Cryptic Rac-binding and p21Cdc42HS/Rac-activated Kinase Phosphorylation Sites of NADPH Oxidase Component p67phox*

Sohail AhmedDagger §, Elena PrigmoreDagger §, Sheila GovindDagger , Claire VeryardDagger , Robert KozmaDagger §, Frans B. Wientjesparallel , Anthony W. Segalparallel , and Louis LimDagger §

From the Dagger  Department of Neurochemistry, Institute of Neurology, 1 Wakefield Street, London WC1N 1PJ, Great Britain, the § Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 10 Kent Ridge Crescent, Singapore 119076, and the parallel  Department of Medicine, University College London, University Street, London WC1E 6JJ, Great Britain

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67phox, but not with the other oxidase components: cytochrome b, p40phox, or p47phox (Prigmore, E., Ahmed, S., Best, A., Kozma, R., Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67phox has been characterized further. Rac1 and Rac2 can bind to p67phox amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67phox can be used as a specific inhibitor of Rac signaling. Deletion of p67phox C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by ~8-fold. p21Cdc42Hs/Rac-activated kinase (PAK) phosphorylates p67phox amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67phox in control of the NADPH oxidase.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Members of the Rho family, Rac1, Cdc42Hs, and RhoA, play essential roles in growth factor-mediated changes in cell morphology associated with the formation of actin microfilaments and "focal complexes" (1-4). They also act downstream of Ras in distinct parts of the transformation process (5-8) and activate Jun N-terminal protein kinase (9, 10) and entry into the G1 phase of the cell cycle (11). Rho family interacting proteins that may play a role as effectors in these signaling pathways include kinases (12-15), GTPase-activating proteins (e.g. n-chimaerin) (16), and adaptors (e.g. p67phox) (17, 18).

Rac1, cytochrome b (gp91phox and p22phox), p67phox, and p47phox comprise the minimal components necessary for superoxide formation by the NADPH oxidase in vitro (19). Another oxidase component, p40phox (20), interacts with both p67phox and p47phox (21, 22), but its function is not clear. Rac1 binds p67phox (17, 18), and p67phox can bind p47phox, which in turn can bind p22phox through an SH3 domain-polyproline interaction (23-25). Reconstitution of the oxidase components in vitro does not accurately reflect complex formation in vivo. For example, the p67phox deletion mutant (amino acids 1-246) can replace p67phox in vitro, but not in vivo (26). Both p47phox and p67phox are phosphorylated during oxidase activation (27-30), and the isolation and identification of the kinases involved are essential to gain a better understanding of the mechanism by which complex formation and oxidase activity are controlled in vivo. PAK has been shown to be activated by fMet-Leu-Phe in neutrophils and to phosphorylate p47phox in vitro (31). Hitherto, kinases that can phosphorylate p67phox have not been identified.

We have been using the NADPH oxidase as a model protein complex to investigate the mechanism by which Rho family GTPases activate cellular pathways. In this study, the Rac1-p67phox interaction has been investigated in more detail. Using binding assays, we show that the Rac1-binding site of p67phox is cryptic, located between amino acids 170 and 199, and that the N-terminal fragment (amino acids 1-192) can be used as an inhibitor of the Rac signaling pathway. The binding sites in p67phox for Rac1 and p40phox are distinct. Recombinant PAK purified from Escherichia coli can phosphorylate p67phox; the phosphorylation site(s) are cryptic and located adjacent to the Rac1-binding site. Deletion of either the polyproline-rich sequence (aa1 226-236) or the C-terminal SH3 domain (aa 460-526) led to increases in Rac1 binding and PAK phosphorylation, suggesting that there is an intramolecular interaction between these two domains of p67phox that gives rise to the crypticity. Taken together, these data suggest that unfolding of p67phox, via disruption of a potential intramolecular "SH3 domain-polyproline" interaction, may play a role in control of the NADPH oxidase.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture and Microinjection-- Swiss 3T3 fibroblast cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotic/antimycotic (Life Technologies, Inc.) at 37 °C and 5% CO2. Swiss 3T3 cells were serum-starved for 24-48 h before being microinjected with p67phox protein (0.1-1 mg/ml) and observed by phase-contrast microscopy as described previously (4, 16).

Purification of p67phox from Insect Cells-- Sf9 cells in IPL-41 medium (Life Technologies, Inc.) containing 10% fetal calf serum were grown in suspension in an orbital shaker. 800 ml of cell culture were infected to saturation with p67phox cDNA containing baculovirus (or control, not infected) and were harvested after 72 h. The cells were washed with 50 mM Tris, pH 7.5, 50 mM NaCl, and 1 mM EDTA supplemented with 1 mM dithiothreitol, 1 mM diisopropyl fluorophosphate, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml Nalpha -p-tosyl-L-lysine chloromethyl ketone. The cells were disrupted by sonication (MSE Soniprep), and cell debris was removed by centrifugation (30,000 × g, 15 min). At this stage, cell extracts were used either for experiments or for purification of p67phox by column chromatography; a Hi-load Q-Sepharose column was used with a 50-500 mM NaCl gradient, followed by a phenyl-Sepharose column eluted with a 1 to 0 M NH4SO4 gradient, resulting in a p67phox preparation of ~95% purity as judged by SDS-PAGE.

