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The
B
L
J
#{128}D
The
American
Journal
Society
of
of Hematology
VOL 69, NO 3
MARCH
1987
REVIEW
Protein
Kinase
C and
the Activation
of the Human
Neutrophil
NADPH-Oxidase
By Alfred I.Tauber
T
HE
NEUTROPHIL
Attuned
leads
to the vagaries
it has developed
a complex
functional
is, adherence/aggregation,
oxidative
burst
which,
focus
of current
phil has become
various
activities
tional
review
in cell
nism
of the
respiratory
against
crucial
role
tory
burst
host
is dependent
on
That
appropriate
basic
for various
B4.’5 A
protein
lipid-dependent
esters
nously
pase
kinase
kinase,
binding
(FMLP)
C (PK-C),
is the correlation
A
in the
between
Patients
reported
with classically
to have abnormal
chemotactic
evidence
ligand
for
and
binding,18
responses
Vol 69. No 3 (March),
PMA
described
receptor
phagocytic
but certain
to various
1987:
been
CGD
affinities
phorbol
that
receptor-
of the
metabolic
and biochem-
linkage
has
is the
phospho-
and endogeof phospholi-
binding
receptor-binding
activation
peptides,’4
exogenous
activation
are
receptors,
a calcium,
by
complex
impor-
receptor
to
NADPH-
established.’79
have not been
as inferred
by
responses3’8
or phorbol
patients
have demonstimuli
and thus exhibit
pp 711-720
may
be
apparatus2#{176}22; those
defects3
in PMA-induced
particular
interest.
of the oxidative
divided
into
two
mechanisms
and abnormalities
The activation
of the human
of responses,
Thus,
with
CGD,
metabolic
types:
which
effector
abnormal
sys-
activating
of NADPH-oxidase.3
neutrophil
by PMA
including
either
oxidase
aggregation,23
leads
to a
stimulation
of the respiratory
burst,’79’226
depolarization
of the plasma
membrane,27
enhanced
phospholipid
turnover,283#{176} and
degranulation
of specific
granules.3’
The
same
constituent
of
the NADPH-oxidase
found in the plasma
membrane
have
been putatively
reported
in the specific granule
membrane;
upon PMA stimulation,
a subset of these granules
is translo-.
cated
to the plasma
membrane,
the site of the
active
respira-
tory burst enZyme.32’33 Of the various
stimulus-responsecoupled functions
defined for the normal neutrophil,
I wish to
focus on those biochemical
phenomena
related
to the PMA
receptor
linked to the expression
of NADPH-oxidase.
In
bypassing
sites
immunoglo-
bacterial
described
The
is critical
expression.
ester
receptor
strated
selected
function,
sequence
of C3,’2
activated
oxidase
Blood,
activation
well-defined
recently
a
steps required
for
made.
By tracing
of the NADPH-
components
or phosphodiesterase)6
burst
normal
respira-
(eg, phorbol
myristate
acetate,
PMA),
by diacyiglycerol
liberated
by the action
ligand
ical
serves
has several
formyl-methionyl
leukotriene
ubiquitous
elements
neutrophil
those
the
and thus
a normal
arrest
the
cell systems,
of the
and
principal
receptor
oxidase,
bulin,’3
the
of
in
of
all defects
spectrum
adenine
pathways,
and an integral
NADPH-oxidase
multicomponent),7”
predicts
multiple
ofactivation
have been
stimulation
including
is
of bacteria,
defense.3’7
tant insights into the sequence
normal
neutrophil
response
exogenous
agonist-induced
now discerned.
The human
neutro-
as a paradigm
activity,
defective
a biochemical
lesion
may
With
recent
studies
in broken
the
in
I wish to
mecha-
nicotinamide
(CGD),
a wide variety
activating
in itselfis
at which
cascade.
enzyme,
disease
in normal
burst
intact
(which
tem,
is a
the
this context,
of the activation
(NADPH)-oxidase,
systems.
This
granulomatous
defense
be studied
transduction
are
includes
of response
and
a defect
in the activation
congenital2’
or acquired
activation
and
a model
of such inquiry,
as attention
to its
has discerned
the outlines
of its informa-
phosphate
secretory
chronic
also
biology,
response
pathways.
