A for 3.0 min (Caly A+) and blotted with the pPak(AL) Ab. No bands were observed when nonimmune serum (C-Ab) was used. Conditions for immunoprecipitation and Western blotting are referenced in Materials and Methods. The positions of the 50- to 60-kDa proteins that react with the pPak(AL) Ab are designated by asterisks.

 
In addition to Thr402, {gamma}-Pak activated in vitro also exhibited enhanced phosphorylation on Ser141 and Ser165 (34). These serine residues do not reside in the optimal consensus sequence for Pak (e.g., Ref. 34 ; Fig. 5Go) and may not be recognized by the pPak(AL) Ab. However, if these serines are phosphorylated in stimulated neutrophils and recognized by the Ab, they must also undergo transient phosphorylation to account for the data in Fig. 4GoB.

Effects of various compounds on the phosphorylation of {gamma}-Pak in neutrophils

The ability of various compounds to effect the phosphorylation of {gamma}-Pak in neutrophils was monitored with the pPak(AL) Ab (Fig. 6Go). PMA is an activator of PKC and a potent stimulus for superoxide production by neutrophils (e.g., Ref. 36). However, PMA did not trigger activation of {gamma}-Pak in neutrophils but, in fact, reduced the basal activity of {gamma}-Pak in these cells (1, 30). As was the case in the renaturation assay, neutrophils treated with PMA consistently exhibited a reduction in the basal reactivity of {gamma}-Pak toward the Ab (Fig. 6GoA, lanes c–f).



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  		FIGURE 6. Effects of various reagents on the phosphorylation of {gamma}-Pak in neutrophils. Western blots show the effects of PMA (A), naphthalenesulfonamides (W-7 and W-5), calyculin A (B), and wortmannin (C) on phosphorylation of {gamma}-Pak in neutrophils monitored with the pPak(AL) Ab. Stimulation of the cells and Western blotting were performed as described in Materials and Methods. A, Neutrophils were treated with: (lane a) 0.25% (v/v) DMSO for 15 s; (lane b) 1.0 µM fMLP for 15 s; (lane c) 100 nM PMA for 15 s; (lane d) 100 nM PMA for 30 s; (lane e) 100 nM PMA for 1.0 min; (lane f) 100 nM PMA for 3.0 min, and (lane g) 0.25% DMSO for 3.0 min. B, Neutrophils were treated with various inhibitors for 10 min at 37°C and then stimulated with 1.0 µM fMLP for 15 s. Lane a, Unstimulated cells. Lane b, Stimulated cells. Lanes c–e, Stimulated neutrophils treated with: (lane c) 50 nM calyculin A; (lane d) 50 µM W-7, and (lane e) 50 µM W-5. C, Lanes a and b are as described in B. Lane c, Cells treated with 200 nM wortmannin for 10 min at 37°C and then stimulated with 1.0 µM fMLP for 15 s. The positions of {gamma}- and {alpha}-Pak are designated by an arrow and filled arrowhead, respectively.