Expression and Purification of Recombinant Proteins in E. coli-- Cdc42Hs, Rac1, p40phox, p67phox, beta PAK, and p67phox deletion mutants were expressed in pGEX plasmids or derivatives, and protein was purified using glutathione-Sepharose as described previously (4, 32, 33) or as detailed below. p67phox cDNA was cloned into pGEX-2T as described previously (19). The C-terminal truncated forms of p67phox (aa 1-58, 1-192, and 1-238) were made by digestion of full-length p67phox in pGEX-2T with two enzymes: EcoRI plus StuI, Bpu11021, or BglII. The DNA was then purified, blunt-ended with Klenow fragment, and religated with T4 DNA ligase. The resulting plasmids contained the first 174, 376, and 714 nucleotides, respectively, of the p67phox coding sequence. C-terminal p67phox was made by digestion of full-length cDNA in pGEX-2T with BamHI and EcoRI, followed by purification of the DNA fragment starting from the internal BamHI site to the EcoRI site (nucleotide 967 to the end) and ligation into BamHI/EcoRI-digested pGEX-3X. The other constructs were made by polymerase chain reaction amplification using full-length p67phox cDNA in pGEX as a template. In all cases, the sense primer contained a BamHI site, and the antisense primer contained an EcoRI site. DNA encoding aa 170-238 was digested with AvrII and EcoRI, blunt-ended with Klenow fragment, and religated. This new plasmid encoded aa 170-199. Mutations were made with the USE kit (Amersham Pharmacia Biotech). In all cases, the correct sequence was confirmed by nucleotide sequencing using an Applied Biosystems automated sequencer. p67phox fragment 1-199 was a kind gift from A. Hall (University College London, London). p67phox deletion mutant protein fragment 1-192 was unstable and had to be used immediately after purification. Protein was quantified by the method of Bradford (34). p67phox deletion proteins were used as GST fusions unless otherwise stated.

PAK fragments were subcloned from beta PAK cDNA into pGEX vectors (35). The NcoI fragment of beta PAK cDNA was cloned into the NcoI site of pGEX-3X derivative p261, encoding aa 37-149 (112 aa). This NcoI clone was digested with BamHI and BglII and religated, generating a plasmid that encoded aa 70-149 (79 aa). This clone starts at the first residue, isoleucine, of the CRIB motif. The NcoIDelta BamHI/BglII plasmid was then digested with EcoRI and religated, deleting aa 93-149. The plasmid NcoIDelta BamHI/BglIIDelta EcoRI encoded aa 70-93 (24 aa).

Cdc42Hs and Rac1 Binding Assays-- p21 probes were prepared by incubating Cdc42Hs or Rac1 (5 µg) with 1 µl of [gamma -32P]GTP (6000 Ci/mmol, 10 mCi/ml, 1.6 µM; NEN Life Science Products) in 50 µl of exchange buffer (50 mM NaCl, 25 mM Mes, pH 6.5, 25 mM Tris-HCl, pH 7.5, 1.25 mM EDTA, 1.25 mg/ml bovine serum albumin, and 1.25 mM dithiothreitol) for 10 min at room temperature. Protein was directly applied to nitrocellulose filters (5-20 µg) and then incubated with p21 probes in Petri dishes containing 4 ml of binding buffer (50 mM NaCl, 25 mM Mes, pH 6.5, 25 mM Tris-HCl, pH 7.5, 1.25 mM MgCl2, 1.25 mg/ml bovine serum albumin, 1.25 mM dithiothreitol, and 0.5 mM GTP). Filters were analyzed as described previously (18). Background binding was variable, and therefore, it was essential to make comparisons of p21-target interactions within the same experiment. p21 probes were used uncleaved and cleaved.

Kinase Assays-- beta PAK was expressed as a GST fusion protein and purified as described previously (35). beta PAK-GST (96 kDa) was not cleaved to allow visualization of potential p67phox phosphorylation (67 kDa). In vitro kinase assays were carried out as follows. beta PAK protein (0.125-0.25 µg) was incubated with 5 µg of either full-length p67phox or fragments in kinase buffer (50 mM Hepes, pH 7.0, 5 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, and 0.5% Triton X-100) with 20 µM ATP/2 µCi of [gamma -32P]ATP (5000 Ci/mmol, 10 mCi/ml; Amersham Pharmacia Biotech) for 15 min at 30 °C. The reaction was stopped by the addition of 5× sample buffer, followed by boiling for 5 min. The proteins were then separated by SDS-PAGE. The dye front was removed, and the gels were stained with Coomassie Blue to visualize proteins. Gels were then destained, fixed, and dried before being exposed to Kodak film for between 10 min and 5 h.