Within
the current
understanding
dinucleotide
neutrophil
modes
of signal
study
Chemotax-
degranulation,
may
problem
life.
destruction,
repertoire:
integrated,
The
dynamic
and
multi-component
highly
form.
major
but
phagocytosis,
require
although
dissected
a short
ofsurveillance
PK-C
many
system
activation,
associated
at a site
the respiratory
is not the
sole
other
soluble
bypass
PK-C
complex
is a promising
burst
just
to the
This PK-C
for oxidase
agonists
and
altogether.34’35
activities,
for elucidating
proximal
enzyme.
cascade
metabolic
avenue
final
activation,
Although
expression
dependent
phagocytosed
FMLP
of parallel
and
cross-over
points
in the
of
pathway
however,
since
particles
may
bypasses
C in elicited degranulation
and oxidase activation,
motactic
response
is reduced by PK-C inhibition.3638
tion
the
oxidase
complex
PK-
the cheDefinihierar-
chy of the activation
process have yet to be discerned,
and
there is only general
acknowledgment
that more than one
activation
pathway
may generate
an active oxidase.
Recent studies have focused on the specificity
of PMA for
PK-C in eliciting
a given biologic
response.
The synthetic
diacylglycerol,
I -oleoyl-2-acetylglycerol
(OAG),
directly
From the William B. Castle Hematology
Research
Laboratory.
Boston City Hospital,
and the Departments
of Medicine,
Biochemistry, and Pathology,
Boston University
School of Medicine.
Supported
in part by grants No. A120064
and HL33565from
the
National
Institutes
ofHealth,
Bethesda,
MD.
Submitted
April 4, 1986; accepted
October 7, 1986.
Address
reprint requests
to Dr Alfred I. Tauber, FGH-l,
Boston
City Hospital,
Boston, MA 02! /8.
© 1 987 by Grune
& Stratton,
Inc.
0006-4971/87/6903--000l$3.OO/O
711
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ALFRED
712
.
stimulates
PK-C and mimics PMA stimulation
of oxidative
burst
activation.39
Although
both PMA#{176}and OAG4’
at
submaximal
concentrations
exhibit
synergism
with the calcium ionophore,
A23187,
OAG has an apparent
calcium
dependence
that
PMA
lacks.42
Another
difference
in the
HL-60
differentiation
model,
in which
:
Fig 1 .
Schema
of human neutrophil
NADPH-oxidase
activation
as discussed
in text. PDE. phosphodiesterase;
DG, diacylglycerol;
PLC, phospholipase
C; PLA2, phospholipase
A2; AA. arachidonic
acid; PS, phosphatidylserine;
Ins 1 .4.5PO-inositoI
triphosphate;
02-. superoxide;
ER. endoplasmic
reticulum.
The NADPH-oxidase
is depicted
as two adjoining
square boxes.
transiently
produced
by certain
surface
turnover
intensively
has
and the
studied
been
shows
RECEPTOR
Correlation
of phorbol
ester’s affinity
characteristics
to
activation
of the burst established
that PMA and its analogue,
phorbol
12,13
dibutyrate
(PDBu),
stimulated
NADPH-oxidase
through
a specific receptor)7”8
Extension
of studies
with intact cells to the subcellular
assessment
of
the oxidase demonstrated
a similar dose-response
activation
profile.’8 This evidence,
linking
ligand-receptor
binding
to
initiation
of the
respiratory
burst,
is important
support
that
NADPH-oxidase
is indeed the principal
enzymatic
activity
of the human neutrophil
oxidative
metabolic
response.
Characterization
of the phorbol ester receptor
showed that it was
not susceptible
to sulfhydryl
reactive reagents,
stilbene disulfonic acids, or neuraminidase,
which explains
the earlier
findings of a lack of effect of these agents on the generation
of 02_ in PMA-stimulated
cells, in contrast
to cells stimulated with opsonized
zymosan
(OZ).49
The stimulus
specificity demonstrated
that the inhibition
acted
on distinct
plasma
membrane
receptors
and not on an oxidase exposed
to the extracellular
medium.
