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A p21-Activated Kinase-controlled Metabolic Switch Up-regulates Phagocyte NADPH Oxidase

2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês

10.1074/jbc.m206650200

1083-351X

Tali Shalom‐Barak, Ulla G. Knaus,

Receptor Mechanisms and Signaling

Chemoattractant-stimulated phagocytes increase their glucose uptake and divert energy production from glycolysis to the pentose phosphate pathway to generate NADPH. NADPH is a required cofactor for the NADPH oxidase to produce reactive oxygen metabolites, an important microbicidal tool in host defense. p21-Activated kinases (Paks) are regulated by the GTPases Rac and Cdc42 and control actin dynamics and phosphorylation of the oxidase component p47phox. Here we report the interaction of Pak with phosphoglycerate mutase (PGAM)-B, an enzyme of the glycolytic pathway. Activated Pak1 inhibits glycolysis by association of its catalytic domain with PGAM-B and subsequent phosphorylation of the enzyme on serine residues 23 and 118, thereby abolishing PGAM activity. Leukocyte activation through chemoattractant receptors leads to Pak activation and transient inhibition of endogenous PGAM-B activity. Consistent with these observations, treatment of neutrophils with phosphoglycolic acid, a competitive PGAM-B inhibitor, increases upstream intermediates, thereby amplifying the respiratory burst. These results demonstrate that Rho GTPases regulate the glycolytic pathway through Pak and suggest a link between chemoattractant signaling and metabolic responses to enhance host defense. Chemoattractant-stimulated phagocytes increase their glucose uptake and divert energy production from glycolysis to the pentose phosphate pathway to generate NADPH. NADPH is a required cofactor for the NADPH oxidase to produce reactive oxygen metabolites, an important microbicidal tool in host defense. p21-Activated kinases (Paks) are regulated by the GTPases Rac and Cdc42 and control actin dynamics and phosphorylation of the oxidase component p47phox. Here we report the interaction of Pak with phosphoglycerate mutase (PGAM)-B, an enzyme of the glycolytic pathway. Activated Pak1 inhibits glycolysis by association of its catalytic domain with PGAM-B and subsequent phosphorylation of the enzyme on serine residues 23 and 118, thereby abolishing PGAM activity. Leukocyte activation through chemoattractant receptors leads to Pak activation and transient inhibition of endogenous PGAM-B activity. Consistent with these observations, treatment of neutrophils with phosphoglycolic acid, a competitive PGAM-B inhibitor, increases upstream intermediates, thereby amplifying the respiratory burst. These results demonstrate that Rho GTPases regulate the glycolytic pathway through Pak and suggest a link between chemoattractant signaling and metabolic responses to enhance host defense. The invasion of pathogens presents an acute challenge to the host organism, which requires an immediate defensive response. Innate immune cells, mainly macrophages, neutrophils, Kupffer cells, and microglia, are specialized in the physical destruction of the invading pathogen via phagocytosis and oxidative burst. These activities are elicited by the binding of chemoattractants including bacterial-derived formylated peptides (formyl-Met-Leu-Phe) to G protein-coupled receptors on the cell surface. The chemotactic stimulus leads to activation of the low molecular weight GTP-binding proteins Rac and Cdc42 and their downstream mediators p21-activated kinases (Paks) 1The abbreviations used are: Pak, p21-activated kinase; PGAM, phosphoglycerate mutase; PPP, pentose phosphate pathway; G6P, glucose 6-phosphate; ROS, reactive oxygen species, fMLF, formyl-Met-Leu-Phe; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GST, glutathioneS-transferase; PGA, phosphoglycolic acid. (1Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Google Scholar, 2Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Google Scholar). Rac and Pak signaling is essential in cytoskeletal reorganization including membrane ruffling and formation of filopodia and lamellipodia, thus enabling cell motility during the chemotactic response (3Hall A. Br. J. Cancer. 1999; 80 Suppl. 