Immunoblot Analysis-- Proteins were separated on SDS-polyacrylamide gel, transferred to nitrocellulose by semidry blotting, and incubated with blocking buffer, followed by incubation with either anti-p40phox (20) or anti-Ste20 (Upstate Biotechnology, Inc.) antibodies. The blots were developed using ECL reagents (Amersham Pharmacia Biotech) to visualize the antibodies as described previously (18).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Localization of the Rac1/2-binding Site of p67phox-- To localize the Rac1-binding site of p67phox, different fragments of this protein were expressed as GST fusions and purified on glutathione affinity columns (Fig. 1A, arrowheads mark full-length protein). p67phox proteins (~5 µg, based on the percentage of full-length protein present in the sample) were then dot-blotted onto nitrocellulose and probed with Rac1 Q61L-[gamma -32P]GTP. Rac1 binding to full-length p67phox was weaker than to aa 1-238 (Delta 239-526) (Fig. 1B). Rac1 binding to N-terminal fragments of p67phox (aa 1-238, 1-199, and 1-192) was similar. However, Rac1 did not bind p67phox fragment 1-58 or 1-131 (Fig. 1B, dots 9 and 10). This suggests that aa 131-192 of p67phox are essential for Rac1 binding. Next, deletions of p67phox fragment 1-238 were examined. Rac1 bound aa 170-238 and 170-199 of p67phox, although binding was significantly weaker then that obtained with aa 1-192 (see below). Rac1 did not bind to p67phox fragment 192-238 or the C-terminal part of the protein (aa 300-526) (Fig. 1B). To confirm that the Rac1-binding site of p67phox was localized to aa 170-199, a deletion of 6 aa of full-length p67phox was made (Delta 178-184). This protein did not bind Rac1 (data not shown). Taken together, these data suggest that the Rac1-binding site of p67phox is located between aa 170-192 and that sequence 1-170 may stabilize the Rac1-p67phox interaction, possibly through the presence of tetratricopeptide repeats (TPRs) (see "Discussion"), but is not essential for binding.


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Fig. 1.   Rac1 binding to p67phox and protein fragments. A, full-length p67phox or protein fragments (1-5 µg) were separated on SDS-polyacrylamide gels (10 and 12% SDS for lanes 1-13 and lanes 14 and 15, respectively) and visualized by Coomassie Blue staining. p67phox protein was purified in E. coli as GST fusions, except for lane 1, which is full-length p67phox (aa 1-526) purified from insect cells. Protein are as follows: full-length aa 1-526 (lane 2), aa 1-238 (lane 3), aa 1-192 (lane 4), aa 126-238 (lane 5), aa 170-238 (lane 6), aa 192-238 (lane 7), aa 300-526 (lane 8), aa 1-58 (lane 9), aa 1-131 (lane 10), Delta 226-236 (lane 12; see Fig. 6 for use), aa 1-460 (lane 13), aa 170-199 (lane 15), and GST (lanes 11 and 14). B, shown is a schematic of p67phox primary structure illustrating positions of SH3 domains and the polyproline-rich motif (P). Lines represent protein fragments expressed, with numbers on the left corresponding to the lanes in A. Numbers on the right of the dot blots indicate amino acids of p67phox expressed. Proteins were purified on glutathione-agarose, dot-blotted onto nitrocellulose (~5 µg), and processed as described under "Materials and Methods." Blots were incubated with Rac1 Q61L-[gamma -32P]GTP, washed, and exposed to autoradiographs. The arrowheads mark the positions of the full-length protein and fragments. ns, not shown. Proteins were purified to ~95%. Each p67phox protein had differing degrees of stability, with aa 126-238 being the most unstable. All proteins are GST fusions, except for full-length p67phox and deletion Delta 226-236 (lanes 1, 2, and 12). Note that fragment 1-192 became unstable on cleavage from GST and lost the ability to bind Rac1.

Rac2 is the major Rac isoform present in human neutrophils. To localize the Rac2-binding site p67phox proteins, aa 1-192, 170-238, and 170-199 were immobilized on nitrocellulose and probed with Rac2-[gamma -32P]GTP (with GST and fragment 300-526 as controls). Fig. 2 shows that Rac2 binds to the same site in p67phox as Rac1.


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Fig. 2.   Rac2 binding to p67phox fragments. p67phox proteins (lanes 1-3, 1, 5, and 10 µg of protein, respectively) were immobilized on nitrocellulose as described in the legend to Fig. 1 and probed with Rac2-[gamma -32P]GTP. GST was present as a control. Rac2 bound more strongly to p67phox than did Rac1.

