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J. Biol. Chem., Vol. 279, Issue 33, 34250-34255, August 13, 2004

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Nox3 Regulation by NOXO1, p47^phox, and p67^phox*

Guangjie Cheng, Darren Ritsick, and J. David Lambeth¶

From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine and the Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, Georgia 30322

Received for publication, January 20, 2004 , and in revised form, May 10, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
gp91^phox (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47^phox and p67^phox. Nox1, a homolog of gp91^phox, is regulated by NOXO1 and NOXA1, homologs of p47^phox and p67^phox, respectively. For both Nox1 and gp91^phox, an organizer protein (NOXO1 or p47^phox) cooperates with an activator protein (NOXA1 or p67^phox) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91^phox, was activated by p47^phox plus p67^phox. Whereas gp91^phox activity required the protein kinase C activator phorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated 30% by PMA. gp91^phox was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91^phox regulation required an intact activation domain in the activator protein, as neither p67^phox(V204A) nor NOXA1(V205A) were effective. In contrast, p67^phox(V204A) was effective (along with p47^phox) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67^phox. Nox3 activity was regulated by PMA only when p47^phox but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47^phox but not in NOXO1. Deletion of the autoinhibitory region from p47^phox rendered this subunit highly active in the absence of PMA toward both gp91^phox and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Nox family of NAD(P)H oxidases has recently been described (1–10). These enzymes are structural homologs of gp91^phox (a.k.a. Nox2), the catalytic subunit of the phagocyte NADPH oxidase, and are distributed in a variety of non-phagocytic tissues such as colon, kidney, vascular smooth muscle, and brain. Reactive oxygen species generated by these novel enzymes are proposed to function in signal transduction related to cell growth and cancer (1, 11–17), angiogenesis (18), and in innate immunity (15), e.g. in barrier cells such as epithelium. In addition, reactive oxygen species from these enzymes are proposed to be causally linked to pathological states including atherosclerosis (19), cancer (11, 15, 20, 21), and diabetes (22).

The human Nox enzymes are encoded by at least 7 genes. NOX1, NOX3, and NOX4 encode proteins that are similar in size and domain structure to gp91^phox. These consist of a C-terminal flavoprotein domain containing both an FAD-binding site and an NADPH-binding site, and an N-terminal membrane-associated hydrophobic domain consisting of 6 transmembrane helices that provide binding sites for 2 hemes (3). Nox5 consists of these same domains along with an N-terminal calcium-binding domain (9); this enzyme is dormant when expressed in cells, but is activated by calcium. Duox1 and Duox2 build upon the Nox5 structure with an N-terminal peroxidase homology region (3, 23) and are also thought to be regulated by calcium.

The activation of gp91^phox by regulatory subunits has been extensively investigated and is reviewed in Refs. 24 and 25. gp91^phox is located in membranes of phagocytes in association with p22^phox, together forming flavocytochrome b₅₅₈. p22^phox functions as an adapter or "organizer" protein that provides a binding site for regulatory subunits. The latter assemble with the membrane proteins upon cell activation, e.g. by phorbol 11-myristate 12-acetate (PMA).¹ p47^phox and p67^phox are in the cytosol of unstimulated cells and, upon cell activation by microbial products or immune mediators, translocate to the membrane and assemble with the flavocytochrome. Another cytosolic component, p40^phox, is associated with p67^phox and also translocates upon cell activation; this protein, while not essential, stimulates activity 2-fold or more (26). The small GTPase Rac, upon cell stimulation, becomes GTP-loaded and also translocates to the membrane. p67^phox, the "activator" subunit, contains an activation domain that regulates the rate-limiting hydride transfer from NADPH to FAD in flavocytochrome b₅₅₈, thereby regulating overall activity (27, 28). An essential function of both p47^phox and Rac is to bind and orient the p67^phox activation domain in such a way as to stimulate electron flux from NADPH through the enzyme to oxygen to form superoxide.

