From the
Cdc42Hs and Rac1 are members of the Ras superfamily of small
molecular weight (p21) GTP binding proteins. Cdc42Hs induces filopodia
formation in Swiss 3T3 fibroblasts while Rac1 induces membrane
ruffling. Rac1 also activates superoxide production by the components
(cytochrome b, p40, p67, and p47) of the neutrophil oxidase.
To isolate target proteins involved in these signaling pathways, we
have probed proteins from neutrophil cytosol immobilized on
nitrocellulose with Cdc42Hs labeled with
[
The Ras superfamily of small molecular (p21) GTP binding
proteins play fundamental roles in cellular physiology, including the
control of cell proliferation, protein trafficking, and actin-based
cell morphology (Hall, 1990). These GTP binding proteins cycle between
GTP-bound (on) and GDP-bound (off) states. Regulatory proteins such as
GTPase activating proteins ( e.g.n-chimaerin)
(Diekmann et al., 1991) and GDP/GTP exchange proteins
( e.g. Dbl) (Hart et al., 1991) determine the degree
to which any particular p21 is in the on state. Cdc42Hs and Rac1 are
members of the Rho family, which is thought to be involved in
controlling cell morphology (Hall, 1992). Cdc42Hs microinjection
induces filopodia formation in Swiss 3T3 cells (Kozma et al.,
1995), and Rac1 microinjection induces membrane ruffling (Ridley et
al., 1992). Rac1 has also been shown to activate the neutrophil
oxidase (Abo et al., 1991; Knaus et al., 1991),
possibly by mediating complex formation of p67
To identify Cdc42Hs and Rac1
targets that may be involved in the pathways described above, we have
probed proteins in neutrophil cytosol immobilized on nitrocellulose
with p21-[
p21 probes
were prepared by incubating Cdc42Hs and Rac1 (1µg) with 1 µl of
[
Neutrophil cytosol contains Rac1 binding proteins of
66-68 and p48 kDa that bind preferentially to the GTP-bound state
of this p21. The possibility that these binding proteins were p67 and
p47 was addressed by probing purified proteins immobilized on
nitrocellulose with Rac1-[
As with Rac1, Cdc42Hs was found to bind specifically to the
66-68-kDa proteins in neutrophil cytosol and purified p67 but not
the other oxidase components. Cdc42Hs does not activate the oxidase
in vitro (Kwong et al., 1993), and this suggests that
p21 binding to p67 is not in itself sufficient to cause activation.
What then is the function of the Cdc42Hs-p67 interaction? One
possibility is that p67 could have a function in neutrophils other than
being a component of the oxidase. Microinjection of Cdc42Hs induces
distinct morphological effects, such as filopodia formation and loss of
stress fibers in Swiss 3T3 fibroblasts (Kozma et al., 1995).
These events are associated with a redistribution of actin polymers. It
is therefore possible that p67 could be a target for Cdc42Hs in a
morphological pathway of neutrophils. If this is the case, p67 could
link activation of the neutrophil oxidase with morphological events
that are essential for phagocytosis. The demonstration that p67 is a
Cdc42Hs binding protein suggests novel functions for this protein that
will require further investigation.
The experiments presented here
investigating the ability of Cdc42Hs or Rac1 to bind to purified
oxidase components utilized microgram quantities of p67. Since the
abundance of p67 in neutrophil cytosol is approximately 0.2%,
The relationship between the p66- and
p68-kDa proteins in neutrophil cytosol and p65 was investigated by
carrying out a two-dimensional gel analysis of Cdc42Hs binding
proteins. Two major protein spots were detected with similar
isoelectric points. An almost identical pattern of Cdc42Hs binding
proteins was seen when brain cytosol was examined, suggesting that
neutrophil protein(s) may be related if not identical to the brain p65.