Studies of the PMA receptor
suggested
new approaches
to
defining
the unknown
pathways
leading from receptor
binding to activation
the
phorbol
of the
oxidase,
with
the
demonstration
that
ester
receptor
is the calcium/phospholipiddependent
PK-C.’6
In the neutrophil,
PK-C
exists as a
cytosolic
aporeceptor,
with high phospholipid
affinity
for
phosphatidylserine
(PS).50’51 Phorbol esters mimic the elaboration of endogenous
diacylglycerol
required
for an active
PK-C complex’6’52
(Fig I ). Diacylglycerol
is generated
as
part of the complex
phosphatidylinositol
(P1) catabolism,
which
leads
to the elaboration
of inositol
triphosphate,
important
for the release of intracellular
calcium,
a second
messenger
in diverse stimulus-coupled
reactions.53
Normally,
diacylglycerol
is almost
absent
from membranes
but is
from
P1 in a signal-dependent
membrane
receptors.54’55
accumulation
in several
described
manner
manner
The
of diacylglycerol
tissues,
and a similar
in neutrophils.55’
multifunctional
PK-C
catalytic
has been
pathway
activated
activity
of P1
role
in this
and
appears
to play roles in transmembrane
control of protein
phosphorylation in a variety of mammalian
tissues. Phorbol esters are
able to substitute
for the diacylglycerol,
and with PS and
Ca2,
PMA
directly
activates
the kinase
and probably
bypasses
ESTER
c.’
MtboIfl#{149}
OAG
totally
fails to mimic the PMA-induced
maturation
of this
tumor
cell line45; this apparent
dissociation
of PMA and
PK-C has been confirmed
with Bryostatin,
another
activator
of PK-C that blocks PMA-induced
HL-60 differentiation.
Interpretation
of these data are difficult,
however,
because
another
target in addition
to PK-C may be involved
in this
system,
modulating
the PK-C effect. Bryostatin,
however,
like OAG, does induce
human
neutrophil-specific
granule
release and oxidative
burst similar to that of PMA stimulation.#{176}Phosphorylation
products
were also similar
when
PMA and Bryostatin
were compared
in the same system (see
below); thus, the ambiguity
of the role of PK-C in HL-60 is
not evident in the neutrophil.47
Furthermore,
the evidence
for
the role of PK-C in PMA-stimulated
neutrophils
rests most
firmly on the reconstitution
studies discussed
(sic infra).
PHORBOL
Uo.0
in
response
is the enhancement
with calcium
and arachidonate
products
observed
with OAG stimulation,
but not found with
PMA.43-”
Dramatic
functional
differences
have
been
observed
I. TAUBER
cyclic
AMP-
and
cyclic
GMP-modulated
ways.’652#{176}Thus,
it is expected
that pertussis
blocks the dissociation
of regulatory
G proteins
inhibits
their
role
in signal
PMA-stimulated
cells,
transduction,6’
in contrast
with FMLP.62 A caution
is raised
second undefined-G-protein-sensitive
cytosis
in PMA-activated
cells,63
pathtoxin,
which
and thereby
has
no effect
to neutrophils
on
activated
by the observation
that a
site may regulate
exosuggesting
the
complexity
of neutrophil
response
to this single agonist.
In the presence
of optimal
phospholipid
and phorbol
esters,
the kinase
is fully active at l0
mol/L
of Ca2,
implying
increase
that the enzyme
may be activated
without
a net
in intracellular
Ca2 concentration
when the appro-
priate
receptors
are
stimulated.
Indeed,
PK-C
has
been
reported
as active at vanishingly
low Ca2 concentrations,TM’65
and in PMA-stimulated
neutrophils,
a rise in intracellular
calcium
is not
This
2.68
indicator
observed
observation
probes
that
fact.6’ Stimulation
teleocidin)
other
with
the
fluorescent
probe
Quin-
is based
on the use of chemical
may introduce
an observational
arti-
of PK-C only with
than diacyglycerol
agents
(phorbol
demonstrates
esters,
this low
Ca2 dependence.