Recently, homologs of p47^phox and p67^phox were reported and shown to be required for activation of Nox1 (29, 30, 31, 34). The p47^phox homolog is referred to as NOXO1 (NOX Organizing protein 1), whereas the p67^phox homolog is referred to as NOXA1 (NOX Activating protein 1). With some exceptions, NOXO1 and NOXA1 show nearly the same domain organization as p47^phox and p67^phox, respectively. Functional (32) and structural (33) studies have shown that p47^phox contains adjacent SH3 domains that cooperate to bind to a proline-rich region at the C terminus of p22^phox. In resting cells, this bis-SH3 domain is fully occupied by an autoinhibitory region (AIR), preventing its interaction with p22^phox. Phosphorylation of residues in this region by protein kinase C and Akt release binding and allows p47^phox to translocate from the cytosol to the membrane, which also involves membrane association through a lipid-binding PX domain. The single largest difference between p47^phox and NOXO1 is that NOXO1 lacks the AIR domain. Consistent with this finding, NOXO1 is fully associated with membranes without PMA activation (34).

Nox3 is expressed in fetal tissues, particularly in kidney, and in several cell lines (2), and was recently shown to be expressed in the inner ear of mouse where it participates in otoconia morphogenesis (35). Otoconia are tiny mineralized structures that are associated with perception of motion and gravity, and mouse mutations in Nox3 result in a head-tilt phenotype in which these perceptions are defective because of an absence of otoconia. The activity and regulation of Nox3 have not been reported. The present study was undertaken to characterize the requirements of Nox3 for regulatory subunits including p47^phox, p67^phox, NOXO1, and NOXA1. Nox3 shows unusual flexibility in its ability to be activated by both phox and NOX regulatory proteins. Moreover, Nox3 is unique in its ability to be activated by NOXO1 alone in the absence of activator subunits. This finding has important implications, discussed herein, regarding the mechanism of regulation of Nox family members by regulatory subunits.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cells and Reagents—HEK293H (Invitrogen) and COS7 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Luminol, horseradish peroxidase, and PMA were purchased from Sigma, Hanks' solution containing 1.26 mM calcium and 0.91 mM magnesium from Invitrogen, FuGENE 6 from Roche Diagnostics, DNA polymerase from Clontech, restriction endonucleases and ligase were from New England Biolabs. Anti-NOXA1 antibody was an IgY fraction raised from chicken against keyhole limpet hemocyanin-conjugated peptide NOXA1-(458–475) and purified from egg yolk by Lampire Biological Laboratory, Pipersiville, PA. The peptide was synthesized and purified by reverse phase high performance liquid chromatograph by Emory's Microchemistry Core Facility. Chicken anti-NOXO1 antibody and rabbit anti-p47^phox antibody were previously described (34, 36). Anti-p67^phox antibody was from Santa Cruz. Anti-tubulin I monoclonal antibody (clone SAP.4G5) and secondary anti-mouse, anti-chicken, and anti-rabbit immunoglobins were purchased from Sigma. The chemiluminescence kit was from Pierce.

cDNAs of Nox Enzymes and Regulatory Proteins—The cDNA clones encoding human Nox1, Nox2 (gp91^phox), Nox3, NOXO1, NOXA1, p47^phox, and p67^phox were described previously (2, 34). p67^phox(V204A) (27) was subcloned into pCMV-Tag4A. V12RAC1 in pcDNA3.1 was obtained from Gluthrie cDNA Resource Center (Sayre, PA).