1: 25-27Google Scholar, 4Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Google Scholar, 5Dharmawardhane S. Brownson D. Lennartz M. Bokoch G.M. J. Leukocyte Biol. 1999; 66: 521-527Google Scholar), as well as in pathogen-induced respiratory burst activity mediated by a Rac2-regulated multimeric NADPH oxidase enzyme (2Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Google Scholar, 6Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Google Scholar). The NADPH oxidase converts molecular oxygen to superoxide anion by electron transport from NADPH, a process that involves translocation and association of active Rac, p67phox, and phosphorylated p47phox with membrane-bound cytochromeb 558 (7Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Google Scholar). Pak participates in this pathway by phosphorylation of several serine residues on p47phox, which are required for oxidase activity and possibly for assembly of the active oxidase (8Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Google Scholar). Neutrophils contain at least three Pak isoforms whose fMLF-induced activation kinetics coincide with Rac2 activation, p47phox phosphorylation, and oxidant production (8Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Google Scholar, 9Huang R. Lian J.P. Robinson D. Badwey J.A. Mol. Cell. Biol. 1998; 18: 7130-7138Google Scholar). Uptake and destruction of pathogens during phagocytosis are accompanied by simultaneous elevation of glucose transporter 1 translocation, glucose uptake, and oxygen consumption (10Stjernholm R.L. Sbarra A.J. Straus R.R. The Reticuloendothelial System. 2. Plenum Press, New York1980: 73-85Google Scholar, 11Zatti M. Rossi F. Biochim. Biophys. Acta. 1965; 99: 557-561Google Scholar, 12Hagi A. Hayashi H. Kishi K. Wang L. Ebina Y. J. Med. Investig. 2000; 47: 19-28Google Scholar). In resting neutrophils, 1.3% of the total glucose is utilized via the pentose phosphate pathway (PPP), and this figure increases to 30% when activated by bacterial products (13Sbarra A.J. Karnovsky M.L. J. Biol. Chem. 1959; 234: 1355-1362Google Scholar). The PPP is connected to the glycolytic pathway by branching off at the level of glucose 6-phosphate (G6P), generating NADPH, ribose-5-phosphate, and, finally, glyceraldehyde-3-phosphate, which then can be shunted back to glycolysis for oxidation to pyruvate. Phosphoglycerate mutase (PGAM) is an enzyme in the lower part of the glycolytic pathway that catalyzes the interconversion of 3- and 2-phosphoglycerate. In this study, we show a direct physical and functional connection between the Rac/Cdc42 effector Pak and the glycolytic enzyme PGAM, causing a switch from glycolysis to PPP in order to increase oxidative and microbicidal phagocyte responses. Isolation of human neutrophils from healthy donors, cytosol preparation, and four-step chromatography were performed as described previously (14Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Google Scholar). Briefly, combined polymorphonuclear neutrophil cytosols were concentrated, followed by DEAE-Sephacel, gel filtration on Sephacel S-200, ion exchange chromatography on Mono Q, and hydrophobic interaction chromatography on heptylamine-Sepharose. Kinase reaction with purified recombinant Pak on column fractions or on recombinant PGAM was performed in kinase buffer (50 mm Hepes, pH 7.5, 10 mm MgCl2, 2 mmMnCl2, 0.2 mm dithiothreitol, and 50 μm ATP) with 10 μCi of [32P]ATP/reaction. The reactions were analyzed using gel electrophoresis and autoradiography. Combined hydrophobic interaction chromatography fractions containing a M r 30,000 Pak substrate (p30) were resolved on 20% SDS-PAGE, and the excised, Coomassie Blue-stained protein band was trypsin-digested and analyzed by mass spectrometry (W. M. Keck Biomedical Mass Spectrometry Laboratory). Monoclonal anti-His and 9E10 anti-Myc antibody (Covance, Richmond, CA), polyclonal anti-His and anti-Myc antibodies (Santa Cruz Biotechnology), polyclonal anti-Pak1 (8Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Google Scholar), and polyclonal anti-PGAM antibodies were prepared in rabbits by immunization with full-length affinity-purified PGAM or were gifts of Dr. K. Uchida (anti-PGAM-B) or Dr. F. Climent. Reagents for measurements of glycolysis intermediates, enzymes, and phosphoglycolic acid (PGA) were