(Received for publication, January 16, 1997, and in revised form, March 28, 1997)
From the The respiratory burst oxidase of phagocytes and B
lymphocytes catalyzes the reduction of oxygen to superoxide anion
(O The respiratory burst oxidase, or
NADPH1 oxidase, of neutrophils and B
lymphocytes is a multicomponent enzyme that catalyzes the
NADPH-dependent reduction of oxygen to superoxide anion
(O Activation of neutrophils by formyl-methionyl-leucyl-phenylalanine
(fMLP) or phorbol myristate acetate (PMA) leads to a marked increase in
the phosphorylation of multiple proteins on serines, threonines, and
tyrosines (8, 9). The functional significance of these phosphorylations
and the relevant protein kinases is unclear (2, 10). One of the
phosphorylated proteins is the cytosolic oxidase subunit
p47phox, which is phosphorylated on several serines (11-14).
This phosphorylation is required for the translocation of
p47phox to the plasma membrane and for the activation of NADPH
oxidase (15, 16). Translocation of p67phox is also essential
for the activation of NADPH oxidase, as the oxidase from CGD patients
deficient in this protein fails to produce O PMA, fMLP, phosphatases inhibitors, proteases
inhibitors, phosphoserine, phosphothreonine, and phosphotyrosine were
from Sigma. Protein kinase C was from Calbiochem or Promega (Madison,
WI). GF109203X was from Calbiochem. Sequencing grade trypsin was from Boehringer Mannheim (Germany). SDS-PAGE reagents were from Bio-Rad. [32P]Orthophosphate and [ Neutrophils were obtained from
normal subjects by dextran sedimentation and Ficoll-Hypaque
fractionation of freshly drawn citrated blood (6). The cells were
resuspended at 1 × 108/ml in phosphate-free buffer
(10 mM Hepes, 137 mM NaCl, 5.4 mM KCl, 5.6 mM D-glucose, 0.8 mM
MgCl2, and 0.025% bovine serum albumin) and treated with
2.5 mM diisopropyl fluorophosphate (DFP) on ice for 20 min.
Neutrophils were incubated in phosphate-free buffer
containing 1 mCi of [32P]orthophosphate/108
cells/ml for 1 h at 30 °C. The cells were then washed and
activated with PMA (1 µg/ml/108 cells) for 8 min or with
fMLP (1 µM/108 cells) for 2 min in the
presence of 1 mM MgCl2 and 1 mM
CaCl2. Activation was terminated with 10 volumes of
ice-cold buffer. The cells were pelleted by centrifugation (400 × g for 10 min at 4 °C) and resuspended at 1 × 108 cells/ml in ice-cold lysis buffer (20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 15 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1.5 mM phenylmethylsulfonyl fluoride, 1 mM DFP, 0.5% Triton X-100, 25 mM NaF, 5 mM NaVO4, 5 mM
Gamma Bind G-Sepharose beads (Pharmacia Biotech Inc.)
were equilibrated with lysis buffer containing 1 mg/ml bovine serum albumin for 1 h at 4 °C. The cleared lysate was incubated with p67phox antibody (1/150 dilution) or p47phox antibody
(1/200 dilution) or their respective IgG controls in the presence of 50 µl of Sepharose beads overnight at 4 °C with gentle mixing. Then,
the beads were washed extensively with lysis buffer without DFP or
DNase I. The immunoprecipitated proteins were eluted by boiling in
electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2.3% SDS, 2% 2-mercaptoethanol). The beads were
pelleted by brief centrifugation, and the supernatant was subjected to
10% SDS-PAGE according to Laemmli (20). Proteins were blotted onto
nitrocellulose using Towbin buffer (21) and detected by autoradiography
or antibody labeling (p67phox antibody = 1/1000 dilution,
p47phox antibody = 1/10,000 dilution), using ECL or
colorimetric detection.
P67phox cDNA was prepared from
EBV-transformed B lymphocytes mRNA as described previously (19).