Generation of Mutants of Nox3 and Regulatory Proteins—NOXA1(V205A) in pCMV-Sport6 was constructed by amplifying NOXA1 in pCMV-Sport6 using a primer pair of SP6 and primer 1 (5'-AGGCCACGGCCTTGGCCTTGCCCAGGAAATCC-3') and primer T7 and primer 2 (5'-AGGCCACGGCCTTGGCCTCTGCCATCCCCGAC-3'). SP6 and T7 are located at the 5'- and 3'-ends of the multiple cloning site of pCMV-Sport6, respectively. Primers 1 and 2 contain the BstXI site, which is underlined. The bases in italics encode changes to mutate valine 205 to alanine in NOXA1. The first PCR product was doubly digested using XhoI and BstXI, whereas the second was digested with BstXI and HindIII. The digested fragments were inserted into XhoI and HindIII sites of pCMV-Sport6.

p47^phox(298–340) in pCMV-Tag4A was constructed by amplifying p47^phox in pCMV-Tag4A using a primer set of T3 and primer 3 (5'-CGGGGGCCCCCGCTTGATCTGGCGTTGGGCCTG-3') as well as a primer set of T7 and primer 4 (5'-GCGGGGCCCGGACCGCAGAGCCCCGGGAGCCCG-3'). T3 and T7 are located at 5'- and 3'-ends of the multiple cloning site of pCMV-Tag4A, respectively. Primers 3 and 4 contain the ApaI site, which is underlined. The first PCR product contained the DNA fragment encoding p47^phox-(1–297) and was digested with EcoRI and ApaI, whereas the second contained the DNA fragment encoding p47^phox-(341–371) and was digested with ApaI and HindIII. The digested fragments were inserted into EcoRI and HindIII sites of pCMV-Tag4A. All cDNA clones were sequenced in both strands using an ABI model 377 sequencer.

Transient Transfections—HEK293H cells were placed in 6-well plates and grown overnight to reach to 40–50% confluence. Plasmids (pcDNA3.1 or vector encoding Nox1, Nox2, Nox3, NOXO1, NOXA1, NOXA1(V205A), p47^phox, p47^phox(298–340), p67^phox, p67^phox(V204A), and/or V12Rac1 alone or in combinations) were transfected in HEK293H cells using FuGENE 6 according to manufacturers' instructions. After 48 h, cells were removed from the well and washed twice with cold Hanks' solution containing calcium and magnesium. The cells then were pelleted at 600 x g for 5 min and re-suspended in Hanks' solution.

Measurement of Reactive Oxygen Species—Reactive oxygen was measured using luminol luminescence according to Ref. 37 as modified in Ref. 34. For each well in a 96-well plate, 2.5 x 10⁵ cells in Hanks' buffer with calcium/magnesium were mixed with 200 µM luminol and 0.32 units of horseradish peroxidase in a 200 µl total volume. PMA was added and incubated at 37 °C for 10 min. Luminescence was quantified using FluoStar^TM luminometor (BMG Labtech, Durham, NC) and luminescence was recorded every 1–4 min for a total of 1 to 2 h. The peak luminescence was reached at about 20 min, and this value was reported. Use of values at other time points or integrated areas gave qualitatively identical results. Specificity for H₂O₂ was demonstrated by elimination of signal when catalase was included. In addition, the Diogenes assay (National Diagnostics), which measures superoxide, was used for some of these experiments and used the protocol provided by the company; the assay typically gave 10% of the signal compared with luminol, but gave qualitatively similar results. Activity was too low to be reliably measured by cytochrome c reduction, and was previously estimated to be 5% or less of that in activated neutrophils (34).