Pontremoli
and colleagues
recently
suggested7#{176}
that PMA indeed mobilizes
intracellular
Ca2 but,
because
of concomitant
activation
of a Ca2
extrusion
pump,7’ a net change
in intracellular
Ca2 is not observed.
The role of Ca2 mobilization
in PMA-stimulated
neutrophils
has
generation
also
been
observed
suggested
with
by
agents
the
that
inhibition
inhibit
of
02
intracellular
Ca2
mobilization72
or calmodulin,73’74
a Ca2-dependent
regulator
of diverse function.
These studies have been difficult to evaluate
because of the nonspecificity
of the inhibitors
used67’75;
conclusions
as
to
the
role
of
ancillary
Ca2-
dependent
activities
on PMA stimulation
of the burst cannot
as yet be made. Finally,
Dougherty
and Niedel showed that
agents which alter cytosolic
Ca2 levels modulate
binding of
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NEUTROPHIL
phorbol
PROTEIN
diesters,
synergism
KINASE
C
suggesting
between
713
a mechanism
subthreshold
for the observed
activation
of
Ca2
the
burst
by PMA,
Phospholipid
and
in contrast
metabolism
to particulate
accompanies
PK-C pathways.76
In this regard,
the low level of recovery
from broken cells of NADPH-oxidase
with the reconstituted
PK-C system
(see below)
may be due to omission
of other
and likely contributes
to membrane
the stimulated
cell.3’85’86 Changes
metabolic
dramatic
tributaries
contributing
to
PMA-initiated
position
burst
In the case
physiologic
cytosolic
form
calpain,
of micromolar
concentrations
rise in Ca2
induced
of PK-C and a Ca2
becomes
associated
with
of calcium
(a
by many
agonists),
the
requiring
thiol proteinase,
the neutrophil
The activation
of calpain converts
PK-C to a proteolytically
modified
Ca2/phospholipid-independent
form, both in broken cell preparations
and in intact
cells stimulated
with
PMA.78 Once the translocated
PK-C has undergone
proteocleavage
to a 65-kd
the cytosol
protein
species
in an active
substrates.
(from
form,
85 kd),
presumably
Whether
it is released
accessible
oxidase
activation
requires
membrane-bound
PK-C51’7#{176}remains
to be demonstrated
the
system.
thus
reconstituted
The
PK-C
may be activated
different
substrate
targets,
ity to the control
tions.
Furthermore,
data
to
to other
far
in
suggest
that
by two separate
systems
with
adding a second level of complex-
modulation
of C-kinase-mediated
the stability
of PK-C
binding
functo the
of PS
for the
cells
with
thetic
diacylglycerols.8#{176}
Of a series
of synthetic
diacylglycerols,
didecanoylglycerol
(diC10)
induced
mal
enzyme
burst.
phorbol
secretion
Unlike
ester
but
other
binding,
binding
sites.
PK-C binding
The
suggested
that
activation
of
failed
to stimulate
diacylglycerols
diC10 competed
data
showed
that
the
sn 1,2lysoso-
respiratory
competed
for only 45%
a correlation
syn-
for all
of the
with a limited
cellular
response,
and further
a discrete
pool of PK-C
was responsible
for
the
NADPH-oxidase.
These
findings
are
of
particular
interest
in regard
to the recent identification
of
multiple,
distinct
forms of PK-C by cloning techniques.83
The specificity
and regulation
of response
has thus acquired
a new
specific
molecular
complexity.788#{176} Determination
PK-C
isoenzymes,
with possibly
differing
characteristics
ticated
and
dimension
nous
PK-C
tase)
associated
target
proteins,
to the
study
inhibitory
characterized.84
with
promises
of PK-C.
(non-protease,
the specific
granules
the substrates
the putative
selective
target
lar localization
of activated
to add
Finally,
factor
Its role,
of typeactivation
has
been
pools of PK-C,
PK-C
require
partially
system,
and the subcelluextensive
coordi-
nated study to define physiologic
control
mechanisms
of
phorbol ester stimulation.