Indeed, purified p68 protein from neutrophil cytosol had kinase
activity and could be immunoprecipitated in an autophosphorylated form
by anti-p65 antibodies. To confirm the relationship between p65 and
p68, affinity-purified antibodies were used in Western analysis. p68
was detected by p65 antibodies but not by p67 antibodies, demonstrating
that p68 is closely related to brain p65. We are presently sequencing
peptides of p68 to establish the amino acid sequence identity of this
protein to p65. Unlike p68, p48 was not detected by anti-p65
antibodies, and therefore further work will be necessary to establish
its identity. In conclusion, we have been able to identify two targets
for the GTP binding proteins Cdc42Hs and Rac1 in neutrophils: a p68
kinase and p67.
We thank members of the Segal laboratory for the
supply of purified proteins and the Glaxo-Singapore Research Fund and
Wellcome Trust for financial support.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-
P]GTP. Cdc42Hs probe detected binding
protein(s) of 66-68 kDa in neutrophil cytosol. Rac1 probe also
detected the 66-68-kDa proteins, suggesting the possibility that
p67 may be a binding protein for both of these p21 proteins. Indeed,
Cdc42Hs and Rac1 were found to bind specifically to purified
recombinant p67 but not the other oxidase components. A 68-kDa Cdc42Hs
binding protein was purified from neutrophil cytosol and found to be
related to the recently described p65 kinase from brain. These results
suggest that the p68 kinase and p67 are targets for Cdc42Hs and Rac1 in
neutrophils.
(
)
and p47 (and p40 (Wientjes et al., 1993)) with the
membrane-bound cytochrome b (Segal and Abo, 1993). The
downstream target(s) for Cdc42Hs or Rac1 in the morphological pathways
is not known. However, recent work has identified specific targets for
p21 signaling pathways. Ras binds to Raf kinase, relocalizing Raf to
the plasma membrane upon cell stimulation (Stokoe et al., 1994
Leevers et al., 1994). Manser et al. (1994) have
isolated a brain protein kinase, p65, by purifying a
Rac1/Cdc42Hs-[
-
P]GTP binding activity.
Brain p65 kinase is activated by Rac1 and Cdc42Hs and is therefore a
novel target for these p21 proteins.
-
P]GTP. Using this technique,
66-68-kDa proteins were detected. We speculated that one of these
proteins might by either p67 or a p65-related protein.
Cdc42Hs-[
-
P]GTP was found to bind
specifically to purified recombinant p67, as did Rac1. During the
course of the present work, Rac1 was shown to bind specifically to p67
(Diekmann et al., 1994). The 66-68-kDa proteins were
found to be related to the brain p65. Thus, this study identifies two
targets for Cdc42Hs and Rac1 in neutrophils: a 68-kDa kinase and p67.
Expression and Purification of Recombinant
Proteins
Rac1, Cdc42Hs, p47, p40, p67, p65
C-terminal deletion (amino acids 1-151) containing a p21
binding site (Manser et al., 1994), from here onward referred
to as p65, and p85 phosphatidylinositol kinase subunit (amino acids
129-273) were expressed in the pGEX plasmids or derivatives and
protein purified as previously described (Abo et al., 1992;
Ahmed et al., 1991, 1993). Cdc42Hs and Rac1 mutations were
made using Clontech mutagenesis kit and will be described
elsewhere.(
)
p65 cDNA (Manser et al.,
1994) was cloned by introducing a BamHI site immediately
preceding the initiating methionine and then cloning a
BamHI- EcoRI fragment into pGEX-2T. Protein was
quantified by the method of Bradford (1976).