purchased from Sigma. A clone expressing PGAM-B was obtained from the IMAGE clone collection. PGAM-B was cloned into pPROEX htb with an amino-terminal His tag, into pCVM6 with a carboxyl-terminal His tag (pCMV6-PGAM-His), or untagged into pCMV6. Serine to alanine and serine to aspartic acid mutants were prepared using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). PGAM-B deletion mutants were prepared as pCMV6M-PGAM (amino acids 1–130), pCMV6M-PGAM (amino acids 21–130), pCMV6M-PGAM (amino acids 24–130), and pCMV6M-PGAM (amino acids 131–254). GST-Pak1, pCMV6M Pak1, pCMV6M Pak1 (T423E, L107F), pCMV6M Pak1 (amino acids 1–205), and pCMV6M Pak1 (amino acids 206–545) have been described previously (4Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Google Scholar, 15Zenke F.T. King C.C. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 32565-32573Google Scholar). Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, HEPES (10 mm), l-glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μm). Cells were transiently transfected using LipofectAMINE Plus reagent (Invitrogen). Cells were lysed in Tris-HCl (25 mm, pH 7.5), EDTA (1 mm), MgCl2 (5 mm), EGTA (0.1 mm), NaCl (100 mm), 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μm okadaic acid, 1 mm sodium pyrophosphate, and 5 mm NaF. For immunoprecipitations, cell extracts were incubated with primary antibody for 2 h at 4 °C, followed by incubation for 1 h with 15–25 μl of protein G-Sepharose beads. Samples were washed and analyzed by SDS-PAGE and immunoblotting using enhanced chemiluminescence (Pierce). GTPγS labeling of cell lysates was performed according to Ref. 16Mira J.P. Benard V. Groffen J. Sanders L.C. Knaus U.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 185-189Google Scholar. For phosphoamino acid analysis, PGAM-B was separated on 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and detected by autoradiography. The bands were excised, hydrolyzed in 6.0 N HCl acid at 110 °C for 4 h, lyophilized, and analyzed according to Boyle et al. (17Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Google Scholar). Two-dimensional phosphopeptide mapping was done after expression of PGAM-B and Pak1 (amino acids 206–545) in 293T cells labeled with [32P]H3PO4 (50 μCi/ml) overnight. PGAM was immunoprecipitated with anti-His antibody, separated on 10% SDS-PAGE, transferred onto nitrocellulose, and detected by autoradiography. Band intensities were quantified by liquid scintillation counting. Tryptic digestion and phosphopeptide mapping were performed as described previously (17Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Google Scholar, 18King C.C. Reilly A.M. Knaus U.G. Methods Enzymol. 2000; 325: 155-166Google Scholar). Full-length wild-type Pak1 fused to GST (GST-Pak1) and His-tagged PGAM-B were expressed and affinity-purified as described previously (18King C.C. Reilly A.M. Knaus U.G. Methods Enzymol. 2000; 325: 155-166Google Scholar). 10 μg of GST-Pak1 or GST were immobilized on glutathione-Sepharose beads in binding buffer (50 mm Hepes, pH 7.5, 10 mm MgCl2, and 2 mm MnCl2). 10 μg of His-PGAM-B were added and incubated for 1 h at 4 °C, followed by three washes in binding buffer. Samples were boiled in SDS sample buffer and separated on SDS-PAGE gels for immunoblotting. PGAM activity was determined by coupling the formation of 2-phosphoglycerate from 3-phosphoglycerate with the enolase-, pyruvate kinase-, and lactate dehydrogenase-catalyzed reactions. Assays were performed at 37 °C and measured at 340 nm according to Beutler (19Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton, Orlando, FL1984Google Scholar). Cell lysates for enzymatic activity were prepared with lysis buffer. The reaction mixture contained 100 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, 2 mm MgCl2, 100 mm KCl, 0.2 mm NADH, 1.5 mm ADP, 10 μm 2,3-bisphosphoglycerate, lactate dehydrogenase (0.6 unit/ml), pyruvate kinase (0.5 unit/ml), enolase (0.3 unit/ml), and 1 mm 3-phosphoglycerate. Experiments were performed at least three times in triplicates. Protein was determined by using the BCA protein assay kit (Pierce). Preparation of perchloric acid cell extracts and measurement of glucose 6-phosphate were performed according to Ref. 19Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton, Orlando, FL1984Google Scholar in triplicates. Briefly, 108neutrophils in 1 ml were lysed with 500 μl of 8% perchloric acid and centrifuged. 