To characterize further the relationship between p67phox fragments 1-192, 170-238, and 170-199, we quantified the amount of Rac1 binding to these proteins. Fig. 3 shows that Rac1 binding to fragment 1-192 was ~18-fold higher than binding to fragment 170-199. Since the latter (minimal) domain of p67phox showed significantly reduced Rac1 binding compared with aa 1-192, we investigated whether this was also true for CRIB motif2 proteins (36). N-terminal fragments of PAK expressed as GST fusions were analyzed in dot-blot assays, and Cdc42Hs binding was quantified. Cdc42Hs binding to PAK fragment 37-149 (112 aa of beta PAK) was ~25-fold higher than binding obtained with a minimal fragment of 24 aa (Fig. 3). In addition, a clone (CBP5) isolated in a Cdc42Hs two-hybrid screen, which possesses similarity to the CRIB motif, also showed a reduction in Cdc42Hs binding as the polypeptide was reduced in size.3 Thus, it appears that additional as yet undefined primary sequences are required for strong Cdc42Hs/Rac1 binding to these targets.


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Fig. 3.   Comparison of p21 interaction with p67phox and PAK fragments. A, schematic of p67phox and PAK primary structure showing domains and positions of protein fragments expressed. T, TPRs of 34 aa (42); P, polyproline-rich stretches (potential SH3-binding sites). PAK has four polyproline-rich sequences that may represent protein-binding sites (e.g. Nck). Lines represent fragments expressed, with numbers on the left referring to the bars in Fig. 2B and those on the right to amino acids expressed. B, p67phox (bars 1-3) and PAK (bars 4-6). p67phox and PAK fragments were purified and probed with Rac1 Q61L-[gamma -32P]GTP or Cdc42Hs Q61L-[gamma -32P]GTP, respectively, on dot blots as described under "Materials and Methods." Interactions were quantified by assessing radioactivity present on 1-cm2 nitrocellulose squares with GST as a control. Binding activity is expressed relative to that obtained with the smallest p67phox (bar 3) and PAK (bar 6) fragments, arbitrarily set at 1. Results were obtained from two to three experiments. Error bars are means ± S.D. (n >=  6).

The Rac1-binding site (RBD) of p67phox has no obvious similarity to the CRIB motif. In addition, we were unable to align the RBD of p67phox with the Rac1-binding proteins POR1 (37), tubulin (38), and p140Sra-1 (39).

p67phox Fragment 1-192 Can Be Used as an Inhibitor of Rac Signaling-- The RBD of p67phox is highly specific for Rac1 in binding assays; it did not interact at all with Cdc42Hs or RhoA (see below). Thus, the RBD could serve as a specific inhibitor of signaling by preventing Rac1 from interacting with targets. To examine this possibility, the effect of p67phox fragment 1-192 on Rac1-dependent phorbol 12-myristate 13-acetate (PMA)-induced ruffling was investigated. Fig. 4 shows a time-lapse experiment of two cells, under phase-contrast microscopy, with the cell in panel A injected with p67phox protein fragment 1-192, showing that p67phox fragment 1-192 does indeed inhibit PMA-induced membrane ruffling. Furthermore, p67phox fragment 1-192 is specific in its effect on ruffling, as it did not inhibit the formation of either filopodia/retraction fibers (Cdc42Hs-mediated) or stress fibers (RhoA-mediated) (data not shown). We also examined the effects of the smaller fragments of p67phox, 170-199 and 170-238, which bind to Rac1, but much more weakly than fragment 1-192 (Fig. 3). These proteins gave variable results, although a degree of inhibition of PMA-induced ruffling did occur. A quantitative assay may be required to determine the effect of these smaller peptides on Rac1 signaling.


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Fig. 4.   Effect of p67phox fragment 1-192 on PMA-induced morphology changes in Swiss 3T3 cells. Swiss 3T3 were treated with PMA (10 nM), and their morphology was monitored using phase-contrast microscopy. A, cell injected with fragment 1-192 (1 mg/ml); B, control cell. Swiss 3T3 cells were starved for 24 h and recorded for 5 min prior to and then for 25 min following treatment/microinjection. The time-lapse sequence shows one surface of a selected cell. Arrowheads in B show areas of membrane ruffling and lamellipodium formation. Similar results were obtained in four other experiments.

Crypticity of the Rac1-binding Site-- Deletion of the C terminus of p67phox (aa 193-526) resulted in an 8-9-fold increase in Rac1 binding (Fig. 5). One possible explanation for this enhancement is that the C-terminal SH3 domain may bind the polyproline-rich sequence (at aa 226-236) and hinder Rac1 binding to aa 170-199. To examine this possibility, p67phox proteins with either the polyproline-rich sequence or the C-terminal SH3 domain deleted were tested for their ability to bind Rac1. Fig. 5 shows that, in both cases, there was an ~8-fold increase in Rac1 binding to the p67phox deletion mutants. These results are consistent with the idea of a cryptic binding site in p67phox for Rac1 due to the presence of an intramolecular SH3-polyproline interaction.


