©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping the Domains of Interaction of p40 with Both p47 and p67 of the Neutrophil Oxidase Complex Using the Two-hybrid System (*)

(Received for publication, December 21, 1994; and in revised form, January 20, 1995)

Alexandra Fuchs (§) Marie-Claire Dagher Pierre V. Vignais

From the Commissariat à l'Energie Atomique/Laboratoire de Biochimie (URA 1130/CNRS), Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, 17 rue des Martyrs, 38054 Grenoble, Cedex 9, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67, p47 and p40 as well as a small G protein Rac (for review, see (1, 2, 3) ). A truncated form of the p40 cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47 as target. This truncated form of p40 consisting of the Src Homology 3 (SH3) domain to the 3` stop codon was also shown to interact with p67 in the same system. A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47 or p67. Results show that the SH3 domain of p40 is sufficient for interaction with p47, whereas the C terminus of p40 but not its SH3 domain is involved in the interaction with p67.


INTRODUCTION

The NADPH oxidase of phagocytic cells is an enzymatic complex producing superoxide anions, necessary for microbicidal activity. The activated complex is a multimeric membrane bound enzyme consisting of a heterodimeric b-type flavocytochrome and a number of cytosolic components which translocate to the membrane on the onset of activation (for review, see (1, 2, 3) ). Among these cytosolic factors are p47 and p67, both containing Src homology 3 (SH3) (^1)domains and polyproline motifs. A Rac monomeric G protein is also involved in oxidase activation and seems to exert its role at the membrane level (4, 5) . Recently, a new cytosolic component, p40, also containing a SH3 domain has been identified(6, 7, 8) . This component coimmunoprecipitated (6, 8) and copurified (7) with p47 and p67, and a primary association with p67 was postulated(6) . Although not required for activity of the oxidase in a cell-free system consisting of the five other purified proteins(9) , p40 might have a specific role in the regulation of the activation complex. Transient complexes occurring between p47, p67, and other proteins have been recently described in the cytosol of non-activated neutrophils(6, 7, 8, 10, 11, 12) . Interactions between various SH3 domains and polyproline motifs were considered as a plausible molecular basis to the formation of these complexes(13, 14, 15) . This possibility was tested by in vitro binding assays in which the cytosolic factors were brought as glutathione S-transferase fusion proteins(16, 17, 18, 19) . These in vitro techniques show their limits when dynamic or transient interactions take place. In this report, we describe interactions between cytosolic proteins through the use of a genetic method: the two-hybrid system(20, 21) , which monitors interactions in conditions close to those found in native cells. A library screen was carried out using this method, with p47 as the target protein versus proteins of lymphocyte B cells immortalized by the Epstein-Barr virus (LB-EBV), which possess a functional NADPH oxidase(22) . Among the clones interacting specifically with p47, a truncated form of p40 was identified and shown to interact with p67 as well in the two-hybrid system. Specific domains of interaction with either protein were further investigated.


EXPERIMENTAL PROCEDURES

Constructions

Full-length clones of p47 and p67 isolated as described (4) were inserted into the pBTM116 vector (23) in frame with the LexA DNA binding protein yielding the Lex47 and Lex67 constructs, respectively. The LexA-Rac2 construct was a kind gift of Dr. Linda Van Aelst. (^2)

Screen of a B Lymphocyte Library Using the Two-hybrid System

The two-hybrid system was developed originally by Fields and Song(20) . In the system used here, one hybrid is a fusion between the LexA-DNA binding protein and one protein of interest, and the other hybrid is a fusion between the activation domain of Gal4 and a second protein of interest. Transactivation of the HIS3 and lacZ reporter genes occurs only if both proteins of interest interact when coexpressed in the appropriate Saccharomyces cerevisiae strain. Interaction can therefore be monitored by beta-galactosidase activity or by prototrophy for histidine. The two-hybrid system used was obtained through Clontech (Yeast Matchmaker). The L40 yeast reporter strain used (23) has the genotype: MATa, trp1, leu2, his3, LYS2::lexA-HIS3, URA3::lexA-LacZ. Yeast were grown in synthetic minimal medium consisting of 2% glucose (w/v), 0.67% nitrogen base without amino acids (w/v) (Difco) with the appropriate supplements: amino acids, uracil, and adenine of cell culture grade (Sigma). To identify proteins interacting with p47, the S. cerevisiae L40 reporter strain containing Lex47 was transformed with a LB-EBV library of hybrid proteins constructed in the pACT vector (Clontech). Approximately 510^5 transformants were plated onto selective medium. His colonies appeared from 3 to 5 days after plating and were tested for beta-galactosidase activity by the color filter assay using the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as described(24) . Of the 20 His clones, all were LacZ. A fraction of the yeast plasmid minipreparation prepared from each His/LacZ clone was used to electroporate HB101 Escherichia coli. The library plasmid was rescued by complementing the leu phenotype of HB101 on minimal medium. This plasmid was transformed again into either the L40 strain carrying Lex47 to confirm interaction or a L40 strain carrying an unrelated hybrid protein, LexLamin to test for false positive clones. All 20 His/LacZ clones were specific for Lex47. Clones were sequenced using the U. S. Biochemical Corp. sequencing kit with Sequenase version 2.0, and a search for homology with known sequences from GenBank was carried out with the DNAstar software.