10 μl of 0.05% methyl-orange, a pH indicator, were added to the supernatant, which was then neutralized by slowly adding 3 m K2CO3. Glucose 6-phosphate was assayed at 340 nm. The reaction mixture contained 100 mm Tris-HCl, 0.5 mm EDTA, 10 mmMgCl2, 0.2 mm NADP, and glucose-6-phosphate dehydrogenase (0.33 unit/ml). Serial dilutions of glucose 6-phosphate were used as standard. Protein was determined by using the BCA protein assay kit (Pierce). 3 × 105neutrophils were incubated in 25 mm Hepes, pH 7.5, 118 mm NaCl, 4.7 mm KCl, 1.2 mmKH2PO4, 1.2 mm MgSO4, 5.5 mm glucose, and 1.0 mm CaCl2(KRHG) at 37 °C with 71 μm luminol and 50 μg/ml horseradish peroxidase and stimulated with fMLF at 1 μmfinal concentration. Chemiluminescence was recorded every 20 s for 15 min. Experiments were performed at least three times in triplicates using a Wallac Berthhold plate luminometer. The data accumulated thus far implicate Pak in several signaling events that mediate phagocyte functions. To gain further insight into additional functional roles of Paks in innate immune cells, the identification of novel Pak target proteins and their contribution to microbicidal activity was initiated. Cytosol from unstimulated human neutrophils was fractionated by sequential column chromatography, assessing putative target proteins in each fraction by in vitro kinase reaction with and without the addition of recombinant GST-Pak1. Fusion to GST renders Pak1 constitutively active by releasing the carboxyl-terminal kinase domain from the inhibitory effect exerted by the intramolecular folding of the amino-terminal regulatory domain (15Zenke F.T. King C.C. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 32565-32573Google Scholar). Two proteins served as major phosphorylation substrates of the recombinant kinase during fractionation: p47phox, the cytosolic NADPH oxidase component previously identified as a Pak1 target; and an unidentified protein of relative molecular weight 30,000. This p30 protein was further resolved by heptylamine-Sepharose chromatography to become the sole Pak1 phosphorylation target in the unbound column fraction (Fig.1, A and B,C7 fraction). No phosphorylation of p30 was detected without addition of active Pak. After SDS-PAGE, Coomassie Blue staining, and tryptic digest of p30, 12 peptide fragments were identified by mass spectroscopy. All of them corresponded to the ubiquitously expressed brain isoform of phosphoglycerate mutase (PGAM-B), together covering 61% of the total protein sequence. The identity of p30 as PGAM-B was confirmed using a brain isoform-specific antibody (20Uchida K. Kondoh K. Matuo Y. Clin. Chim. Acta. 1995; 237: 43-58Google Scholar), which detected a single band in neutrophil cytosol and in the highly purified C7 fraction (Fig. 1 C). The full-length coding sequence of PGAM-B was cloned into expression vectors to prepare recombinant PGAM-B. Comparison with endogenous PGAM revealed that carboxyl-terminal epitope tagging of PGAM did not alter Pak-induced phosphorylation, dimerization, or enzymatic activity. Recombinant PGAM-B was efficiently phosphorylated by kinase active Pak1 (Fig. 1 D). Similar results were obtained with purified PGAM-M, the predominant form of PGAM in muscle (data not shown). These results identify PGAM, an enzyme in the glycolytic pathway, as a novel effector of Pak1. Phosphoamino acid analysis of either endogenous neutrophil or recombinant PGAM-B phosphorylated by Pak1 revealed that phosphorylation of PGAM is exclusively on serine residues (Fig.2 A). To locate the phosphorylated serine residue(s), we used a combined approach of deletion analysis, proteolytic fragment electrophoresis, mass spectrometry, and site-specific mutagenesis. Ser23 and Ser118 of PGAM-B were identified as equally potent phosphorylation sites. To confirm these sites, single or double serine to alanine PGAM mutants were prepared, expressed together with the constitutively active Pak kinase domain, and immunoprecipitated from metabolically labeled 293T cells. Mutation of either Ser23 or Ser118 to alanine (PGAM-BA23, PGAM-BA118) resulted