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Fig. 5.   Rac1-binding site of p67phox is cryptic. A, dot blots; B, quantification of Rac1 binding to p67phox. E. coli-expressed full-length p67phox (bar 1) was compared with fragments 1-192 (bar 2), 300-526 (bar 3), Delta 226-236 (bar 4), and 1-460 (bar 5). Binding activity is expressed relative to that obtained with full-length p67phox, arbitrarily set at 1. Protein fragments of p67phox (equimolar) immobilized on nitrocellulose were probed with Rac1 Q61L-[gamma -32P]GTP, and binding was quantified as described under "Materials and Methods." Results were obtained from two to three experiments. Error bars are means ± S.D. (n >=  6). P, polyproline-rich stretches.

Distinct Rac1- and p40phox-binding Sites on p67phox-- p40phox has been proposed to interact with two parts of p67phox: in the N-terminal region, where Rac1 also binds (22, 40), and in the C-terminal region between the two SH3 domains (21). Therefore, the possibility exists that Rac1 binding may be affected by p40phox. To investigate this, Rac1 binding to full-length p67phox or aa 1-192 was measured in the presence of increasing amounts of p40phox. The presence of p40phox at up to 10 times the concentration of p67phox did not affect Rac1 binding (Fig. 6A), thus making it unlikely that the binding sites of Rac1 and p40phox overlap.


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Fig. 6.   Distinct binding sites for p40phox and Rac1 on p67phox. A, full-length p67phox or aa 1-192 were incubated with increasing amounts of p40phox, and the mixture was dot-blotted and then probed with Rac1 Q61L-[gamma -32P]GTP as described under "Materials and Methods." Lane 1, no addition; lanes 2-4, p40phox present at 1, 5, and 10 times the amount of Rac1, respectively. B, different p67phox-GST fusion proteins were bound to glutathione-agarose and incubated with purified p40phox, and the amount of this protein retained by the beads was determined by immunoblots. Approximately equal amounts of p67phox protein were used, except for fragment 1-238, which was in 5-fold excess. Similar results were obtained in at least two other experiments.

Next, we used a more direct protein-protein interaction assay to determine which portion of p67phox bound to p40phox. Different p67phox-GST fusion proteins (fragments 1-526, 1-526 with polyproline-rich deletion, Delta 58K, 1-238, 300-526, and 300-460) were bound to glutathione-Sepharose beads and incubated with purified p40phox. The amount of p40phox retained after washing the beads was assessed by immunoblotting following SDS-PAGE. p67phox Delta 58K is a mutation found in a chronic granulomatous disease patient that disrupts Rac1 binding (41), whereas the other proteins are fragments that have been used to examine Rac binding. p40phox bound clearly to p67phox proteins containing aa 300-460, with little if any binding (above background) to aa 1-238 (Fig. 6B). These results suggest that the major binding site for p40phox on the p67phox protein is in the C-terminal half of p67phox between the two SH3 domains and confirm data obtained with the yeast two-hybrid system (21).

A PAK-like Kinase Copurifies with Insect Cell p67phox-- We have previously reported that Cdc42Hs can apparently bind p67phox purified from insect cells (18). To localize the Cdc42Hs-binding site of p67phox, deletion mutants were used in dot-blot assays as described for Rac1. Cdc42Hs did not bind any of the deletion mutants or the full-length p67phox protein produced in E. coli (data not shown). Nevertheless, we observed good binding of Cdc42Hs to p67phox purified from insect cells. A possible reason for this difference between E. coli-expressed and insect cell-expressed p67phox could be that a Cdc42Hs-binding protein copurifies with the latter p67phox protein. To examine this, extracts from insect cells infected with control virus were compared with extracts infected with p67phox virus and with purified p67phox protein from insect cells. Interestingly, a 68-kDa Cdc42Hs-binding band was present in all three protein preparations (Fig. 7, lanes 1-3). This band was found to migrate slightly above p67phox when a Coomassie Blue-stained gel was overlaid with the x-ray film from the Cdc42Hs binding experiment. The level of this protein was reduced in the insect cell extracts expressing p67phox. The 68-kDa Cdc42Hs-binding protein did not bind wild-type Rac1 under the conditions of the assay (18). To determine whether this 68-kDa protein could be a Ste20/PAK-like serine/threonine protein kinase present in insects cells, an immunoblotting analysis was carried out using anti-Ste20 antibody. This antibody is raised against the kinase domain of Ste20 and does cross-react with mammalian neutrophil PAK proteins (alpha , beta , and gamma  isoforms).4 The anti-Ste20 antibody did indeed react against the 68-kDa protein present in insect cell purified p67phox (Fig. 7, lane 9). Furthermore, the purified p67phox preparations from insect cells possessed a 68-kDa kinase activity (data not shown). Thus, the Cdc42Hs binding to p67phox that we initially observed (18) is an artifact due to the presence of a 68-kDa Cdc42Hs-binding protein in insect cell p67phox preparations.