Construction of a Library of Fragments of p40

The truncated cDNA of p40 (base pair (bp) 565 to the poly(A) as numbered in the GenBank data base) isolated from the above screen was amplified by PCR using primers of the pACT vector on both sides of the cloning site (GAAGATACCCCACCAAACCCA and CAGTATCTACGATTCATAGATC, respectively). The PCR product (approximately 1000 bp in length) was purified by low melting agarose gel electrophoresis and extracted from the agar by phenol extraction. Partial digestion by the Sau3AI enzyme (Boehringer Mannheim) was carried out for 30 min at 37 °C using a ratio of 0.1 unit of enzyme/µg of DNA. Arrows in Fig. 1A show the Sau3AI restriction sites used to construct the library. The fragments were then ligated in both the pGAD424 and pGADGH vectors (25) digested by BamHI (Boehringer Mannheim), thus providing both reading frames necessary for all p40/Sau3A digests as fusion proteins with the activation domain of Gal4. The library was amplified on plates, and DNA was prepared by classical methods. The library was transformed into the L40 strain carrying either Lex47 or Lex67, and clones with the His/LacZ phenotype were sequenced. A full-length clone of p40 was obtained by screening the LB-EBV library with a digoxygenin-labeled probe (labeling kit from Boehringer Mannheim) consisting of the 600 last base pairs of p40 and by PCR analysis using primers from the 5`-non-coding sequence. The correct frame was determined by sequencing.


Figure 1: Mapping the interacting domains of p40 with p47 and p67. In A, under the full-length clone of p40 (insert 1), are given the domains required for interaction with either p47 or p67 (inserts 2-5) isolated from either screen (see ``Experimental Procedures''). In B is shown a photograph of the corresponding X-gal filter assay (24) carried out on the cotransformants with the corresponding insert and either Lex47 or Lex67. Darkpatches are relevant for positive protein interaction.




RESULTS AND DISCUSSION

In a preliminary experiment, p47 expressed as a fusion protein with LexA (Lex47) was assayed in the two-hybrid system against a number of hybrid proteins to test for background activity. Thus, we detected no transactivating activity of Lex47 alone or when coexpressed with the Gal4 activation domain alone or as a hybrid with an unrelated protein. To identify partners of p47, we therefore transformed yeast expressing Lex47 with a cDNA library of LB-EBV fused to the activation domain of Gal4 and tested the double transformants for beta-galactosidase activity and prototrophy for histidine. Among the 20 positive clones obtained by the screen, one copy of a truncated form of the p40 cDNA was identified by sequencing and comparison with the known sequence in the GenBank data base (DNAstar software). The sequence corresponded to base +565 to the stop codon, covering the putative SH3 domain of the protein and the entire C terminus. Two other unrelated clones were obtained, and a sequence homology search revealed no significant homology to any published sequence. Other clones were highly homologous to mRNA splicing factors and were discarded as false positives. All positive clones from this screen were tested against p67 and Rac2 expressed as fusion proteins with LexA (Lex67 and LexRac2, respectively). The truncated form of p40 showed an interaction with Lex67 and thus revealed at least two partners for p40, namely p67 and p47. No positive clone showed an interaction with LexRac2.

To identify the specific domains of interaction of p40 with either p47 and p67, a library of smaller fragments of p40 was constructed (see ``Experimental Procedures'' and Fig. 1). The library was transfected into the L40 yeast strain carrying either Lex47 or Lex67. Over 800 cotransformants were screened for His and LacZ phenotypes, and 13 and 59 positive clones were obtained for the Lex47 and Lex67 screen, respectively. Inserts were sequenced and results analyzed as follows (Fig. 1). An insert covering exclusively the SH3 domain (bp 565 to 910) of p40 was sufficient for interaction with p47, whereas interaction with p67 required the C-terminal extremity (bp 910 to the stop codon) and not the SH3 domain of p40. Clones containing the SH3 domain and only part of the C terminus of p40 showed no interaction with p67. A full-length clone of p40 obtained from the LB-EBV library showed interaction with p47 as well as with p67.