in a 50% decrease in Pak1-induced phosphorylation, whereas PGAM-BA23, A118 showed no incorporation of phosphate (Fig. 2 B). Comparative phosphopeptide mapping of in vivo-labeled wild-type and S→A mutant PGAM confirmed Ser23 and Ser118 as Pak phosphorylation sites. Two distinct spots corresponding to phosphorylated tryptic fragments were observed with wild-type PGAM, which were selectively lost, singly or conjointly, upon mutation of the respective serine residue (Fig. 2 C). The proximity of the amino-terminal catalytic domain of the mutase (His11) to Ser23 did not influence the extent of Pak phosphorylation of this residue because polypeptide fragments spanning either residues 1–130 or 21–130 of PGAM-BA118were equally phosphorylated (data not shown). A bona fide phosphorylation substrate often physically interacts with the respective protein kinase. Immunoprecipitation of Pak1 or PGAM-B, both expressed in 293T cells, revealed that PGAM-B co-precipitated with Pak1. The interaction was strongly enhanced when either a full-length constitutively active PAK1 mutant, Pak1F107, E423 (Fig. 3 A), or the carboxyl-terminal catalytic domain of PAK1 (206–545) was used (Fig.3 B). This demonstrates that only the active form of Pak kinase interacts with PGAM-B and that amino-terminal regulatory elements of Pak including the GTPase-binding domain or proline-rich motifs are not necessary for Pak-PGAM-B association. The complex formation between Pak1 and PGAM-B was entirely dependent on Pak activation because only GTPγS-treated lysates of cells expressing wild-type Pak and PGAM-B showed Pak-PGAM-B interaction (Fig.3 C). In vitro GTPγS loading of whole cell lysates renders Rac and Cdc42 GTPases constitutively active, thereby enabling continuous activation of the effector kinase Pak. We used affinity-purified PGAM-B and active GST-Pak1 bound to glutathione-Sepharose beads to demonstrate direct complex formation between Pak1 and PGAM-B (Fig. 3 D). PGAM activity can be assessed by coupling the last three steps of glycolysis to measure the oxidation of NADH to NAD (19Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton, Orlando, FL1984Google Scholar). To determine the consequences of Pak-mediated PGAM-B phosphorylation on leukocyte function, we evaluated the impact of Pak activation on PGAM-B activity. Neutrophils were stimulated with the chemotactic peptide fMLF, which activates Pak through a heterotrimeric G protein signaling cascade (8Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Google Scholar). Under conditions in which fMLF stimulates superoxide generation and Pak activation, the enzymatic activity of endogenous PGAM-B was transiently inhibited, with an initial 30% decrease after 30 s and a maximal inhibition of up to 60% within 15–20 min after fMLF administration (Fig. 4 A). The immediate and early onset of PGAM inhibition correlates with Pak activation (8Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Google Scholar, 9Huang R. Lian J.P. Robinson D. Badwey J.A. Mol. Cell. Biol. 1998; 18: 7130-7138Google Scholar), whereas PGAM dephosphorylation and reactivation are much slower and most likely dependent on an unidentified phosphatase. The direct involvement of Pak in this inhibitory response was assessed by analyzing the effect of increasing amounts of active Pak on the enzymatic activity of either endogenous or transfected PGAM-B in 293T cells. We found a direct dose-response relationship between the amount of activated Pak and the extent of PGAM-B inhibition, which reached 60% at the highest Pak levels (Fig. 4 B). Subsequently, a PGAM-B double mutant mimicking serine phosphorylation, PGAM-BD23, D118 was prepared and tested for enzymatic activity. Mutation of serines to aspartic acids led to complete loss of PGAM-B activity, whereas the counterpart PGAM-BA23, A118, which represents phosphorylation-deficient PGAM-B, retained high enzymatic activity (Fig. 4 C). The enzymatic activity of the PGAM-BA23, A118 mutant was not inhibited by active Pak1, confirming that inhibition of PGAM activity indeed relies on the ability of Pak to phosphorylate PGAM-B on those residues. Single and double mutations of serine 23 and/or 118 to aspartic acid in PGAM showed that serine 23 is the predominant inhibitory phosphorylation