p21 Binding Assays
For one-dimensional
analysis, recombinant, neutrophil, and brain proteins were prepared in
sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol,
0.14 M -mercaptoethanol, 0.1% bromphenol blue) and boiled
for 2-3 min before being separated on 10% SDS-PAGE. For
two-dimensional analysis, proteins were mixed with an equal volume of
lysis buffer (9.5 M urea, 10 mM lysine, 5%
-mercaptoethanol, 2% Nonidet P-40 containing 4% ampholines, pH
5-7, and 1% ampholines, pH 3.5-10) and separated on tube
gels containing the same ampholines by electrophoresis overnight at 300
V and then for 1 h at 1000 V. Tube gels were then equilibrated in
sample buffer for 1 h and loaded onto 10% SDS-PAGE. One- and
two-dimensional gels were transferred to nitrocellulose using a Bio-Rad
semi-dry blotter in transfer buffer (4.8 mM Tris, 3.9
mM glycine, and 0.375% SDS). For dot blotting, proteins
(5-20 µl) were applied directly on 1-2-cm
nitrocellulose filters. After protein transfer, filters were
incubated in renaturation buffer (3% BSA, 0.1% Triton X-100, 0.5
mM MgCl
, 5 mM DTT) for 2 h.
-
P]GTP (DuPont NEN; 6000 Ci/mmol, 10
mCi/ml, 1.6 M) or 2 µl of
[
-
P]GTP (DuPont NEN; 3000 Ci/mmol, 10
mCi/ml, 0.8 M) 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 BSA, 1.25 mM DTT) for 10 min at
room temperature. [
-
P]GDP probes were
generated by loading with [
-
P]GTP and
allowing the p21-GTP complex to hydrolyze the
-phosphate group for
60-90 min at room temperature. Probes were used immediately on
preparation by diluting into 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 MgCl
, 1.25 mg/ml BSA,
1.25 mM DTT, 0.5 mM GTP). Filters were incubated with
p21 probes for 5 min at room temperature before being washed six times
for 5 min each in wash buffer (50 mM NaCl, 25 mM Mes,
pH 6.0, 5 mM MgCl
, 0.25% Triton X-100) on ice.
After washing, the filters were quickly dried by placing on Whatman 3MM
paper and then exposed to Kodak X-Omat film for 5-25 h.
Quantitative p21 binding analysis was carried out by cutting out 1
cm
of nitrocellulose and determining the amount of
radioactivity by scintillation counting.
Kinase Assay
p68 protein purified as
described below (100 ng) was incubated for 10 min with 50 µCi of
[-
P]ATP (Amersham Corp., 5000 Ci/mmol, 10
mCi/ml) in kinase buffer (50 mM Hepes, pH 7.0, 5 mM
MgCl
, 5 mM MnCl
, 1 mM DTT,
0.05% Triton X-100), and the reaction was stopped by the addition of 2
sample buffer and boiling for 3 min. Samples were then loaded
onto 10% SDS-PAGE and electrophoresed for 50 min at 180 V or until the
dye front containing free label was run off the bottom of the gel. The
gel was then dried and exposed to Kodak X-Omat film for 10 min.
Purification of p68 Cdc42Hs Binding
Protein
p68 protein was purified in five steps using the
Cdc42Hs binding assay described above. (i) Neutrophil cytosol (280 ml,
containing 20 mg/ml protein) was applied to a Q-Sepharose column
(Pharmacia Biotech Inc.) equilibrated with 10 mM Pipes, 100
mM KCl, 3 mM NaCl, 4 mM MgCl (pH
7.0) containing 6% sucrose and protease inhibitors (1 mM
pepstatin and 100 µM N
- p-tosyl-L-lysine
chloromethyl ketone). Protein was eluted with a linear gradient of NaCl
(0.1-1 M) in the above buffer, and 10-ml fractions were
collected. Binding protein eluted at between 0.2-0.3 M
NaCl. Six fractions were pooled and diluted 3-fold in ice-cold water.