The cDNA was cloned in pGEX-3X plasmid at the EcoRI site
then transformed into competent XL-Blue I Escherichia coli
bacteria. E. coli expressing GST-p47phox (22) was a
kind gift from Dr. Bernard M. Babior (The Scripps Research Institute,
La Jolla, CA). Recombinant proteins were obtained as glutathione
S-transferase GST-fusion proteins expressed in bacteria and
purified on glutathione-agarose beads (22). Briefly, E. coli
was grown at 37 °C overnight in 50 ml of ampicillin broth medium.
The overnight culture was diluted into 450 ml of fresh medium and grown
for an additional hour at 37 °C. The culture was then made up to 0.1 mM in isopropyl- Five micrograms of recombinant
GST-p67phox or p47phox was incubated with 0.5 µg of
protein kinase C in 40 mM Hepes, 10 mM
MgCl2, 2 mM MnCl2, 0.5 mM CaCl2, 1 mM dithiothreitol, 50 µM (1 µCi) [ Tryptic
digestion of p67phox and p47phox blotted onto
nitrocellulose was performed as described elsewhere (13, 23). The dried peptides were dissolved in water and lyophilized three times, redissolved in electrophoresis buffer (88% formic acid/water = (3:17)), and applied to one corner of a 20 × 20-cm cellulose
thin-layer plate. Electrophoresis was carried out at 6 °C for 25 min
at 1100 V on an LKB-flat gel apparatus. Chromatography was performed as described previously (13, 23).
Phosphoamino acid analysis of
p67phox was performed using one-dimensional analysis by the
method of Boyle et al. (23). The polyvinylidene difluoride
membrane containing the radiolabeled p67phox band was located
by autoradiography, excised, and transferred to a microcentrifuge tube.
Protein was then hydrolyzed in 0.25 ml of 5.7 N HCl for
1 h at 110 °C. The sample was dried using a Speed-Vac
concentrator and then resuspended in 10 µl of a buffer composed of
100:10:1890 (v/v) acetic acid:pyridine:water. The sample was spotted on
a thin-layer cellulose plate (Merck) and subjected to electrophoresis
at 1100 V for 45 min with water cooling. Phosphoserine,
phosphothreonine, and phosphotyrosine (1 mg/ml) were used as markers.
The plate was then dried, sprayed with ninhydrin to localize the
phosphoamino acid standards, and used for autoradiography.
To determine if p67phox is phosphorylated,
neutrophils were loaded with [32P]inorganic phosphate and
activated with fMLP or PMA; p67phox was immunoprecipitated with
a specific antibody as described under "Experimental Procedures."
Fig. 1A shows the autoradiography of the
corresponding gel. While p67phox was weakly phosphorylated in
resting cells, its state of phosphorylation clearly increased after
stimulation of human neutrophils with PMA (1 µg/ml for 8 min) or fMLP
(1 µM for 2 min). The absence of the phosphorylated
protein after immunoprecipitating with the control IgG showed that the
presence of this phosphorylated protein was not the result of
nonspecific binding to the beads. Corresponding Western blot analysis
(Fig. 1B) identified this phosphoprotein as p67phox.
Furthermore, as conflicting results have been reported on the phosphorylation of p67phox, possibly due to the use of
different and not very specific antibodies, we checked that our
antibody did not recognize other proteins than p67phox. Fig.
2A shows that in EBV-transformed B cells from
a CGD patient deficient in p67phox, the anti-p67phox
antibody did not cross-react with another protein around the 67-kDa
area. However p47phox is normally expressed in these cells and
both phox (phagocyte oxidase)
proteins are expressed in normal lymphoblasts. In addition, the
32P-labeled protein was not immunoprecipitated from
p67phox-deficient cells but was immunoprecipitated from normal
lymphoblasts (Fig. 2, B and C). These results
clearly show that p67phox is phosphorylated in activated
neutrophils and EBV-transformed B lymphocytes.
As in resting cells p47phox and p67phox
form a complex, we wondered if these proteins remain in the complex
after being phosphorylated. Immunoprecipitation with either
p67phox antibody or p47phox antibody resulted in the
isolation of both phosphorylated p67phox and p47phox
(Fig. 3). However, more of each phosphorylated protein
was precipitated when the corresponding antibody was used.