site with respect to the enzymatic activity of PGAM (Fig.4 D). PGAM mutations did not affect the association of PGAM-B with the carboxyl-terminal, kinase active Pak1 fragment or its propensity to homodimerize, as determined by native gel electrophoresis (data not shown). These observations rule out the possibility that the loss of enzymatic activity was due to nonspecific conformational effects or the inability of mutant PGAM-B to associate with Pak. Respiratory burst onset is associated with a sudden and transient increase in the activity of the PPP. Therefore, phosphorylation of PGAM-B by Pak could be part of a glycolysis "shut-off" mechanism, serving to divert energy production to the pentose phosphate pathway. Such a scenario may increase the conversion of glucose via G6P into NADPH, the obligatory energy source for the oxidative burst in phagocytes. The levels of glycolytic intermediates after fMLF stimulation of neutrophils were measured. Indeed, whereas the concentrations of lactic acid and pyruvic acid were reduced as expected if glycolysis is inhibited (data not shown), glucose 6-phosphate accumulated rapidly. Moreover, specific competitive inhibition of PGAM-B activity by the substrate analog PGA (21Sasaki R. Hirose M. Sugimoto E. Chiba H. Biochim. Biophys. Acta. 1971; 227: 595-607Google Scholar) further increased both basal and fMLF-induced levels of G6P in intact neutrophils (Fig.5 A). G6P is the substrate of the first and rate-limiting enzyme (glucose-6-phosphate dehydrogenase) in the pentose phosphate pathway. When glycolysis is inhibited at the PGAM-B level, G6P accumulates in the cell. G6P will then be catabolized more readily via the pentose phosphate pathway, increasing the cellular supply of NADPH and thus facilitating the production of reactive oxygen species (ROS) by NADPH oxidase, a requirement for the microbicidal activity of phagocytes. This concept was tested by analyzing chemoattractant-stimulated ROS production by neutrophils after preincubation with the PGAM inhibitor PGA. PGAM inhibition increased the maximal peak of fMLF-stimulated superoxide generation of human neutrophils by 70–100% (Fig.5 B), coinciding with the rapid rise in G6P concentration that is dynamically catabolized at the first critical minute. These observations strongly imply that Pak-induced PGAM-B phosphorylation generates the metabolic environment required for an efficient oxidative burst by transiently diverting the energy production from the glycolytic to the pentose phosphate pathway. Phagocytic cells are critical components of the innate immune response, serving as a first line of defense against pathogens. Upon challenge, leukocytes migrate to the site of infection and engulf the microorganism into the phagolysosome. Consecutive destruction of internalized bacteria is accomplished by release of degrading proteases and reactive oxygen metabolites. The activation of the Rac effector protein kinase Pak is dependent on chemoattractant receptor signaling and integrin ligation, both early events in stimulated phagocytes. Pak has been implicated in NADPH oxidase activation through phosphorylation of the NADPH oxidase component p47phox on several carboxyl-terminal amino acid residues. This phosphorylation is necessary for ROS generation in cell-free assay systems and in ROS-producing model cell lines (22Ago T. Nunoi H. Ito T. Sumimoto H. J. Biol. Chem. 1999; 274: 33644-33653Google Scholar, 23Inanami O. Johnson J.L. McAdara J.K. Benna J.E. Faust L.R. Newburger P.E. Babior B.M. J. Biol. Chem. 1998; 273: 9539-9543Google Scholar). Our data now show that active Pak kinase fulfills at least one additional essential role for respiratory burst activity—the inhibition of PGAM activity—and the subsequent transient rise of PPP-generated NADPH, an absolute requirement for NADPH oxidase activity. The Pak-mediated phosphorylation of PGAM occurs on two serine residues in the amino-terminal part of the enzyme. The critical importance of the phosphorylation state of both PGAM-B serine residues is evident from structural information obtained from Saccharomyces cerevisiae PGAM. S. cerevisiae PGAM has 64% amino acid homology to mammalian PGAM-B, with close to 100% identity at the catalytically active sites. The high-resolution crystal