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Fig. 7.   A PAK-like protein copurifies with p67phox in insect cells. Sf9 cell extracts were prepared from uninfected (lanes 1, 4, and 7) or p67phox-infected cells (lanes 2, 5, and 8) and compared with insect cell purified p67phox (lanes 3, 6, and 9). Lanes 1-3, Cdc42Hs binding activity; lanes 4-6, anti-p67phox antibody reactivity ; lanes 7-9, anti-Ste20 antibody reactivity. Cdc42Hs binding analysis and immunoblotting were carried out as described under "Materials and Methods."

PAK Phosphorylation Sites of p67phox Are Cryptic-- Since a 68-kDa PAK-like protein copurified with p67phox in insect cells, we examined whether PAK could phosphorylate p67phox in vitro. Recombinant beta PAK purified from E. coli was fully active and nonresponsive to the presence of Rac1 or Cdc42Hs in either autophosphorylation (Fig. 8A) or myelin basic protein phosphorylation (data not shown) assays. However, in some experiments, we did observe the appearance of a novel phosphorylated band at around 68 kDa when Cdc42Hs or Rac1 was added (Fig. 8A, lanes 2'-3'). This 68-kDa band is probably a breakdown product of PAK-GST. Interestingly, if the recombinant PAK protein was treated with alkaline phosphatase, its ability to phosphorylate substrate was reduced significantly (data not shown), suggesting that an E. coli kinase can activate PAK. The recombinant PAK protein was able to phosphorylate p67phox (Fig. 8B). In attempts to localize the PAK phosphorylation sites of p67phox, we observed, as for the Rac1 binding, that deletion of the C-terminal part (aa 239-526) stimulated PAK phosphorylation (Fig. 8C). p67phox fragments 1-58, 1-131, and 300-526 were not phosphorylated by PAK. The best p67phox protein fragment substrate for PAK was aa 170-238 (Fig. 8C, lane 6), which has four potential phosphorylation sites located between aa 203 and 233 (Fig. 9). Since aa 192-238 were not phosphorylated by PAK, the peptide sequence between aa 170 and 191 must be essential for PAK interaction. PAK phosphorylation of p67phox was also stimulated by deletion of either the C-terminal SH3 domain (aa 1-460) or the polyproline-rich sequence (Delta 226-236), suggesting that an SH3-polyproline interaction gives rise to cryptic PAK phosphorylation sites (Fig. 8D, compare lanes 2 and 6).


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Fig. 8.   Phosphorylation of p67phox by PAK. A, Coomassie Blue stain of purified PAK-GST (5 µg; lane 1) and kinase activity of PAK in the absence (lanes 2 and 2') or presence of Rac1 Q61L-GTP (lanes 3 and 3') or presence of Cdc42Hs Q61L-GTP (lanes 4 and 4'). Lanes 2-4 and 2'-4' show the variation obtained in PAK autophosphorylation assays between experiments. B, kinase activity of PAK in the absence (lane 1) or presence (lane 2) of full-length p67phox (arrowhead marks the position of p67phox). C, PAK phosphorylation of p67phox fragments. PAK autophosphorylation was carried out with no additions (lane 1) or with GST (lanes 2 and 11) and in the presence of p67phox fragments 1-238 (lane 3), 1-192 (lane 4), 126-238 (lane 5), 170-238 (lane 6), 192-238 (lane 7), 300-526 (lane 8), 1-131 (lane 10), and 1-58 (lane 11). D, PAK phosphorylation in the absence (lanes 1 and 5) or presence (lane 2) of p67phox and deletions Delta 226-236 (lane 3) and 1-460 (lanes 4 and 6). Arrowheads mark the positions of the full-length protein. Note that lower exposure times were used for lanes 3 and 4 to show positions of phosphorylated band(s). Bands below the arrowhead in lane 6 are breakdown products. Kinase assays were carried out as described under "Materials and Methods." Full-length p67phox and protein fragments were present at ~1-2 µg. p67phox fragments are GST fusions, except for fragments 1-238 and 1-192.