 
Activation of {gamma}-Pak in fMLP-stimulated neutrophils is blocked by antagonists of phosphoinositide 3-kinase (PI 3-K; wortmannin), calmodulin (CaM; W-7), and type 1 and 2A protein phosphatases (e.g., calyculin A) (1, 30, 36, 37). Treatment of neutrophils with 200 nM wortmannin, 50 nM calyculin A, or 50 µM W-7 for 10 min at 37°C before stimulation with 1.0 µM fMLP for 30 s either blocked or partially reduced the ability of {gamma}-Pak to react with the pPak(AL) Ab (Fig. 6Go, B and C). In contrast, W-5 did not affect this reaction (Fig. 6GoB). W-5 is an unchlorinated/inactive analog of W-7 (e.g., Ref. 37). The ability of these compounds to effect the reactivity of {gamma}-Pak toward the pPak(AL) Ab was estimated by densitometry by comparing the heights of the {gamma}-Pak peak in the absence and presence of the inhibitors. Treatment of neutrophils with wortmannin, calyculin A, W-7, or W-5 as described above reduced the {gamma}-Pak peak by 68 ± 10%, 45 ± 6%, 98 ± 2%, and 23 ± 21%, respectively (mean ± SD, n = 4). It is noteworthy that while calyculin A was highly effective in blocking the activation of {gamma}-Pak in neutrophils (1, 2), this reagent was, at best, only marginally effective in reducing the reactivity of this kinase toward the pPak(AL)Ab (Fig. 6GoB, lane c). It is possible that calyculin A promotes phosphorylation of {gamma}-Pak at sites other than Thr402 and these modifications participate in the inactivation/inhibition of this kinase. The reactivity of {alpha}-Pak with the pPak(AL) Ab was always much lower than that of {gamma}-Pak. However, when significant amounts of {alpha}-Pak were observed it behaved in a manner identical with that of {gamma}-Pak in terms of its sensitivity to the inhibitors used above.

Previous studies have shown that antagonists of type 1 and 2A protein phosphatases (e.g., okadaic acid, calyculin A) trigger autophosphorylation and activation of a group of renaturable protein kinases in neutrophils with molecular masses in the 50- to 60-kDa range (1, 2, 30). Treatment of neutrophils with calyculin A also resulted in the appearance of proteins in this region that displayed a striking reactivity toward the pPak(AL) Ab (Fig. 6GoB, lane c). Studies presented below identify two of these proteins as MST-1 and MST-3.

Identification of the 50- to 60-kDa proteins that react with the pPak(AL) Ab as MSTs

The MSTs exhibit masses in the 50- to 60-kDa range and contain the sequence KRXTXXGTP within their ALs (27, 28, 29). These kinases undergo activation in certain cell types treated with calyculin A, okadaic acid, stress conditions, or apoptotic agents (28, 29). Immunological studies were undertaken to determine whether the 50- to 60-kDa proteins that reacted with the pPak(AL) Ab in calyculin A-treated neutrophils were members of the MST family (Fig. 7Go).

Western blotting experiments were performed first to determine whether the MSTs are present in neutrophils (Fig. 7Go). A very prominent immunoreactive band in the 50- to 60-kDa region was observed when neutrophil lysates were blotted with a mAb generated to residues 331–483 of human MST-1 (Fig. 7GoB). The position of this band corresponded closely to the top two bands that reacted with the pPak(AL) Ab (Fig. 7Go, A and B). MST-1 and -2 have predicted masses of 56.3 and 55.6 kDa, respectively (27, 28). In contrast, a mAb generated to residues 275–393 of human MST-3 recognized only a single protein in neutrophils that exhibited a mass very similar to that of the lowest band recognized by the pPak(AL) Ab (Fig. 7CGo). MST-3 has a predicted mass of 48 kDa (29). Similar results were observed with goat polyclonal antipeptide Abs to these kinases (Santa Cruz Biotechnology) (data not shown).

Treatment of lysed calyculin A-treated neutrophils with the monoclonal MST-1 or MST-3 Ab resulted in the immunoprecipitation of only one protein in the 50- to 60-kDa region that reacted with the pPak(AL) Ab (Fig. 7GoD) and partial removal of these proteins from the lysate (data not shown). The protein that was immunoprecipitated by the MST-3 Ab exhibited a lower mass than that precipitated by the MST-1 Ab (Fig. 7GoD), which is also consistent with these proteins being MST-1 and MST-3. Neither of these bands were observed when nonimmune serum was used (Fig. 7GoD). In contrast, MST-1/3 immune complexes obtained from cells not treated with calyculin A did not exhibit any proteins that reacted with the pPak(AL) Ab (Fig. 7GoD) but did contain proteins that reacted with either the MST-1 or MST-3 Abs with the expected molecular masses (data not shown). These data strongly suggest that at least two of the proteins that reacted with the pPak(AL) Ab in calyculin A-treated neutrophils were MSTs.