(ii) The above pool (180 ml) was applied to a heparin-agarose column,
equilibrated as above, and proteins eluted with a linear NaCl gradient
(0.1-1 M) in the same buffer. Binding protein eluted
between 0.3 and 0.4 M NaCl. Five fractions of 5 ml were
pooled, and the volume was reduced to 5 ml using polyethylene glycol by
dialysis for 3 h at 4 °C. (iii) After filtration to clear the
protein sample, it was loaded onto a Superose 12-gel filtration column
(Pharmacia), equilibrated as before, and 1.3-ml fractions were
collected in the above buffer. (iv) A 1-ml chelating Sepharose column
was prepared by applying 10 column volumes of 100 mM
ZnCl
; the protein sample was applied, the column was washed
in buffer Z (100 mM NaCl, 0.5 mM MgCl
,
0.05% Triton X-100) containing 25 mM Tris, pH 7.5, and elution
was carried out in buffer Z containing 25 mM Mes, pH 6.0. (v)
Protein was loaded onto a Cdc42Hs-G12V-GTP glutathione-Sepharose
affinity column in the presence of 1 mM GTP. For these
experiments, Cdc42Hs was not cleaved with thrombin, leaving the GST
component as an affinity tag. The column was washed in 5 ml of buffer Z
containing 25 mM Mes, pH 6.0, and proteins were eluted in 1
column volume fractions of buffer Z containing 25 mM Tris, pH
8.5, or freshly prepared 5 mM glutathione, 50 mM
Tris, pH 8.0.
Antibodies and Immuoblot Analysis
p67
antibody was a gift from Dr. Franz Wientjies. p65 and GST antibodies
were raised in rabbits using purified recombinant protein from
Escherichia coli. The immunization procedure consisted of
injection of fusion protein (100 µg) in complete Freund's
adjuvant, followed by boosts (every 4 weeks) of fusion protein (100
µg) in incomplete Freund's adjuvant. Rabbits were bled 1 week
after the third boost and after subsequent injections. Antibodies were
purified on affinity columns of the recombinant protein.
Affinity-purified p67 antibodies have been previously described
(Wientjes et al., 1993). Proteins were transferred to
nitrocellulose using a semi-dry blotter as described above. Filters
were then blocked and washed as previously described (Ahmed et
al., 1994) before incubation with primary antibody (1 in 10,000
dilution for the p67 antibody and 1 in 500 for p65 in PBS, 0.1% Tween,
1% non-fat milk). Filters were washed in PBS, 0.1% Tween (5 5
min) before incubation with secondary antibody; DAKO
peroxidase-conjugated Swine Anti-rabbit immunoglobulins, diluted 1 in
1,000 in PBS, 0.1% Tween, 1% non-fat milk, and staining visualized
using enhanced chemiluminescence (Amersham) was as described by the
manufacturer.
Immunoprecipitation
After
phosphorylation, both recombinant GST-p65 and p68 were incubated for 60
min at 4 °C with either 5 µl of affinity-purified p65 antibody
or preimmune serum (in 50 mM Tris, pH 7.5, 25 mM
NaCl, 0.5 mM MgCl, 0.05% Triton X-100, 1
mM DTT; final volume, 100 µl). A 50% slurry in the same
buffer (v/v) of protein A-Sepharose beads (Sigma, 100 µl) was then
added, and incubation continued for a further 60 min. The protein
A-Sepharose beads were sedimented by packing in a spin column, and the
unbound material and radioactivity was removed by discarding the
supernatant. The column was then washed three times with 750 µl of
PBS, 0.1% Triton X-100. 50 µl of sample buffer (2
) was then
added to the beads followed by incubation at 95 °C for 10 min. The
beads were then spun down, and the supernatant containing protein was
analyzed by one-dimensional SDS-PAGE.
Cdc42Hs Binding Proteins in
Neutrophils
To detect potential Cdc42Hs targets, proteins
in neutrophil cytosol were separated on SDS-PAGE, transferred to
nitrocellulose, renatured, and probed with
Cdc42Hs-[-
P]GTP. Proteins with molecular
masses of 66-68 kDa were detected (Fig. 1). Partial
purification of Cdc42Hs binding activity by DEAE chromatography
revealed two distinct protein bands (not shown).
Cdc42Hs-[
-
P]GTP detects proteins in brain
cytosol of a similar size, which are most probably the recently
characterized p65 kinase (Manser et al., 1994). This
interaction was specific to the GTP-bound state of Cdc42Hs as (i)
Cdc42Hs-GDP at 10-fold higher concentration did not compete with the
probe, and (ii) Cdc42Hs-[
-
P]GDP did not
detect the neutrophil (or brain) binding proteins (Fig. 1).