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Fig. 9.   Location of the major putative PAK phosphorylation sites of p67phox. A schematic of p67phox is presented showing the location of its domains. The amino acid sequence between positions 126 and 238 contains (boxed), TPR4, RBD, a polyproline-rich motif (P), and the potential PAK phosphorylation sites (S and T residues in boldface). The major in vivo phosphorylation site, Thr-233 (see Footnote 5), is marked with an asterisk.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rac1/2-binding Site of p67phox-- Rac1 interacts directly with p67phox in a GTP-dependent manner (17, 18). Amino acid residues 1-199 are sufficient for Rac1 binding (17). In this study, we have localized the Rac1/2-binding site to aa 170-199. Rac1 did not interact with the C-terminal portion (aa 300-526), excluding the possible presence of more than one Rac1-binding site. However, sequences N-terminal to the binding site are required for full activity, possibly for stabilizing the binding site (similar observations were made for Cdc42Hs binding to fragments of PAK and a novel Cdc42Hs interacting protein, CBP5). The p67phox deletion mutants Delta 58K (41) and Delta 178-184 no longer bind Rac1. Since the Rac-binding site is located between aa 170 and 199, we believe that the former mutation destabilizes the protein possibly by disrupting the second TPR located between aa 37 and 70 of p67phox. TPR domains are thought to form amphipathic helices that self-associate and stabilize protein structures (42). The Rac1-binding sequence of p67phox does not align with the Rac1-binding regions of POR1, tubulin, or p140Sra-1 (37-39). Isolation of more Rac1-specific targets will be required before a Rac1-specific binding motif can be generated.

p40phox did not affect Rac1 binding to p67phox and was found to interact predominantly with the C terminus of this protein, between the two SH3 domains, which agrees with data obtained by a yeast two-hybrid analysis (21, 43). Thus, it is possible for both Rac1 and p40phox to bind to p67phox at the same time.

The use of the dominant-negative (N17) Ras (44, 45) and Rac1 (1) mutation has been very important in the investigation of small GTP-binding protein function in cells. However, the specificity of these N17 proteins is problematic, particularly in transfection-based assays. p67phox aa 1-192 inhibited the PMA-induced membrane ruffling in Swiss 3T3 cells. This inhibition was specific, as Cdc42Hs-induced morphology, including the formation of filopodia/retraction fibers (via stimulation by bradykinin) and RhoA-associated stress fibers, was not affected by this protein fragment. Thus, aa 1-192 of p67phox can be used as a specific inhibitor of the Rac1 signaling pathway, and this will be useful to supplement the use of Rac1 T17N. The smaller p67phox fragments (170-199 and 170-238) did not have a clear-cut effect on PMA-induced ruffling, and this possibly reflects the significantly lower Rac1-binding capacity of these proteins and/or the instability of these peptides in the cellular environment.

PAK Phosphorylation-- p47phox phosphorylation has been shown to be important for oxidase activity (46). In particular, the observation that mutation of residue 379 destroys activity suggests that isolation of the kinase(s) involved would be an important step in understanding control of NADPH oxidase activity. Recent work has shown that alpha PAK1 and gamma PAK2 immunoprecipitated from fMet-Leu-Phe-activated neutrophils can phosphorylate full-length p47phox and a peptide covering aa 324-331 (31). Constitutively active recombinant PAK also phosphorylates p47phox, but not p67phox (31). p67phox is phosphorylated in vivo (29), and in this study, we found that recombinant beta PAK was able to phosphorylate p67phox in vitro. These results suggest different substrate specificity for PAK isoforms.

The major PAK phosphorylation site(s) of p67phox was localized adjacent to the Rac1-binding site and the polyproline-rich sequence. Fragment 170-238 was phosphorylated, whereas the protein fragment of p67phox aa 192-238, containing the major putative phosphorylation site(s), was not phosphorylated, suggesting that the sequence between aa 170 and 192 is required for PAK interaction (Fig. 9).

Role of Cryptic Rac1-binding and PAK Phosphorylation Sites in p67phox-- In resting neutrophils, the components of the oxidase are present as one membrane and two cytosolic complexes. Membrane-bound cytochrome b has two subunits, p22phox and gp91phox, both transmembrane proteins. p67phox and p40phox are in tight association in the cytosol, whereas p47phox associates with the p67phox-p40phox complex more loosely (20, 22). The small molecular mass GTP-binding protein Rac1 (or Rac2 in human neutrophils) associates with the Rho GDP dissociation inhibitor also in the cytosol (47). We propose that p67phox, like p47phox (24, 25), exists in a closed conformation via SH3 domain-polyproline interactions (Fig. 10). Activation of the oxidase is induced by the unfolding of p47phox and p67phox, leading to new SH3-polyproline interactions coupled with dissociation of Rac1 from its complex with the Rho GDP dissociation inhibitor (Fig. 10, step 1). These initial events (priming) could be induced, for example, by receptor activation of phospholipase A2, and this is supported by the observation that arachidonic acid disrupts an SH3 domain-polyproline interaction in p47phox (24). This does not exclude other potential mechanisms for unfolding of p67phox and p47phox, for instance, by phosphorylation. Indeed, there is evidence that phosphorylation of p47phox is required for its translocation to the membrane (27). Protein unfolding would allow interaction between the p67phox C-terminal SH3 domain and the p47phox polyproline-rich region (aa 360-380), thus exposing both SH3 domain(s) of p47phox. Translocation of the p67phox-p40phox-p47phox complex to the membrane would follow due to interaction of the exposed N-terminal SH3 domain of p47phox with the polyproline-rich region of p22phox.