Western Blot Analysis—Transfected or control HEK293H cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA) with protease inhibitor mixture (Sigma). Lysate (60 µg of protein) was resolved by 12% SDS-PAGE and transfered to polyvinylidene difluoride membrane using the semi-dry electrophoretic transfer cell (Bio-Rad) at 15 V for 1 h. The proteins were detected using standard Western blotting and visualized by chemiluminescence. Blots were stripped and re-probed as necessary.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Activation of Nox3 by p47^phox and p67^phox—As reported previously, activation of gp91^phox in cells required both p47^phox and p67^phox. Fig. 1 demonstrates that this activation is absolutely dependent upon the presence of the protein kinase C activator PMA. Like gp91^phox, Nox3 was strongly activated by the combination of p47^phox and p67^phox (Fig. 1), but activity was less tightly regulated. For Nox3, whereas optimal activity required both p47^phox and p67^phox, p67^phox by itself produced moderate activation of Nox3 in the absence of p47^phox. Unlike gp91^phox, significant PMA-independent Nox3 activity was seen with p47^phox and p67^phox, although PMA produced some further activation (about 30–40%) when p47^phox was present. Reactive oxygen species generation by gp91^phox but not by Nox3 required co-expression of V12Rac1, because in the absence of V12Rac we were unable to detect any activity.² V12Rac1 is a constitutively activated mutant form of Rac1 that lacks GTPase activity, thereby trapping Rac in its GTP-bound form. These results suggest either that Nox3 does not require Rac1 for activation, or that endogenous levels of Rac1-GTP are sufficient for Nox3 activity. These possibilities as well as the role of Rac1 in Nox1 activation are under investigation in our laboratory.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Activation of Nox3 and gp91phox by p47phox and p67phox. gp91phox (left panel) or Nox3 (right panel) were co-expressed in HEK293 cells along with p47phox, p67phox, and/or p67phox V204A as indicated and as described under "Experimental Procedures," either in the absence (open bars) or presence (filled bars) of 200 nM PMA. Luminol luminescence was measured as under "Experimental Procedures." In all experiments using gp91phox, V12Rac was co-expressed along with other components. Error bars show the standard error of four incubations representing four independent transfections. Western blots inferior to each bar graph indicate expression in one of the incubations of p47phox, p67phox, and tubulin as a control.

gp91^phox required an intact activation domain, because p67^phox mutated in this domain, p67^phox(V204A), was inactive (Fig. 1). This site was previously shown to be essential for activation of gp91^phox (27, 28, 38). For Nox3, p67^phox(V204A) was less active than wild type p67^phox in the absence of PMA, but the activation domain mutant was fully active when PMA was used.

Activation of Nox3 by NOXO1 and NOXA1—Nox1-dependent activity is highly stimulated by the combination of NOXO1 plus NOXA1 (Refs. 29–31 and 34; and see below). To investigate the regulation of Nox3 by NOXO1 and NOXA1, co-transfection experiments were carried out as in Fig. 2. Unexpectedly, high activity was seen with the organizing protein NOXO1 alone in the absence of NOXA1. A small stimulation of activity was seen with NOXA1 alone, and this was absent when NOXA1(V205A) was used. This mutation is analogous to the V204A mutation in the activation domain of p67^phox. When NOXA1 was included along with NOXO1, only a very small further increase in activity was seen. In addition, PMA had little or no effect on activity in the presence of NOXO1, whether or not NOXA1 was present. This is consistent with the absence of regulatory phosphorylation sites on NOXO1, and on the recent finding (34) that NOXO1 (in contrast to p47^phox) is pre-assembled at the plasma membrane along with its Nox target in the absence of cell activation.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Activation of Nox3 and gp91phox by NOXO1 and NOXA1. gp91phox (left panel) or Nox3 (right panel) were co-expressed in HEK293 cells along with NOXO1, NOXA1, or NOXA1(V205A) as in Fig. 1, in the absence (open bars) or presence (filled bars) of PMA. Luminol luminescence was measured as under "Experimental Procedures." Error bars show the standard error of four incubations representing four independent transfections. Western blots inferior to each bar graph indicate expression in one of the incubations of NOXO1, NOXA1, and tubulin.

View larger version (32K): [in this window] [in a new window]   FIG. 3. Activation of Nox1 and gp91phox by heterologous combinations of phox and NOX regulatory subunits. Nox1 (left panel) or gp91phox (right panel) were co-expressed in HEK293 cells along with p47phox, p67phox, NOXO1, and NOXA1 as described in the legend to Fig. 1, either in the absence (open bars) or presence (filled bars) of 200 nM PMA. Luminol luminescence was measured as under "Experimental Procedures." Error bars show the standard error of four incubations representing four independent transfections. Western blots inferior to each bar graph indicate expression in one of the incubations of NOXO1, NOXA1, p47phox, p67phox, and tubulin.