Phosphoamino acid analysis (Fig. 4)
showed that p67phox was phosphorylated on serine residues; no
phosphothreonine or phosphotyrosine was detected with appropriate
markers. In addition, no detectable staining was observed with an
anti-phosphotyrosine antibody (data not shown). These results suggest
that a Ser/Thr protein kinase, not a tyrosine kinase, phosphorylates
p67phox.
The chemotactic peptide fMLP
activates neutrophils via a membrane receptor that triggers a multitude
of signaling pathways involving phospholipases and protein kinases.
However, PMA, which bypasses the receptor, is believed to be more
specific for PKC activation. To determine if fMLP and PMA induced the
phosphorylation of the same or different phosphopeptides in
p67phox, we used two-dimensional tryptic peptide mapping. Fig.
5 shows the presence of one major phosphorylated peptide
after neutrophil activation and that the phosphopeptide map of
32P-labeled p67phox from PMA-activated neutrophils
was identical to that of labeled p67phox from fMLP-activated
neutrophils. In resting cells the same peptide was weakly
phosphorylated (data not shown). It is not clear if this peptide
contains one or several phosphoserines that could be phosphorylated by
different protein kinases.
The phosphorylation of p67phox is induced by
PMA, a direct activator of PKC, suggesting the involvement of this
kinase in the phosphorylation of p67phox. To assess this
possibility and to determine whether or not PKC is involved in
fMLP-induced phosphorylation of p67phox, neutrophils were
incubated with the PKC antagonist GF109203X. As seen in Fig.
6, p67phox phosphorylation induced by PMA was
strongly inhibited by GF109203X; in contrast, fMLP-induced
p67phox phosphorylation was minimally affected by this
inhibitor. This suggests that, in addition to the GF109203X-sensitive
PKC isoforms, other protein kinases (insensitive to GF109203X) are
involved in p67phox phosphorylation induced by fMLP. In
comparison with p67phox, the p47phox phosphorylation
induced by PMA or fMLP was inhibited by GF109203X.
To determine
if PKC phosphorylates p67phox directly, recombinant
GST-p67phox was incubated with [
The link between respiratory burst oxidase activation and protein
phosphorylation is believed to be exclusively mediated by p47phox phosphorylation. Recent and conflicting results (17,
18) have raised the possibility of p67phox phosphorylation:
Dusi and Rossi (17) have reported that p67phox is
phosphorylated during neutrophil activation, while Heyworth et
al. (18) observed no such phosphorylation. The difficulties in
p67phox phosphorylation analysis are due mainly to the very low
level of the protein in cells (24), the fact that p67phox is
very sensitive to proteolysis (25), and the lack of a strongly specific
antibody for immunoprecipitation studies. We used an antibody that
specifically immunoprecipitates p67phox, as shown by the lack
of an immunoprecipitant p67 protein in EBV-transformed B cells from a
CGD patient deficient in this protein, as well as the single band
obtained by Western blotting of the immunoprecipitate with another
anti-p67phox antibody. Using this antibody, we observed that
the p67phox component of NADPH oxidase clearly became
phosphorylated on stimulation of neutrophils with either fMLP, a
receptor-dependent chemotactic activator, or PMA, a PKC
activator. The weak phosphorylation observed in nonstimulated cells
could correspond to basal phosphorylation or to slight activation
during neutrophil isolation.
In resting cells, p47phox and p67phox exist in a
complex (22) that translocates to associate with cytochrome
b558 during oxidase activation. In this report
we show that p47phox and p67phox remain, at least
partially, complexed after their phosphorylation. Partial dissociation
cannot be ruled out, as more phosphorylated protein was precipitated by
the corresponding specific antibody than by the antibody directed
against the other member of the complex. In addition, the observed
dissociation could occur naturally or be induced by the antibodies. It
has recently been shown that the cytosolic oxidase complex contains, in
addition to p67phox and p47phox, a protein named
p40phox that participates in oxidase activation (26). We found
that p40phox copurified with the phosphorylated
p47phox/p67phox complex but was not
phosphorylated.2
Our results show that p67phox undergoes phosphorylation on
serine residues during neutrophil activation, without threonine or tyrosine phosphorylation. These results suggest that a Ser/Thr protein
kinase, not a tyrosine kinase, induces the phosphorylation of
p67phox. Our observation that GF109203X, a PKC inhibitor,
strongly inhibited PMA-induced phosphorylation and barely modified
fMLP-induced phosphorylation, points to both PKC-dependent
(isoforms sensitive to GF109203X) and PKC-independent (or
GF109203X-insensitive) pathways in the phosphorylation of
p67phox. Indeed, human neutrophils express in addition to the
Several lines of evidence support a role of PKC in NADPH oxidase
activation. PMA, an activator of PKC, is a strong stimulus of
O We are grateful to Dr. Bernard M. Babior from
the Scripps Research Institute for the antibodies, the E. coli expressing p47phox and lymphoblasts.