Rac1-[
-
P]GTP also reacted with proteins of
around 68 kDa from neutrophils (Fig. 1) and brain (not shown).
However, the signals were approximately 5-fold weaker when Rac1 was
used as a probe, suggesting that it binds less tightly than Cdc42Hs
(Fig. 1, lane3; note longer exposure for
Rac1). A very weak signal at 48 kDa was also seen with Rac1
(Fig. 1, lane3) and with Cdc42Hs, but this was
variable.
Figure 1:
Cdc42Hs and Rac1 binding protein in
neutrophils. Lanes1 and 3,
Cdc42Hs-[-
P]GTP and
Rac1-[
-
P]GTP, respectively. Lanes2 and 4,
Cdc42Hs-[
P]GDP and
Rac1-[
P]GDP, respectively. Neutrophil
cytosolic proteins (100 µg/lane) were separated on 10% SDS-PAGE,
transferred to nitrocellulose, and probed with GTP- or GDP-labeled p21
proteins. Filters were exposed to film for 5 h (Cdc42Hs) and 25 h
(Rac1). Arrow shows faint signal at 48
kDa.
p67 Is a Cdc42Hs Binding Protein
On the
basis of these characteristics, we speculated that the p66-68
Cdc42Hs binding proteins in neutrophils might be either p67 or a member
of the p65 family of kinases, which are abundant in brain (Manser
et al., 1994), and p48 might be p47. Purified recombinant
neutrophil oxidase components (5 µg) were immobilized on
nitrocellulose after SDS-PAGE and then probed with
Cdc42Hs-[-
P]GTP. Recombinant p67, but not
p47 or native cytochrome b protein, was detected with this
Cdc42Hs probe. However, p67 was not detected with
Rac1-[
-
P]GTP (Fig. 2 A). To
increase the sensitivity of the assay for Rac1, proteins were directly
dot blotted onto nitrocellulose, thus avoiding the denaturation caused
by running in SDS-PAGE. Using this method of immobilizing protein, with
Rac1-[
-
P]GTP as probe, signals were
obtained for p67 (2.5-7.5 µg) but not for p47, p40,
cytochrome b, BSA, or the recombinant phosphatidylinositol
kinase p85 subunit (Fig. 2 B). Similarly, with Cdc42Hs as
probe, signals were not seen for p47 or p40 (not shown). The strength
of signal obtained with Rac1 as probe increased, with an increase in
p67 protein amounts between 2.5 and 7.5 µg. As with the
66-68-kDa binding proteins in neutrophil cytosol, Rac1-GDP did
not inhibit the Rac1-[
-
P]GTP interaction
with p67, and signals were not obtained for p67 with
Rac1-[
-
P]GDP as probe (not shown). Taken
together, these data establish the use of this dot-blotting technique
to detect protein-protein interactions eliminating the SDS-PAGE step,
which denatures proteins.
Figure 2:
Cdc42Hs
and Rac1 bind to p67. A: cytochrome b, lanes1 and 4; p47, lanes2 and
5; p67, lanes3 and 6. Purified
proteins (5 µg) were separated on 10% SDS-PAGE, transferred to
nitrocellulose, and probed with either
Cdc42Hs-[-
P]GTP ( lanes1-3) or Rac1-[
-
P]GTP
( lanes4-6). B, purified proteins
(2.5-7.5 µg) were dot blotted onto nitrocellulose and probed
with Rac1-[
-
P]GTP. Lane1, 2.5 µg; lane2, 5 µg; and
lane3, 7.5 µg. p47 protein was present at 10,
20, and 40 µg ( lanes1, 2, and
3, respectively). Control protein BSA and p85 were present at
10 µg. Similar results were obtained in at least one other
experiment.