View larger version (48K): [in this window] [in a new window]   FIG. 4. Activation of Nox3 by heterologous combinations of phox and NOX regulatory subunits. Nox3 was co-expressed in HEK293 cells along with the indicated combinations of p47phox, p67phox, NOXO1, and NOXA1 either in the absence (open bars) or presence (filled bars) of 200 nM PMA. Not shown here, the combination of NOXO1 plus NOXA1 showed nearly the same activity as NOXO1 alone (see Fig. 2). Luminol luminescence was measured as under "Experimental Procedures." Error bars show the range of two determinations, and the experiment is representative of four independent transfection experiments. The Western blot inferior to the bar graph indicates expression in one of the incubations of NOXO1, NOXA1, p47phox, p67phox, and tubulin.

As above, Nox3 was activated nearly maximally by NOXO1 alone, and NOXA1 (Fig. 2) or p67^phox (Fig. 4) produced only a modest further activation when expressed along with NOXO1. The heterologous combination of p47^phox plus NOXA1 activated Nox3 in a PMA-dependent manner. In contrast to gp91^phox, Nox3 was regulated in a PMA-dependent manner only when p47^phox was present, consistent with p47^phox as a primary target of regulatory phosphorylation in the Nox3 system.

Role of the Autoinhibitory Region of p47^phox in Nox3 Activation—The domain structure of p47^phox and NOXO1 are diagrammed in Fig. 5A. As above, the two proteins differ in the presence in p47^phox of an AIR that is the target of multiple phosphorylations by protein kinase C and other kinases. To investigate the role of the AIR in p47^phox in regulating Nox3, we made a deletion mutant of p47^phox that lacks AIR, making it identical in domain structure to NOXO1 (Fig. 5A). p47^phox(AIR) used in place of p47^phox to support gp91^phox-dependent activity showed high basal activity in the absence of PMA (Fig. 5B), consistent with AIR as a target for regulation by protein kinase C. Moreover, PMA stimulated activity was 2-fold higher using p47^phox(AIR) compared with wild type p47^phox (Fig. 5B), consistent with a regulatory phosphorylation distinct from those in the AIR.

View larger version (36K): [in this window] [in a new window]   FIG. 5. Negative regulation of gp91phox by the AIR of p47phox. A, the domain structure of p47phox and NOXO1 is diagrammed. Both proteins contain an N-terminal PX domain, a central tandem SH3 domain, and a C-terminal proline-rich region (PRR). In addition, p47phox contains an AIR that is not present in NOXO1 and which contains phosphorylation sites (Ser303, Ser304, and Ser328) that have been implicated in its regulation by protein kinase C and Akt. Also diagrammed is p47phox(298–340) in which the AIR has been deleted. B, gp91phox was co-expressed in HEK293 cells along with p47phox, p47phox(298–340) (indicated here as ), or p67phox as indicated, either in the absence (open bars) or presence (filled bars) of 200 nM PMA. Luminol luminescence was measured as under "Experimental Procedures." Error bars show the standard error of four incubations representing four independent transfections. C, the Western blot from the samples of one of the incubations indicates expression of NOXO1, NOXA1, p47phox, p67phox, and tubulin.

View larger version (33K): [in this window] [in a new window]   FIG. 6. Negative regulation of Nox3 by the AIR of p47phox. Nox3 was co-expressed in HEK293 cells along with p47phox, p47phox(298–340) (indicated here as ), p67phox, or NOXA1 as indicated, either in the absence (open bars) or presence (filled bars) of 200 nM PMA. Luminol luminescence was measured as under "Experimental Procedures." Error bars show the range of two determinations, and the experiment is representative of four independent transfection experiments. Western blots inferior to the bar graph indicate expression in one of the incubations of NOXA1, p47phox, p67phox, and tubulin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When p47^phox and p67^phox are used, Nox3 conforms to a similar activation paradigm, requiring both an adapter and an activator protein for optimal activity. Under these conditions, regulation is less stringent than for gp91^phox, because significant activity is also seen with p67^phox alone (or NOXA1 alone). This is in contrast to the very tight regulation of gp91^phox in cells, which as shown in Fig. 1, absolutely requires both p47^phox and p67^phox.