The rapid flux in lipids, mobilization
of arachidonic
acid
(AA) by the activation
of phospholipases,
and the extensive
turnover
of membrane
phospholipids
during
phagocytosis
represent
associated
metabolic
activities
of the respiratory
burst.3’”’57
Inhibitors
chemotaxis
and
tion
direct
of
phagocytosis
phospholipid
and
sequence
of the
respiratory
evidence
linking
phospholipid
turnover
may
do
interfere
burst,2’3’5
turnover
inhibit
in the activa-
but
there
to activation
first
3 minutes
of stimula-
is cytosolic
and has a
appears
most effective
suggests
that the PS elabofor PK-C
activity,
but the
receptor,’6’5154
role
by which
PS
is elaborated
in PMA-stimulated
biochemical
parameters
of the two agonists
been
defined
(Fig 1 ). PMA differs from OZ stimulation
in its susceptibility to a PK-C
inhibitor
(isoquinolinesulfonamide,87)
lack of PMA-initiated
release
from prelabeled
cells.35 The
pholipase
A2
(PLA2)
has been
in the
implicated,
observed
release
and
with
and
the
of radiolabeled
AA ([3H]AA)
specific
involvement
of phosof AA
in OZ-stimulated
the partial
inhibitors
reduction
of
AA
of 02-
metabolism
(BW755C
and indomethacin)
further
supports
a linkage
between
AA release and 02- generation
in OZ-stimulated
cells.35 Finally,
like 02- generation,
[3H]AA
release in OZstimulated
from
cells
is calcium
dependent,35
calcium-independent
that
PMA
exogenous
AA
another
difference
activation.*
stimulates
the
It is of
respiratory
burst
in intact cells seemingly
by the same PLA2-dependent
pathway as with OZ, but in this case, inhibition
of AA metabolism
has
no
effect
on
particle
stimulation
PMA in that the
calcium
suggest
In
toto,
NADPH-oxidase
stimulus
opsonized
differs
is PLA2
from
mediated,
and independent
of PK-C.
These
physiologic
stimulus,
binding
to
receptors,
distinct
generation.88
particulate
dependent
that
this
membrane
02-
of the
induces
from
that
an activation
generated
when
studies
plasma
cascade
biochem-
PK-C
is directly
stimulated
by phorbol
esters.
PMA,
in bypassing
these
associated
lipid metabolic
activities,
allows examination
of
the activation
cascade
at a point distal to them. This is a
useful juncture
at which
to study
the activation
of the
NADPH-oxidase
directly.
an endoge-
non-phospha-
of the calpain
the
most
which may activate
the P1 cascade
described
above,56
02- release
by a mechanism
distinct
from that of
PMA.35 This has been amply demonstrated
with neutrophils
from patients
with paroxysmal
nocturnal
hemoglobinuria3
and a congenital
neutrophil
dysfunctional
case,2’ in which
response
to OZ was intact, whereas
PMA could not initiate
a
normal
respiratory
burst. Only recently
have some of the
ically
a sophis-
during
the
a net elabo-
Oz,
interest
of selective
dependent,
cells being
stimulates
formation
oxidase
stimulus
seen in
com-
is not known.
demontrated
of the
noted
a functional
mechanism
cells
activation
and
in PMA-stimulated
activated
has
membrane
appears
to vary with the stimulus
used.79
Physiologic
control
mechanisms
have been complicated
further
by the recent findings
of Cox and co-workers,
who
selective
time
process
fluidity
changes
in total phospholipid
tion.29’#{176}
That the PMA aporeceptor
uniquely
high affinity for PS, which
rated
membrane.77
both
change
ration
activation.
lytic
are
stimulation.3”
the activation
is no
of
5A role for Ca2
in PMA-activated
neutrophils
has
been
1mph-
studies, which show that when cells are depleted
of intracellular
Ca2, or when intracellular
Ca2 mobilization
is
blocked,
02 generation
and depolarization
are abolished
in a
Ca2-free
buffer. The effective extracellular
Ca2 concentration
that
restores
50% activity is 100 mol/L
for depolarization
and 1 mol/L
for 02
production,
suggesting
different
requirements
for these
functions.
Considering
the Ca2 requirement
of PK-C, these fIndings
cated
are
in preliminary
expected,
stimulation
and
thus
the
is only relatively
apparent
Ca2-independence
low as compared
to other
of
agonists.’’