structure shows that the entrance to the catalytic cleft site is surrounded by Lys97, Arg113, and Arg114 in S. cerevisiae PGAM (Lys100, Arg116, and Arg117 in human PGAM-B) (24Rigden D.J. Alexeev D. Phillips S.E. Fothergill-Gilmore L.A. J. Mol. Biol. 1998; 276: 449-459Google Scholar). The positive electrostatic potential of these basic residues is thought to be necessary to enable binding of highly negatively charged substrates. Pak-induced phosphorylation of the neighboring Ser118, which is highly conserved in yeast and mammalian M- and B-type PGAM, will change accessibility and electrostatic potential at the cleft. The second Pak phosphorylation site on PGAM, Ser23, is located next to the conserved Gly24 (in human PGAM), which is implicated in substrate binding and substrate specificity. Several amino-terminal PGAM deletion mutants were prepared to confirm Ser23 as a Pak phosphorylation site and to ensure the structural integrity of Ser23 mutants because this residue is located close to the catalytically active His11. Alanine substitutions did not abolish PGAM activity, and both alanine and aspartic acid mutants resembled wild-type PGAM-B in their dimer formation. This is in agreement with conclusions drawn from the binding interfaces of theS. cerevisiae PGAM tetramer, which predict that mainly Pro168 and residues at the carboxyl terminus influence multimer formation (25White M.F. Fothergill-Gilmore L.A. Kelly S.M. Price N.C. Biochem. J. 1993; 295: 743-748Google Scholar). Most glycolytic enzymes, including PGAM, exist in complexes bound to the microtubule network (26Merkulova T. Lucas M. Jabet C. Lamande N. Rouzeau J.D. Gros F. Lazar M. Keller A. Biochem. J. 1997; 323: 791-800Google Scholar, 27Volker K.W. Reinitz C.A. Knull H.R. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1995; 112: 503-514Google Scholar). The GTPases Rac and Cdc42 regulate not only F-actin reorganization but also microtubule dynamics through Pak, which phosphorylates and thereby inhibits the microtubule-destabilizing protein stathmin, leading to microtubule stabilization (28Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Google Scholar). Elongation, assembly, and bundling of microtubules are rapid initial changes during chemoattractant stimulation and phagocytosis and might be connected to oxidant production (29Burchill B.R. Oliver J.M. Pearson C.B. Leinbach E.D. Berlin R.D. J. Cell Biol. 1978; 76: 439-447Google Scholar). It is therefore conceivable that microtubules or other cytoskeletal structures serve as a scaffold to connect chemoattractant signaling to metabolic pathways and facilitate the interaction of active Pak and PGAM. Pak-mediated PGAM phosphorylation may act in concert with the phosphorylation of other Pak targets, such as myosin light chain kinase or p47phox of the NADPH oxidase, to cause structural and biochemical changes required for microbicidal activity. Whereas the specific PGAM inhibitor used in this study led to a highly increased rate of ROS generation, and inhibition of Pak activity decreases the oxidative burst up to 60–70%, 2U. G. Knaus, manuscript in preparation. binding and phosphorylation studies as shown in epithelial cells were not possible in chemoattractant-stimulated neutrophils. Three different anti-PGAM antibodies failed to immunoprecipitate PGAM from human neutrophils. The development of Pak-deficient mice and of specific pharmacological inhibitors of glycolytic enzymes upstream of PGAM will aid to establish the molecular mechanisms of Pak-regulated ROS production. It is important to note that phagocytic cells are not the only mammalian cells relying on NADPH derived from the PPP. The reduction of glutathione by erythrocytes, as well as fatty acid and steroid biosynthesis, utilizes large amounts of NADPH. The potential involvement of Pak as an early signaling mediator in these processes could present the regulation for metabolic changes that accommodate immediate cellular needs. We thank M. Kinter (W. M. Keck Biomedical Mass Spectrometry Laboratory) for liquid chromatography-mass spectrometry analysis, K. Uchida and F. Climent for anti-PGAM antibody, C. C. King and C. DerMardiossian for advice, P. Rutledge for secretarial assistance, and B. Babior for critical reading of the manuscript.