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Fig. 10.   Model for assembly, activation, and turnover of the NADPH oxidase. In resting cells, p67phox and p47phox are in closed conformation via intramolecular SH3-polyproline interactions (in p67phox, a SH3 domain (aa 458-517) binds the polyproline sequence (aa 226-236); in p47phox, a SH3 domain (aa 151-214) binds the polyproline sequence (aa 361-369) (24, 25)) in the cytosol. The C terminus of p40phox binds tightly to p67phox (amino acids between the two SH3 domains). Full-length p40phox does not interact with p47phox in two-hybrid analysis (43). However, interactions between the SH3 domain of p40phox and polyproline regions of p47phox have been observed (43). Cell activation leads to the phosphorylation of p47phox (e.g. Ref. 27) and release of second messengers such as arachidonic acid. These two events cause the unfolding of p67phox and p47phox, triggering conformational changes within the labile complex (priming; step 1). Interaction of the C-terminal SH3 domain of p67phox and the C-terminal polyproline sequence of p47phox (23, 25) exposes the first and second SH3 domains of p47phox, allowing them to interact with the polyproline sequences of membrane-bound p22phox (aa 149-162) and p67phox (aa 226-236), respectively (49). Together, these changes in SH3 domain-polyproline interactions lead to formation of a more stable complex, its translocation to the membrane, and possibly the ability to interact with non-oxidase (possibly cytoskeletal) proteins. In step 2, Rac1 and PAK are recruited to the complex, leading to activation of the oxidase by a combination of conformational changes and protein phosphorylation events (of both p67phox and p47phox). The p47phox PAK phosphorylation sites have been mapped (34). The p67phox PAK phosphorylation sites are adjacent to the Rac1-binding site and polyproline sequence (see Fig. 9), suggesting a role in modification of protein-protein interactions. In step 3, a potential function for the PAK phosphorylation may be to facilitate return of the oxidase to the resting state by inducing hyperphosphorylation of p67phox/p47phox and inhibiting protein-protein interactions (48). Complete return to the resting state will involve phosphatase activity. GDI, GDP dissociation inhibitor; PKA and PKC, protein kinases A and C, respectively.

In this model, the assembly of the p67phox-p40phox-p47phox-p22phox complex at the membrane would precede its interaction with Rac1. Indeed, evidence has been obtained with tyrosine kinase inhibitors that Rac translocates to the membrane independently of the components of the NADPH oxidase (50). Possible functions for Rac1 binding could be to induce conformational changes in p67phox supporting oxidase activity or to recruit PAK kinases to the membrane-bound complex, allowing phosphorylation of p67phox/p47phox. Another possibility is that Rac influences the conformation of cytochrome b while in complex with p67phox (51). The major in vivo phosphorylation site of p67phox has been mapped to Thr-233 5, and this residue is potentially phosphorylated by PAK in vitro (Fig. 9). Since the major PAK phosphorylation sites are adjacent to the RBD and polyproline-rich motif (Fig. 9), PAK phosphorylation may influence the activity of these two domains. It should be noted that the identity of the kinases that phosphorylate p67phox and p47phox in vivo has not been established clearly. Nonetheless, it will be interesting to investigate the effect of mutations, in both p67phox and p47phox, of the PAK phosphorylation sites on in vivo NADPH oxidase activity.

In conclusion, the concept that emerges from this work is that switching between intramolecular and intermolecular SH3-polyproline interactions is one mechanism by which protein complexes can be assembled and disassembled. Work examining intramolecular SH3-polyproline interactions in p67phox and the effect of PAK phosphorylation on them is under way.

    FOOTNOTES

* This work was supported in part by the Glaxo-Singapore Research Fund and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 171-278-1552; Fax: 171-278-7045.

1 The abbreviations used are: aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; Mes, 4-morpholineethanesulfonic acid; CRIB, Cdc42Hs/Rac1 interacting domain; TPR, tetratricopeptide repeat; RBD, Rac1-binding domain; PAK, p21Cdc42Hs/Rac-activated kinase; PMA, phorbol 12-myristate 13-acetate.

2 The CRIB motif, a sequence of 16 aa, first identified in proteins such as PAK, ACK, and WASP, has been suggested as a consensus sequence for Cdc42Hs/Rac1 binding.

3 S. Govind and S. Ahmed, unpublished data.

4 K. Marler and S. Ahmed, unpublished data.

5 L. V. Forbes, O. Truong, S. J. Moss, and A. W. Segal, unpublished data.

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Abstract
Introduction
Materials & Methods
Results
Discussion
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