PMA
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ALFRED
714
PK-C
ACTIVATION
OF
trophils,33
NADPH-OXIDASE
enabled
reconstituted
definition
of the
oxidase
system.
system
was
The respiratory
burst enzyme,
NADPH-oxidase,
is maximally stimulated
by PMA49; the electrons
utilized to produce
reactive
oxygen
species
arise from the activation
of the
hexose monophosphate
(HMP)
shunt, which not only sup-
tuted
Ca2
dependence
plies
demonstrated
with
the addition
the cytoplasm
and
leached
NADPH
(the
reducing
agent)
but
also
stoichiometri-
cally generates
one proton
for each electron
required
to
reduce
molecular
oxygen
to superoxide.89
The protons
are
secreted
largely by a sodium-proton
antiporter,
whose control of cellular pH appears
to regulate
cell activation
in an as
yet
undefined
manner9#{176} In
activation
of
binding
was
[3H]PDBu
the
the
initial
NADPH-oxidase,
measured.9’
binding
Almost
was
found
studies
distribution
95%
in the
of
the
cytosolic
unstimulated,
nitrogen-cavitated
neutrophils,
binding
activity
of 8.6 pmol/mg
protein.
of
PK-C
of
PDBu
of
with a specific
The
Kd for
[3H]PDBu
binding
to the cytosohic
fraction
reconstituted
with PS determined
by Scatchard
analysis
was 1.2 nmol/L
(n
3). This value
is similar
to that
determined
for the
partially
purified receptors
from mouse brain cytosol.92 This
affinity
is considerably
higher
than that observed
in intact
cells (38 nm),’8 presumably
reflecting
the reconstitution
of
the receptor
in PS rather than the actual phospholipids
of the
=
the
binding
activity
was
between
and PMA-stimulated
in other studies.’9’5’
has been
distribution
found
the
in the
distribution
of
the
NADPH-oxidase
ly, the levels
added
It remains
in activating
of the
plasma
membrane-bound
is direct
suggested.
tives
unable
to stimulate
AA,
the
oxidase
in disrupted
neutrophils
PK-C
in
Quantitativewith
fled
from
does
not
or stimulation
The
that
in a
was
not
supporting
initial
are
The
these
observations,
characterization
however,
process.
is used
In both AA
systems,
no signifi-
had
and
SDS
oxidase
of PMA.’#{176}
of the cytosolic
stimulation
of
are Mg2-dependent,
for
in vitro
pathway
of AA
that
pun-
mechanism
metabolic
pathways
from
observa-
in the
by a nonenzymatic
independent
arachidonate
although
they
GTP
with
ester
partially
NADPH-oxidase
broken-cell-stimulated
inhibitor
isoquinohinesulfonamide
effect,
activation
using
by anachidonate
another
occurs
phorbol
unpublished
brain.’05
either
derivaspecific
an ID50 of 190 imol/L
obtained
correlate
that
acid
inhibits
neutrophil
and
factor
as originally
fatty
AA
Tauber,
activation
suggests
SDS
PK-C
A.I.
mouse
NADPH-oxidase
and
the
to the cytosolic
to those
been
oleic
soluble
to be PK-C,
oxygenated
a K, of 38 imol/L
PK-C
setting
the
(SDS)’#{176}’to the membrane-
not appear
and
has
AA,998
an additional
or other
similar
the
factor(s)
oxidase
has
phosphohipid,
not involved.’#{176}’#{176}7
Curnutte
and
shown
ATP,
colleagues
for
that
and
showed
their putative
soluble
factor
had no PK-C
activity,
and
patient
missing
this factor,
the level of neutrophil
PK-C
normal.’#{176} As in their previous
report,’#{176} the patient
did
respond
to PMA,
suggesting
that the cytosolic
factor
was
either
independent
activation.
activator,
of PK-C
tion by AA”#{176}and
preliminary
report
Finally,
the
oxidase
AA and
that
cell
linkage)’4
and
other
has
whereas
depend
its role
distal
soluble
agonists,””3
used a calmodulin
to PK-C
but
inhibitor
AA-stimulated
system
and shown
role of calmodulin
in neutrophil
is Ca2
the
its
relation
correlation
observed
with
dependent
latter
on a putative
to
between
system
PLA2
PK-C,
intact
broken
and
cells
releases
is Ca2
function
only one
in the
is as yet
cell
with
in that
prelabeled
for oxidase
unclear.