However, regulation of Nox3 fails to conform to the adaptor protein/activator protein paradigm in several respects. First, with p47^phox, its regulation by p67^phox does not require an intact activation domain, indicating that other regions of p67^phox are important for regulating Nox3 activity. Second, Nox3 can be maximally activated by NOXO1 alone, and does not require NOXA1. As far as we can determine, there is no sequence in either NOXO1 or Nox3 that is homologous to the activation domain of p67^phox/NOXA1, suggesting that the presence of a p67^phox-like activation domain is not essential for regulation of Nox proteins. Whereas it is possible that Nox3 might be regulated in a fundamentally different manner compared with Nox1 and gp91^phox, it is telling that in a recent study, gp91^phox was activated by MRP8/MRP14 (42), which do not possess a p67^phox-like activation domain. Thus, it seems more likely that a variety of protein-protein interactions can stabilize an activated conformation in Nox enzymes. It seems likely that NOXO1 functions by inducing an activated conformation in Nox3, and that there is no strict requirement for a particular activating signature sequence such as the activation domain in p67^phox. Whereas it is theoretically possible our model cells contain an unknown homolog of p67^phox/NOXA1, we consider this possibility unlikely, because extensive searching of the now complete genomic data base, as well as searching of EST data bases has failed to uncover any additional homologs.²

The present studies indicate a surprising degree of flexibility of regulation of Nox family proteins, particularly Nox3, with regard to utilizing diverse combinations of regulatory subunits, including heterologous combinations of ^phox and non-^phox subunits. The regulatory adaptability may be biologically important, e.g. in providing redundant backup in some tissues for regulatory subunit mutations or genetic deletions. Phagocytes express only the ^phox components, and mutation of any one of these results in chronic granulomatous disease. Functional redundancy of regulatory subunits in other tissues may suppress the expression of disease phenotypes in these sites when ^phox subunits are mutated. These studies also emphasize caution in interpreting phenotypes in mouse knockout studies, for example, p47^{phox(–/–)} mice, wherein it cannot be rigorously established that a ^phox regulatory subunit partners exclusively with gp91^phox.

Finally, the present studies provide evidence that the PMA regulation of Nox3 by p47^phox plus an activator protein (p67^phox or NOXA1) involves the autoinhibitory domain of p47^phox. Deletion of AIR results in a highly active Nox3 system when either p67^phox or NOXA1 is present, and activity is no longer further stimulated by PMA. These data are consistent with functional and structural studies indicating that that AIR combines in resting cells with the tandem SH3 domain of p47^phox to prevent its interaction with p22^phox (32, 33, 43). Phosphorylation of specific serines in the AIR disrupts this interaction, and allows binding of p47^phox to p22^phox and targeting to the membrane. When the autoinhibitory domain is eliminated, the p47^phox(AIR) behaves like NOXO1 with respect to activity in the absence of PMA. However, activation of Nox3 still requires an activator protein, and elimination of AIR fails to provide p47^phox with the same strong activator properties as NOXO1. Thus, unknown features of NOXO1 permit Nox3 to function as a strong activator in the absence of a protein with an activation domain. This is consistent with the idea that regulation of Nox enzymes involves stabilization of an active conformation in the catalytic subunit by interacting regulatory proteins, and that such regulation is not unique to activator proteins containing an activation domain.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant CA84138. The costs of publication of this article were defrayed in part by the payment of page charges. This 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. E-mail: dlambe{at}emory.edu.

¹ The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; SH3, Src homology domain 3; AIR, autoinhibitory region.

² G. Cheng, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Guthrie cDNA Resource Center for providing plasmid expressing V12Rac1. Thanks also to Xiwen (Wendy) Yu for technical help with Western blots.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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