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