activation
is poor
independent
kinase
activa-
a possible
activation,
The
in particular,
former
or played
It is of interest
that the multifunctional
calmoduhin,
has been implicated
in oxidase
broken
In these PK-C reconstitution
studies,
separation
of the
cytosol
fraction
and a light membrane
fraction,
the site of
NADPH-oxidase
in prefractionated
PMA-stimulated
neu-
be
Ca2
from
cytosol,
corresponded
directly,’#{176}2’#{176}
and
binding
with
when tested at concentrations
corresponding
to -50-fold
the
Kd for the phorbol
ester
receptor;
however,
under
specific
conditions
appropriate
for PK-C activation,
namely inclusion
of PMA,
PS, ATP, and a cytosohic
factor,
the NADPHwas activated.9’
only
presumably
activated.
cytosol
studies,
it does
oleate,
activate
receptor,
NADPH-
oxidase or modification
of a cytosolic
regulatory
component
which could then act on the oxidase.
Regardless
of the transport
of PK-C in intact cells, which
appears
to require membrane
binding in physiologic
stimulation,95 in vitro manipulation,
based on these binding studies
of phorbol ester, has reconstituted
a functional
activation
of
the NADPH-oxidase.
Previous studies showed that the membrane-bound
NADPH-oxidase
may be activated
in a broken
cell system by the addition
of AA to a cytosolic
factorY98
Those studies were extended
by characterizing
the pathway
of oxidase activation
through
the PK-C system.
PMA alone
was
but
Blumberg
if the site
was
PK-C
obtained
substituted
for
similarly
sulfate
In these
values
to be determined
Ca2
purified
were
in the
dodecyl
oxidase.
(P.M.
the NADPH-oxidase
could
Residual
enzyme.
was required,
cant
phosphorylation
purified
bound
has
resolved.
membrane
was
of PK-C
acid,’#{176}#{176}
or sodium
and
of PK-C
of EGTA.
As noted,
activation
of NADPH-oxidase
achieved
in broken cell preparations
by adding
under other lysis conditions,
these discrepancies
may reflect
the extractability
of the potentailly
redistributed
receptors
under
certain
fractionation
conditions.
Because
different
methods
of cell breakage
and assay have been used, this issue
not been
NADPH-oxidase
activation
on particuPK-C
studies
in other
of the reaction
preparations.
When
brain
and its co-factors
tions),
changes
in apparent
PK-C
some other
systems5170’7793’94
for
of oxidase
that for
of the
of a reconsti-
sufficient
for reconstitution
of activity.
The dose-response
curve for PMA revealed
a half-maximally
effective
dose of
1.1 nmol/L,
which was consistent
with the high affinity of
PMA for PK-C
and the measured
amount
of PK-C
in the
of
fractions
requirements
component
required
dependence
paralleled
in contrast
to
ester treatment
subcellular
neutrophils,
Because phorbol
reported
to induce
in neutrophils
and
of action
The
[3H]PDBu
neutrophil
membranes.
No significant
difference
control
findings
tissues.92
cytosol
mouse
specific
fraction
PK-C
expression.
The
lar phosphohipids
basic
Each
I. TAUBER
the
[3H]AA
and
does
not
expression.88
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NEUTROPHIL
PROTEIN
KINASE
The
suggest
distinct
data
NADPH-oxidase
fractions
C
715
mechanisms
by which
the
in intact cells and membrane
is stimulated
Although
the
precise
correlates
discerned,
the
activate
stimulatory
of AA
evidence
the
pathway
or SDS
is compelling
Because
that
by
the
have
a
not
AA
and
activation
SDS
groups
is only
I 5% as active
as that obtained
with AA or SDS activation
(which
are comparable),’#{176}
it may represent
either
a less
efficiently
SDS
reconstituted
activation.
system
The
activation
may
suggest
expression.