Specificity of Cdc42Hs Binding to p67
The
specificity of these interactions were determined by using the Q61L
GTPase negative mutants of Cdc42Hs and Rac1, RhoA and p65. Ras-Q61L has
increased effector function in terms of transformation ability and
Ras-GTPase activating protein interaction (Brownbridge et al.,
1993). Rac1-Q61L is a more potent activator of neutrophil oxidase (Xu
et al., 1993) and membrane ruffling(
)
and binds the Rac1-GTPase activating protein
n-chimaerin more tightly than wild-type or Rac1-G12V proteins
(Ahmed et al., 1994). Rac1-Q61L probe gave a stronger signal
than wild-type protein for interaction with p67 (compare
Fig. 3B with 2 B; see below for quantitation),
as did Cdc42Hs-Q61L probe (not shown). These mutant p21 proteins only
generated a signal with p47 when it was present at high amounts. The
Q61L mutation also increased the binding of Rac1 to p66-68 from
neutrophil or brain cytosol (data not shown). RhoA, a member of this
subfamily of Ras-related GTP binding proteins, did not bind p67 under
these conditions (Fig. 3 C). Further evidence for the
specificity of the Rac1 and Cdc42Hs interaction with p67 was obtained
by carrying out dot blots in the presence of p65, which includes the
Rac1/Cdc42Hs binding domain, as a competitor. p65 interacts
specifically with GTP-bound states of Rac1 and Cdc42Hs with high
affinity (nM). 2 µM p65 binding domain inhibited
Cdc42Hs-Q61L (Fig. 3 D) and Rac1-Q61L (not shown) from
interacting with p67, while 2 µM GST was without effect.
Figure 3:
Specificity of Cdc42Hs and Rac1
interactions. Purified proteins were dot blotted onto nitrocellulose
and probed with Cdc42Hs-Q61L-[-
P]GTP ( A and D), Rac1-Q61L-[
-
P]GTP
( B), and RhoA-[
-
P]GTP
( C). For p67, proteins were present at 2.5, 5, and 7.5 µg
( lanes1, 2, and 3, respectively).
For p47, proteins were present at 10, 20, and 40 µg ( lanes1, 2, and 3, respectively). Control
protein BSA and p85 were present at 10 µg. D, competing
proteins (p65 and GST) were present at 500-fold excess over p21-GTP
concentration. Similar results were obtained in at least two other
experiments.
Quantitative Analysis of Rac1 and Rac1-Q61L Binding
to p67
To quantify the interactions between Rac1 and p67,
the nitrocellulose filters were radioassayed rather than being exposed
to film. To detect Rac1 binding by this technique, amounts of p67
greater than 2 µg of protein had to be used. Fig. 4 A shows that there was a linear increase in Rac1-Q61L binding as the
amount of p67 protein was increased. p47 bound low levels, just above
background, of Rac1-Q61L, but this was not dependent on the amount of
p47 over the range studied. To quantify the difference between Rac1 and
Rac1-Q61L in strength of binding to p67, both probes were used in the
same experiment, and the filters were radioassayed. p67 bound
approximately 17-fold more Rac1-Q61L than Rac1
(Fig. 4 B).
Figure 4:
Quantitative analysis of Rac1 binding to
p67. A, p67 () and p47 (
) were dot blotted at
between 3 and 19 µg and probed with
Rac1-Q61L-[
-
P]GTP. Data are presented as a
percentage of cpm obtained with 18 µg of p67. B, p67 (5
µg) was dot blotted on nitrocellulose and probed in parallel with
Rac1-[
-
P]GTP and
Rac1-Q61L-[
-
P]GTP. Data are presented as a
percentage of cpm obtained in A with 5 µg of p67. Rac1 and
Rac1-Q61L were loaded with [
-
P]GTP (see
``Materials and Methods'') and used to probe p67 and p47
proteins on nitrocellulose filters as described in Fig. 2. The amount
of bound radioactivity was estimated by radioassaying 1 cm
nitrocellulose in scintillant. Background binding (cpm) was
determined by using GST as a control protein and subtracted from all
values presented. Rac1 did not bind p47 (data not shown). Error bars
represent ± S.D. ( n =
3).