INSERM U294,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2) at the expense of NADPH. This multicomponent enzyme is
dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms
regulating the activation of the respiratory burst oxidase are unclear,
particularly the phosphorylation status of the cytosolic component
p67phox. In this study, we found that activation of human
neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a
chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator
of protein kinase C (PKC), resulted in the phosphorylation of
p67phox. Using an anti-p67phox antibody or an
anti-p47phox antibody, we showed that phosphorylated
p67phox and p47phox form a complex. Phosphoamino acid
analysis of the phosphorylated p67phox revealed only
32P-labeled serine residues. Two-dimensional tryptic
peptide mapping analysis showed that p67phox is phosphorylated
at the same peptide whether fMLP or PMA is used as a stimulus. In
addition, PKC induced the phosphorylation of recombinant
GST-p67phox in vitro, at the same peptide as that
phosphorylated in intact cells. PMA-induced phosphorylation of
p67phox was strongly inhibited by the PKC inhibitor GF109203X.
In contrast, fMLP-induced phosphorylation was minimally affected by
this PKC inhibitor. Taken together, these results show that
p67phox is phosphorylated in human neutrophils by different
pathways, one of which involves protein kinase C.
2), a precursor of microbicidal oxidants (1, 2). The
importance of the oxidase in host defenses is demonstrated by the
recurrent and life-threatening infections that occur in patients with
chronic granulomatous disease (CGD), a hereditary disorder resulting in defective NADPH oxidase activity (3, 4). Components of this oxidase
include cytochrome b558, a membrane-bound
flavohemoprotein, the cytosolic proteins p47phox,
p67phox, and p40phox, and a small GTP-binding protein
Rac2 or Rac1. In resting cells the enzyme is inactive, and its
components are distributed between the cytosol and membranes. When
cells are activated, the cytosolic components migrate to the membranes
and their cytoskeleton fraction, where they associate with cytochrome
b to form the catalytically active oxidase (5-7).
2 (3). While
the phosphorylation of p47phox has been extensively
investigated, conflicting results have been reported on the
phosphorylation of p67phox (17, 18). Using an antibody that
specifically immunoprecipitates p67phox, we show here that this
protein becomes phosphorylated in human neutrophils stimulated with
fMLP or PMA. In addition, we show that the PKC inhibitor GF109203X
inhibits PMA-induced phosphorylation of p67phox without
affecting fMLP-induced phosphorylation. These results suggest that
phosphorylation of p67phox participates in the regulation of
NADPH oxidase by PKC-dependent and -independent
pathways.
Materials
-32P]ATP were
from DuPont NEN Life Science Products. Rabbit anti-p67phox
polyclonal antibody raised against the synthetic peptide extending from
amino acid 512 to the C-terminal residue was prepared as described
elsewhere (19). The anti-p47phox antibody and EBV-transformed B
lymphocytes from p67phox-deficient CGD patient, and normal
subjects were kindly provided by Dr. Bernard M. Babior (The Scripps
Research Institute, La Jolla, CA).
-glycerophosphate, 1 mM p-nitrophenyl
phosphate, 0.25 M sucrose, and 1 mg/ml DNase I), sonicated
(3 × 10 s), and centrifuged (100,000 × g,
30 min at 4 °C). Lymphoblasts were labeled with 32P as
described previously (16). Briefly, the cells were incubated overnight
in phosphate-free medium, then transferred to fresh medium containing
32P (0.2 mCi/ml) and incubated for 4 h at 37 °C.