It should also be emphasized that neutrophils treated with calyculin A exhibit a dramatic hyperphosphorylation of a very large number of proteins in all regions of the gel (38). However, the pPak(AL) Ab reacted with relatively few of these proteins (Figs. 6GoB, lane c, and 7, A and D), which further demonstrates the specificity of this Ab toward the AL in the Pak/MSTs.

Effects of hyperosmolarity on the proteins in neutrophils that react with the pPak(AL) Ab

Hyperosmolarity triggers rapid apoptosis of neutrophils (39) and activation of {gamma}-Pak in 3T3-LI mouse fibroblasts (25). Cells undergoing apoptosis also exhibit cleavage of {gamma}-Pak to a constitutively active 36-kDa fragment that contains the catalytic domain and AL (23, 24, 40). The effects of hyperosmolarity on {gamma}-Pak and other proteins in neutrophils that react with the pPak(AL) Ab are shown in Fig. 8Go. Incubation of neutrophils in PBS for up to 4 h at 37°C did not result in phosphorylation of the activation loop of {gamma}-Pak or the MSTs (Fig. 8GoA, I). In contrast, exposure of neutrophils to 0.25 M sucrose resulted in a marked increase in the reactivity of the Ab toward two bands with masses of ~63 and 57 kDa along with the appearance of a prominent new band at ~36 kDa (Fig. 8GoA, II). Immunological experiments indicated that the 63- and 57-kDa bands were {gamma}-Pak and MST-1, respectively. An increase in the reactivity of a protein with the same mass as MST-3 was also evident in these experiments. The 36-kDa band is likely to be the catalytic fragment of {gamma}-Pak because a new band with this same mass was also observed when aliquots of these samples were blotted with an Ab that recognized the C-terminal region of {gamma}-Pak ({gamma}-Pak(C-19) Ab) (Fig. 8GoB). The 63- and 57-kDa bands exhibited optimal reactivity within 1 h of exposure of the cells to 0.25 M sucrose followed by a significant diminution of reactivity by 4 h. In contrast, maximal staining of the 36-kDa band with the pPak(AL) Ab and {gamma}-Pak(C-19) Ab was observed at 3–4 h after exposure of the cells to sucrose. Thus, unlike the situation in normal neutrophils, stressed cells exhibit chronic phosphorylation of {gamma}-Pak and MST-1 (cf Refs. 25 and 40).



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  		FIGURE 8. Effects of hyperosmolarity on Pak and other proteins in neutrophils that react with the pPak(AL) Ab. A, Alterations in proteins that react with the pPak(AL) Ab were monitored by Western blotting after neutrophils were incubated in the standard assay mixture in the absence (I) or presence of 0.25 M sucrose (II) for the indicated periods of time. Lane i, Cells treated with 0.25% (v/v) DMSO for 5.0 min before exposure to 0.25 M sucrose for 4 h. Lane j, Cells stimulated with 1.0 µM fMLP for 5.0 min before exposure to 0.25 M sucrose for 4 h. Preparation of cell lysates and conditions for Western blotting are referenced in Materials and Methods. B, Aliquots of the samples described above were blotted with an Ab generated to the C-terminal region of {gamma}-Pak ({gamma}-Pak(C-19) Ab) to demonstrate proteolysis of this kinase in neutrophils under hyperosmotic conditions. The positions of intact and proteolysed {gamma}-Pak are designated by a filled and open arrow, respectively. MST-1 is designated by an asterisk.