Considering these results and those published by other groups(16, 17, 18) , we propose the two following models (Fig. 2). In the first model, p47 in its resting state adopts a closed conformation as suggested (17, 18) by binding one of its SH3 domain to the polyproline motif in its C terminus. p67 binds to the C terminus of p40. Upon activation, p47 changes conformation after phosphorylation and opens the polyproline/SH3 interaction. The polyproline region binds to p40 and brings p47 and p67 into contact: p67 binds to one of the SH3 domains of p47 and the other SH3 domain of p47 binds to the polyproline motif of p22(17) .


Figure 2: Two proposed models of change of conformation and interaction between the three cytosolic factors p47, p67, and p40. Both models are discussed in the text. The arrows show the zone of p47, which undergoes phosphorylation upon activation. PolyP stands for polyproline motif.



In the second model, p47, p67, and p40 form, in the cytosol of resting neutrophils, a complex that takes a new conformation upon activation. This model is in better accord than the previous one with the various reports of the pre-existence in the cytosol of resting neutrophils of a complex containing p47 and p67 as well as other proteins(6, 7, 8, 10, 11, 12) and with our present results with the two-hybrid system. We therefore propose that in the resting state the SH3 domain of p40 interacts with the polyproline motif of p47 and that the C-terminal tail of p40 binds to p67. Upon activation, p47 is phosphorylated on a cluster of serine residues in the C terminus of the molecule(26) , and this opens the p47/p40 interaction and frees the polyproline motif of p47. This change of conformation is postulated on the experimental basis that the SH3 domains of p47 are inaccessible to a monoclonal antibody in the cytosol of resting neutrophils and become accessible on the addition of an amphiphilic agent, such as arachidonic acid or SDS(17) . Arachidonic acid could mimic the effect of phosphorylation that occurs in in vivo activation. The polyproline motif of p47 would then be accessible to the C-terminal SH3 domain of p67(16, 18) . This new interaction changes the overall structure of the complex and makes it able to recognize the flavocytochrome b through the polyproline motif of p22 and one of the SH3 domains of p47, the other being bound to p67(17) . The role of Rac is yet unknown. We have shown that Rac exerts its effect at the membrane level(4) , and Diekmann's group (19) reports that Rac2 in a GTP form binds to the N terminus of p67. Rac may therefore also play a role in the positioning of the complex onto the flavocytochrome b in the activated state. These models do not exclude the possibility that the cytosolic factors may also interact with gp91 by a mechanism other than the SH3/polyproline motif interaction.

The models described here propose mechanisms for the in vivo activation of the oxidase but probably not for the activation occurring in the cell-free system, which requires the addition of an amphiphilic reagent such as arachidonic acid or sodium dodecyl sulfate, as well as p47 and p67 but not p40, to the membrane fraction. A plausible explanation is that p40 probably plays the role of a chaperone for the p47-p67 cytosolic complex but may not be required in the active conformation of the oxidase. In this context, it may be recalled that dramatic differences in the p67 structural requirements for full oxidase activation in the cell-free system versus the intact cell assays were shown in de Mendez's recent report(27) . In vitro, the C terminus region comprising both SH3 domains of p67 was not necessary for full oxidase activation, whereas in vivo the full-length protein was required. The role of the amphiphilic reagents used to promote activation is still unclear, but addition of these reagents could bypass the specific interactions needed in vivo by artificially directing the proteins to the membrane vesicules. Under these in vitro conditions p40 is probably unnecessary or redundant.


FOOTNOTES

*
This work was supported in part by grants from the Centre National de la Recherche Scientifique, from the Association pour la Recherche sur le Cancer, and from the Université Joseph Fourier-Faculté de Médecine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33 76 88 34 76; Fax: 33 76 88 51 85.

(^1)
The abbreviations used are: SH3, Src homology domain 3; LB-EBV, B lymphocyte cells immortalized by the Epstein-Barr virus; bp, base pair(s); PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.

(^2)
L. Van Aelst, unpublished results.


ACKNOWLEDGEMENTS

We thank Linda Van Aelst for the gift of the LexRac2 plasmid.^2 We wish to thank Drs. Richard Benarous, Jacques Camonis, Eric Chang, Stanley Fields, Francisco Romero, Anne Vojtek, and Michael White for all strains and cloning plasmids, as well as for unpublished protocols.