The cells were then activated for 12 min with PMA (1 µg/ml/108 cells) then lysed like the neutrophils.
-D-thiogalactoside and grown
for an additional 3 h at room temperature (p67phox) or
37 °C (p47phox). The bacteria were recovered by
centrifugation at 5000 × g for 20 min at 4 °C. The
pellet was resuspended in 5 ml of ice-cold bacteria lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 20 mM leupeptin, 15 mM pepstatin, and 0.5 mM DFP) and disrupted by sonication (3 × 10 s).
The sonicate was clarified by centrifugation at 20,000 × g for 20 min at 4 °C. The fusion proteins were isolated
from the clarified sonicate by purification over glutathione-agarose
beads as described previously (22).
-32P]ATP, 5 µg/ml
diolein, and 50 µg/ml phosphatidylserine in a final volume of 100 µl. The reaction was run for 30 min at 30 °C and terminated by
adding 5 × Laemmli sample buffer. The proteins were analyzed by
SDS-PAGE.
p67phox Is Phosphorylated in Activated Human
Neutrophils
Fig. 1.
P67phox is phosphorylated in PMA and
fMLP-treated human neutrophils. 32P-Labeled
neutrophils were activated with PMA (1 µg/ml/108 cells
for 8 min) or fMLP (1 µM/108/ml for 2 min).
P67phox was immunoprecipitated and then analyzed by SDS-PAGE
and blotting onto nitrocellulose membranes. The protein was detected by
autoradiography (A) and by use of an anti-p67phox
antibody (B). The preparation shown was obtained from
50 × 106 cells/lane. "Hch" is the
immunoglobulin heavy chain."Ab" is antibody. Data are
representative of three experiments.
[View Larger Version of this Image (34K GIF file)]
Fig. 2.
The phosphorylated protein is absent from
p67phox-deficient B lymphoblasts. Normal and
p67phox-deficient lymphoblasts were lysed (A) or
labeled with 32P, activated with PMA (1 µg/ml for 12 min), lysed, and incubated with anti-p67phox antibody as
described under "Experimental Procedures" (B and C). The proteins were analyzed by SDS-PAGE and detected by
the anti-p67phox antibody (in A and C) or
the anti-p47phox antibody (A) and autoradiography
(B). Each track contains protein from 50 × 106 cells for the immunoprecipitated p67phox.
"Hch" is the immunoglobulin heavy chain. Data are
representative of three experiments.
[View Larger Version of this Image (23K GIF file)]
Fig. 3.
Phosphorylated p67phox forms a
complex with phosphorylated p47phox.
32P-Labeled neutrophils were stimulated with PMA (1 µg/ml/108 cells for 8 min), lysed, and incubated with
anti-p47phox antibody or anti-p67phox antibody. After
SDS-PAGE, proteins were detected by autoradiography and Western blot
using anti-p47phox and anti-p67phox antibodies. Data
are representative of three experiments. IP, immunoprecipitation.
[View Larger Version of this Image (32K GIF file)]
Fig. 4.
P67phox is phosphorylated on serine
residues. Purified 32P-labeled p67phox from
neutrophils was blotted onto polyvinylidene difluoride membranes and
hydrolyzed in HCl. The resulting phosphoamino acids were analyzed by
one-dimensional high-voltage electrophoresis as described under "Experimental Procedures." The position of phosphoserine
(PS), phosphothreonine (PT), and phosphotyrosine
(PY), determined by ninhydrin staining of standards, is
indicated. Data are representative of three experiments.
[View Larger Version of this Image (25K GIF file)]
Fig. 5.
Tryptic phosphopeptide maps of
32P-labeled p67phox from PMA- and fMLP-activated
neutrophils. 32P-Labeled neutrophils were activated
with PMA (1 µg/ml for 8 min) or fMLP (1 µM for 2 min)
as described in the text. 32P-Labeled p67phox was
purified by immunoprecipitation followed by SDS-PAGE, then blotted onto
nitrocellulose membranes and analyzed by tryptic phosphopeptide
mapping. TLC, thin-layer chromatography. TLE,
thin-layer electrophoresis. The point of application of the sample is
indicated by the dot in the lower left corner of
each panel. Data are representative of three experiments.