 
Stimulation of neutrophils with fMLP can inhibit apoptosis of these cells (39). Interestingly, stimulation of neutrophils with 1.0 µM fMLP for only 5.0 min before exposure of the cells to 0.25 M sucrose for 4 h significantly reduced the reactivity of the 36-kDa band toward the pPak(AL) Ab (compare lanes i and j in Fig. 8GoA, II). This diminution was estimated by densitometry and found to be 50 ± 5% (SD, n = 3). A similar decrease in the staining of the 36-kDa protein with the {gamma}-Pak(C-19) Ab was also evident in some experiments.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have generated a phosphospecific Ab to Pak (pPak(AL) Ab) that recognized members of the Ste20/Pak family of protein kinases when they are phosphorylated in their ALs within the sequence KRXTXXGTP. This Ab was used to show that the transient activation of {gamma}-Pak in stimulated normal neutrophils was closely associated with rapid phosphorylation and dephosphorylation of this region of the kinase. In contrast, {gamma}-Pak in stressed neutrophils exhibited chronic phosphorylation. In addition, we report that certain MSTs are present in neutrophils and react with the pPak(AL) Ab only in chemically or osmotically stressed cells. The MSTs are members of the Pak family and contain the sequence KRXTXXGTP in their activation loops. The significance of these and other novel findings are developed below.

As noted above, genetic, molecular biology, and biochemical studies have demonstrated that the N-terminal region of Pak contains an ID that can interact with the KD to maintain Pak in an inactive/low-affinity state toward nonbasic substrates (15, 16, 17, 18, 19, 20, 21). Binding of activated Rac or Cdc42 partially activates Pak by disrupting the ID-KD interaction and subsequent phosphorylation of the AL results in a fully activated kinase that can maintain its activity in the absence of the GTPase (19). This model is supported by the observation that substitution of Glu for Thr423 in the AL of {alpha}-Pak (which corresponds to Thr402 in {gamma}-Pak) results in an activated form of the kinase (20) that is insensitive to a peptide that corresponds to the ID (19, 20). Phosphorylation of Thr423 in {alpha}-Pak may occur as a result of autophosphorylation triggered by activated Rac/Cdc42 (e.g., Refs. 4, 21, 34) or can be catalyzed by 3-phosphoinositide-dependent kinase 1 in the presence of sphingosine (30–100 µM) (41).

We and others have previously noted that the transient activation of Paks in stimulated neutrophils was likely to involve a phosphorylation event because this response was observed in an in-gel renaturation assay and was reversed by phosphatases (1, 30, 42). We now show that this transient activation of {gamma}-Pak is closely associated with the phosphorylation and subsequent dephosphorylation of this enzyme in its AL (Fig. 4GoB). These data are consistent with the in vitro studies described above (19, 20, 21, 40) and strongly suggest that phosphorylation-dephosphorylation of Thr402 in {gamma}-Pak is critically involved in the regulation of this kinase in stimulated neutrophils. A recent study indicates that phosphorylation of {gamma}-Pak on serine residues 192 and 194 (cf Ref. 34) prevents this kinase from interacting with the exchange factor PIX and the adaptor protein Nck (43).

Neutrophils stimulated with fMLP exhibit a number of transient phenomena that display kinetics similar to the activation of {alpha}- and {gamma}-Pak. These include phosphorylation of the 47-kDa subunit of the superoxide generating oxidase complex (p47phox) (1), superoxide release (1), and increased association of actin with the cytoskeleton (44). Pak catalyzes the phosphorylation of recombinant p47phox in vitro (45). We have previously reported that a variety of chemoattractants trigger activation of the Paks in neutrophils at the same concentrations in which these agonists elicit shape changes and directional migration in these cells (3). Transfection of Pak into cells is known to trigger a variety of cytoskeletal alterations (e.g., Refs. 5 and 6) .Thus, there is a growing body of evidence that Paks may participate in some of the major functional responses of neutrophils. Chernoff and colleagues have also recently generated a phosphospecific Ab to the AL of Pak (46). They have reported that this kinase undergoes phosphorylation/activation in fibroblasts stimulated with platelet-derived growth factor or wounding and that phosphorylated Pak accumulates in areas of cortical actin polymerization (46). Their data support a role for Pak in regulating actin dynamics at certain sites (46).