REFERENCES

  1. Morel, F., Doussière, J., and Vignais, P. V. (1991) Eur. J. Biochem. 201, 523-546 [Abstract]
  2. Babior, B. (1992) Adv. Enzymol. Relat. Areas Mol. Biol. 65, 49-95 [Medline] [Order article via Infotrieve]
  3. Thrasher, A. J., Keep, N. H., Wientjes, F., and Segal, A. W. (1994) Biochim. Biophys. Acta 1227, 1-24 [Medline] [Order article via Infotrieve]
  4. Fuchs, A., Dagher, M. C., Jouan, A., and Vignais, P. V. (1994) Eur. J. Biochem. 226, 587-595 [Abstract]
  5. Uhlinger, D. J., Tyagi, S. R., Inge, K. L., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 8624-8631 [Abstract/Free Full Text]
  6. Wientjes, F. B., Hsuan, J. J., Totty, N. F., and Segal, A. W. (1993) Biochem. J. 296, 557-561 [Medline] [Order article via Infotrieve]
  7. Someya, A., Nagaoka, I., and Yamashita, T. (1993) FEBS Lett. 330, 215-218 [CrossRef][Medline] [Order article via Infotrieve]
  8. Tsunawaki, S., Mizunari, H., Nagata, M., Tatsuzawa, O., and Kuratsuji, T. (1994) Biochem. Biophys. Res. Commun. 199, 1378-1387 [CrossRef][Medline] [Order article via Infotrieve]
  9. Abo, A., Boyhan, A., West, I., Thrasher, A. J., and Segal, A. W. (1992) J. Biol. Chem. 267, 16767-16770 [Abstract/Free Full Text]
  10. Park, J. W., Ma, M., Ruedi, J. M., Smith, R. M., and Babior, B. M. (1992) J. Biol. Chem. 267, 17327-17332 [Abstract/Free Full Text]
  11. Iyer, S. S., Pearson, D. W., Nauseef, W. M., and Clark, R. A. (1994) J. Biol. Chem. 269, 22405-22411 [Abstract/Free Full Text]
  12. Jouan, A., Dagher, M. C., Fuchs, A., Foucaud-Gamen, J., and Vignais, P. V. (1993) Biochem. Biophys. Res. Commun. 197, 1296-1302 [CrossRef][Medline] [Order article via Infotrieve]
  13. Ren, R., Mayer, B. J., Ciccheti, P., and Baltimore, D. (1993) Science 259, 1157-1161 [Medline] [Order article via Infotrieve]
  14. Lim, W. A., Richards, F. M., and Fox, R. O. (1994) Nature 372, 375-379 [CrossRef][Medline] [Order article via Infotrieve]
  15. Feng, S., Chep, J. K., Yu, H., Simon, J. A., and Schneiber, S. L. (1994) Science 266, 1241-1247 [Medline] [Order article via Infotrieve]
  16. Finan, P., Shimizu, Y., Gout, I., Hsuan, J., Truong, O., Butcher, C., Bennett, P., Waterfield, M. D., and Kellie, S. (1994) J. Biol. Chem. 269, 13752-13755 [Abstract/Free Full Text]
  17. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y., Ohno, M., Minakami, S., and Takeshige, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5345-5349 [Abstract]
  18. Leto, T. L., Adams, A. G., and de Mendez, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10650-10654 [Abstract/Free Full Text]
  19. Diekmann, D., Abo, A., Johnston, C., Segal, A. W., and Hall, A. (1994) Science 265, 531-533 [Medline] [Order article via Infotrieve]
  20. Fields, S., and Song, O. (1989) Nature 340, 245-246 [CrossRef][Medline] [Order article via Infotrieve]
  21. Chien, C., Bartel, P. L., Sternglantz, R., and Fields, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9578-9582 [Abstract]
  22. Maly, F. E., Cross, A. R., Jones, T. G., Wolf Vorbeck, G., Walker, C., Dahinden, C. A., and Deweck, A. L. (1988) J. Immunol. 140, 2334-2339 [Abstract/Free Full Text]
  23. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214 [Medline] [Order article via Infotrieve]
  24. Van Aelst, L., Barr, M., Marcus, S., Polverino, A., and Wigler, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6213-6217 [Abstract]
  25. Hannon, G. J., Demetrick, D., and Beach, D. (1993) Genes & Dev. 7, 2378-2391
  26. El Benna, J., Faust, L. P., and Babior, B. M. (1994) J. Biol. Chem. 269, 23431-23436 [Abstract/Free Full Text]
  27. de Mendez, I., Garrett, M. C., Adams, A. G., and Leto, T. L. (1994) J. Biol. Chem. 269, 16326-16332 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.