The p68 Cdc42Hs Binding Protein Is a p65-related
Kinase
Initially, to investigate whether p66-68 were
related to p65, we carried out a two-dimensional gel Cdc42Hs binding
analysis of proteins in neutrophil and brain cytosol. The major binding
proteins in brain have been shown to be p65 and possibly related
proteins (Manser et al., 1994). Proteins in neutrophil and
brain cytosol were analyzed on isoelectric focusing tube gels, followed
by SDS-PAGE, blotted onto nitrocellulose, and probed with Cdc42Hs
(Fig. 5). The locations of the binding proteins in brain and
neutrophils were very similar, with the proteins having similar
molecular masses (around 68 kDa) and basic isoelectric points. Two
major protein spots were detected for brain and neutrophil cytosol.
Additional protein spots with more acidic isoelectric points were also
seen with longer exposure (not shown) and probably represent
phosphorylated forms of the same protein or isoforms.
Figure 5:
Two-dimensional gel localization of
Cdc42Hs binding proteins in brain and neutrophil cytosol. Brain and
neutrophil cytosolic proteins (100 µg) were separated on tube gels
followed by 10% SDS-PAGE as described under ``Materials and
Methods.'' Protein was then transferred to nitrocellulose and
probed with Cdc42Hs-[-
P]GTP. Basic to
acidic pH levels ( left to right) and high to low
molecular weights ( top to bottom) are as
shown.
Next, we
purified p68 using column chromatography and
Cdc42Hs-[-
P]GTP binding assays, with
affinity purification as the last step (Fig. 6). The yield of
protein eluted from Cdc42Hs columns was low with the bulk of protein
either flowing through the column or remaining bound to resin
(Fig. 6, lane6). p48 was found to copurify
with p68, and its level varied from batch to batch of neutrophil
cytosol. We believe p48 is a breakdown product of p68, but further work
will be necessary to establish its origin. p68 possessed kinase
activity as seen by its ability to autophosphorylate. The kinase
activity was stimulated by Rac1 (Fig. 7).
Figure 6:
Purification of p68 from neutrophil
cytosol. Lane1, protein from gel filtration column
(starting material). Lane2, flow-through from
Cdc42Hs affinity column. Lane3, wash with buffer Z,
pH 6.0. Lanes4 and 5, elution with buffer
Z, pH 8.5. Lane6, glutathione-Sepharose resin with
Cdc42Hs-GST and bound p68. Neutrophil Cdc42Hs p68 binding protein was
purified on Q-Sepharose/heparin-agarose/gel filtration and then applied
to Cdc42Hs affinity columns as described under ``Materials and
Methods.'' On some occasions a chelating zinc column was used
between the gel filtration and Cdc42Hs affinity column to improve
yield. Proteins (10-20 µl) were separated on 10% SDS-PAGE and
stained with Coomassie Blue. Dot blots of different fractions were
probed with Cdc42Hs-[-
P]GTP and are shown
on top of the SDS-PAGE. The glutathione-Sepharose resin shown
in lane6 was not dot blotted to probe for Cdc42Hs
binding activity ( lane6 shows proteins bound to
resin of which the major band at 48 kDa is Cdc42Hs-GST, the bands
around 26 kDa are Cdc42Hs-GST breakdown products, and the 68-kDa band
is the p21 binding protein). Arrow marks position of p68 in
lanes4-6.
Figure 7:
Kinase activity of p68. Lane1, Coomassie Blue stain of partially purified p68.
Lanes2-4, kinase activity as seen by
autophosphorylation of p68. Lane2, in the absence of
additions. Lane3, in the presence of Rac1-GDP.
Lane4, in the presence of Rac1-GTP. p68 was
partially purified (approximately 80%) by
Q-Sepharose/heparin-agarose/gel filtration. Chelating Sepharose was
incubated with 50 µCi of [-
P]ATP for 10
min, and then the sample was analyzed on 10% SDS-PAGE and the gel was
dried down and exposed to film for 10 min.