Antagonists of type 1 and/or 2A protein phosphatases induce apoptosis in a variety of cells (e.g., Ref. 47) including leukocytes (e.g., Refs. 48 and 49). Treatment of neutrophils with calyculin A resulted in a remarkable increase in the ability of a group of proteins in the 50- to 60-kDa range to react with the pPak(AL) Ab (Fig. 6GoB). Immunoprecipitation experiments identified two of these proteins as MST-1 and -3 (Fig. 7Go). MSTs are members of the Pak family and contain the sequence KRXTXXGTP in their ALs (Fig. 5Go) but lack a CRIB motif (27, 28, 29). One or more MSTs also underwent phosphorylation when neutrophils were exposed to hyperosmotic conditions (Fig. 8Go). Interestingly, the MSTs did not undergo similar phosphorylation or activation when neutrophils were stimulated with fMLP (Figs. 1Go and 4Go). Thus, the MSTs in neutrophils may respond primarily to stress. MSTs in other cell types are thought to amplify the apoptotic response, perhaps by activating stress-activated protein kinase and p38-MAPK (50).

Apoptosis of human neutrophils induced by various agents/conditions can be blocked by antagonists of caspases and p38-MAPK, or by prior stimulation of the cells with fMLP (39). Exposure of other cell types to various stresses can result in the activation of intact {gamma}-Pak (25), MST-1 (e.g., Refs. 28 and 50), and cleavage of {gamma}-Pak to an active 36-kDa fragment (23, 24, 40). Data presented in Fig. 8Go indicate that osmotically stressed neutrophils exhibit phosphorylation of intact Pak and generation of a phosphorylated 36-kDa fragment of this kinase. The generation and phosphorylation of the 36-kDa form of Pak was reduced by prior stimulation of the cells with fMLP (Fig. 8Go). Whether this response reflects, at least in part, phosphorylation/inactivation of caspase 9 by activated Akt/PKB (51) in fMLP-stimulated neutrophils (Fig. 4Go, D and E) is currently under investigation. It is noteworthy that while phosphorylation of the AL of Pak in normal stimulated neutrophils was transient and lasted for only minutes (Fig. 4GoB), that in stressed cells was chronic and persisted for hours (Fig. 8Go). Addressing whether these differences reflect a sustained activation of Rac/Cdc42 (cf Ref. 25) and/or inactivation of the relevant phosphatase(s) may provide new insights into apoptosis in neutrophils.


   Acknowledgments
 
We are grateful to J. Chernoff for suggesting that the 50- to 60-kDa proteins that react with the pPak(AL) Ab in calyculin A-treated neutrophils were MSTs.


   Footnotes
 
1 These studies were supported by National Institutes of Health Grants DK 50015, AI 23323, PO1 DE 13499 (to J.A.B.) and CA 75134 (to A.T.). Back

2 Address correspondence and reprint requests to Dr. John A. Badwey, Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Thorn Building, Room 703, 75 Francis Street, Boston, MA 02115. Back

3 Abbreviations used in this paper: Pak, p21-activated kinase; KD, kinase domain; ID, autoinhibitory domain; MAPK, mitogen-activated protein kinase; CRIB, Cdc42/Rac interactive binding domain; AL, activation loop; pPak(AL) Ab, a phosphospecific Ab generated to Thr402 in the AL of Pak; {gamma}-Pak(N-19) Ab, Ab to the N terminus of {gamma}-Pak; {alpha}-Pak(C-19) Ab, Ab to the C terminus of {alpha}-Pak; PI 3-K, phosphoinositide 3-kinase; p47phox, the 47 kDa subunit of the phagocyte oxidase; PKC, protein kinase C; PKB, protein kinase B; PP2A, protein phosphatase 2A; MST, mammalian sterile twenty-like kinase; CaM, calmodulin; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; W-5, N-(6-aminohexyl)-1-naphthalenesulfonamide. Back

Received for publication December 26, 2000. Accepted for publication March 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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