[View Larger Version of this Image (50K GIF file)]
Fig. 6.
Effect of the protein kinase C inhibitor on
p67phox phosphorylation. 32P-Labeled
neutrophils were incubated with 2.5 µM of the PKC
inhibitor GF109203X for 10 min, before stimulation with 1 µM fMLP for 2 min or 1 µg/ml PMA for 8 min.
Immunoprecipitation of p67phox and p47phox was
performed as described under "Experimental Procedures." Data are
representative of three experiments.
[View Larger Version of this Image (47K GIF file)]
-32P]ATP and
purified protein kinase C. As shown in Fig. 7, PKC
phosphorylated GST-p67phox, although to a lesser extent than
p47phox. Furthermore, tryptic peptide mapping showed that the
serine(s) phosphorylated in vitro by PKC was/were located in
the same peptide as that phosphorylated in intact cells. These results
strongly support the participation of PKC in p67phox
phosphorylation.
Fig. 7.
GST-p67phox is phosphorylated
in vitro by protein kinase C. GST-p67phox
expressed in and purified from bacteria was incubated with purified PKC
and analyzed by SDS-PAGE and autoradiography (top panel). GST-p47phox was used as comparator. Phosphorylated
p67phox was analyzed by tryptic phosphopeptide mapping
(bottom panel). Data are representative of three
experiments.
[View Larger Version of this Image (48K GIF file)]
and
PKC isoforms the PKC
(27). The PKC inhibitor GF109203X
could be more effective against the
and
isoforms than the
one. However, we found that PKC
is not able to phosphorylate
p67phox in vitro.3
Whether or not GF109203X inhibits PKC
, this result suggests that
PKC
is not involved in p67phox phosphorylation.
Two-dimensional phosphopeptide mapping showed that the same
p67phox peptide was phosphorylated after fMLP and PMA
stimulation. It is conceivable that this peptide contains several
serines that are phosphorylated by different protein kinases: the
different sensitivity of p67phox phosphorylation to the PKC
inhibitor (GF109203X) when induced by fMLP or PMA supports this
hypothesis. Whatever the other kinases involved, the phosphorylation of
recombinant GST-p67phox in vitro by purified PKC, on
this same peptide, suggests that PKC participates in the
phosphorylation of p67phox. After neutrophil activation with
PMA or fMLP, p47phox is phosphorylated on several serines (13,
14), the result obtained by tryptic peptide mapping of p67phox
suggests that p67phox has less phosphorylated sites than
p47phox. However, the phosphorylation of both proteins could
have a crucial importance in the regulation of NADPH-oxidase
activation.
2 production in whole cells (28). Purified p47phox is
a good substrate for PKC in vitro (29). Staurosporine, a powerful inhibitor of PKC, inhibits superoxide production and p47phox phosphorylation (15, 30). The data presented here
provide clear evidence that, in addition to p47phox,
p67phox itself could play a role in the regulation of NADPH
oxidase by phosphorylation/dephosphorylation reactions and that the
phosphorylation events involve a PKC-dependent pathway.
Little is known of the possible role of other protein kinases in the
regulation of NADPH oxidase. It has recently been suggested that cyclic
AMP-dependent protein kinase, mitogen-activated protein
kinase (14, 31), and p21-activated kinase (32) could regulate NADPH
oxidase by phosphorylating p47phox. Our findings suggest that
protein kinases other than PKC may participate in p67phox
phosphorylation. The kinases involved in this process are currently under investigation.
*
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.: 33-1-40-25-85-21;
Fax: 33-01 40-25-88-53; E-mail: benna{at}bichat.inserm.fr.
1
The abbreviations used are: NADPH, nicotinamide
adenine diphosphate reduced form; CGD, chronic granulomatous disease;
fMLP, formyl-methionyl-leucyl-phenylalanine; PMA, phorbol
12-myristate 13-acetate; PKC, protein kinase C; PAGE, polyacrylamide
gel electrophoresis.
2
J. El Benna and M. Gougerot-Pocidalo,
unpublished observations.
3
P M.-C. Dang, M.-A. Gougerot-Pocidalo, and J. El
Benna, manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.