To examine whether
p68 was related to p67 or p65, specific affinity-purified antibodies
were used. Neutrophil cytosol, p67, and purified p68 were separated by
10% SDS-PAGE and transferred to nitrocellulose. p67 antibodies detected
recombinant p67 and a protein of similar size in neutrophil cytosol but
not p68. p65 antibodies detected p68 and a band of similar size in
neutrophil cytosol but not p67 or p48 (Fig. 8 A). p65
antibodies also detected a protein of approximately 68 kDa in brain
cytosol. Thus, the p68 protein from neutrophil cytosol is closely
related to p65.
Figure 8:
p65-specific antibodies react with and
precipitate p68. A, purified recombinant p67 (1 µg,
lanes1 and 4), p68 (100 ng, lanes2 and 5), neutrophil cytosol (100 µg,
lanes3 and 6), and brain cytosol (100
µg, lane7). Proteins were separated on 10%
SDS-PAGE, transferred to nitrocellulose, and then probed with either
p67-specific antibodies ( lanes1-3) or
p65-specific antibodies ( lanes4-7) as
described under ``Materials and Methods.'' B,
immunoprecipitation in the presence of anti-p65 antibodies ( lanes1 and 3) or preimmune serum ( lanes2 and 4). Neutrophil p68 ( lanes1 and 2) and recombinant GST-p65 (92 kDa, lanes3 and 4) are as shown. Proteins were incubated
with [-
P]ATP for 15 min,
immunoprecipitated, and analyzed on one-dimensional SDS-PAGE; the gel
was then dried down and exposed to film for 16
h.
To show that the p68 kinase activity was not due to
a contaminating 68 kDa protein, a purified p68 fraction and recombinant
GST-p65 were incubated in parallel with
[-
P]ATP for 15 min followed by incubation
with purified anti-p65 antibodies (or GST antibodies), and proteins
were precipitated using protein A-Sepharose beads. Anti-p65 antibodies
were able to precipitate both the autophosphorylated p68 and GST-p65
(Fig. 8 B). Anti-p65 antibodies also precipitated Cdc42Hs
binding activity with p68 and GST-p65 (not shown).
-
P]GTP.
Interestingly, p67 was found to bind specifically to Rac1 but not to
RhoA. The NADPH oxidase components, p40, p47, and cytochrome
b, did not bind Rac1. p67 bound approximately 17-fold more of
the GTPase negative mutant protein Rac1-Q61L than Rac1, which
correlates with the increased potency of Rac1-Q61L to activate
neutrophil oxidase (Xu et al., 1993). During the course of
this work, Diekmann et al. (1994) demonstrated that Rac1
interacted specifically with p67 by using GST fusion proteins and
glutathione-Sepharose to examine protein-protein interactions. In
general, our results are in agreement with those presented by Diekmann
et al. (1994). However, in their study, Rac1-Q61L did not
interact more strongly with p67 than wild-type Rac1. This could be
explained by differences in the sensitivity of the assays employed or
the exact conditions used. We have also shown here that purified native
cytochrome b and p40 are not Rac1 binding proteins. Thus, out
of all of the components of the neutrophil oxidase, p67 is the sole
target for Rac1. In preliminary experiments, we have mapped a Rac1
binding site of p67 very near its polyproline domain
(
)
(SH3 binding site), which is thought to interact with p47.
This may suggest that one purpose of the Rac1-p67 interaction is to
influence the ability of p67 to bind p47. Further work on the formation
of the complex will be required to elucidate the exact mechanism by
which Rac1 activates superoxide production by the neutrophil oxidase.
it can be estimated that 200 ng is present in the SDS-PAGE
analysis of Cdc42Hs and Rac1 binding proteins. This amount of purified
recombinant p67 is not detected even in our dot-blotting assay with
Rac1-Q61L as probe while 100 ng of p68 or p65 is detected. It is
therefore likely that the p68 kinase is the major component of the
Cdc42Hs and Rac1 binding signals seen in neutrophil cytosol with
separation on SDS-PAGE.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.