One
model
movement
and juxtaposition
tron
transfer
leagues,
may
from
activate
the
intact
locale
by
their
on the
PMA/PK-C
(or possibly
Although
juxtaposition
tive
activate
for electron
the
result-
PK-C
PHOSPHORYLATED
PRODUCTS
The roles of neutrophil
protein phosphorylation
examined
with regard
to the activation
of the
burst,
since
increased
concomitant
or
phosphorylation
proteins
represents
variety
of
human
their
kinase
activity
or dephosphorylation
an important
mammalian
neutrophil
functional
phosphatase
have been
respiratory
regulatory
cells.
Earlier
protein
kinases
roles
have
not
of target
mechanism
work
and
been
has
but
deter-
with our new
of well-defined
broad
substrate
endogenous
unidentified
ly, protein
acceptor
phorylate,
many
suggests
extent,
rylated
specificity
and
proteins
phosphorylates
in most
tissues.
Apparent-
to various
extents,
membrane-bound
that
the
but several
specifically
seryl
proteins.
sites
kinase
threonyl
residues
of PK-C
from
protein
located
C rapidly
threonyl
A
preliminary
of phosphorylation
seryl and
by PK-C.
ity is well illustrated:
residue
(senine-38)
and
overlap
threonyl
residues
are
The relative
reaction
to some
phosphovelocities
used, and the seryl and
and location.
When
H I
the biochemical
specific-
kinase
A greatly
favors
a seryl
near
the N-terminus,
whereas
phosphorylates
both
seryl
and
located
near the C-terminus.”9”#{176}
Isolation
bovine’2’
and
rabbit’22
neutrophils
was
Segal
CGD
of such
mentioned
by Orkin
and
X-hinked
significance
the X-linked
and
tion,
X-linked’24
of 44
associated
remains
CGD
has
CGD
to 48
with
the
been
kd.
a defecmolecular
established.
to be resolved.
is well
known333
co-workers
cytochromes
and
proteins
does not find a phosphorylation
patients,
but finds such a defect
cases,’23
cloning
cases
that
has
even
raised
Hetero-
and
the
recent
protein
found
homology
with
of a missing
no
uncertainty
as to the
of the b cytochrome
as a molecular
disease.’26
The tantalizing
reports
marker
of
of Segal’23
Babior’24
need to be pursued,
and characterization
of the
proteins
in normal
Previously,
a major
with
putative
identification,
isolacrucial
phosphory-
cells.
difficulty
appears
to have
arisen
from
the use of insufficiently
defined
systems
for analysis.’24’2’
Because
the NADPH-oxidase
has been localized
to the
plasma membrane,
its seems appropriate
to study the phosphorylated
purified
products
oxidase
at
this
site,
preparation.
such
a
or
protein
was
preparation
prominently
Sha’afi’s
the
in plasma
previously
also identified
substrate;
while
relation
to
the
elucidated.’30”3’
system
incubated
but
of
50
kd
was not
appears
it correlates
closely
activation
cascade
the
over Ultragel.
but a 3 1 .5-kd
by
an
apparent
that
this
characterized
a 3l-kd
PMA-stimulated
membranes
protein
colleagues
neutrophils;
he suggested
rabbit
plasma
membranes,
yielding
a small,
This protein
was the only one identified
Another
and
and filtered
products,
and
of
group
in
pig
phosphorylated
a component
product
in a partially
Rossi
from
mechanism,’28
was
better,
Recently,
NADPH-oxidase
was solubihized
Rossi found several phosphorylated
rylated
in
previously
has not previously
defects
oxidase.’
analysis
identification
b cytochrome,’#{176}”25
CGD
of CGD
product
residues
for
surprisingly,
the
has been
low potential
PK-C-mediated
many
kinases
C and A often
use the same
phosphate
proteins.”7”8
For example,
both
enzymes
phos-
differ
among
the substrate
proteins
threonyl
residues
vary in amount
histone
is used as a model substrate,
protein
is one
protein
PK-C
techniques
the
(a)
of
CGD
in autosomal
examined
Recognition
of relevant
phosphate
acceptor
proteins
of the major
problems
for defining
the effects
of
kinases
in general.
When
tested
in cell-free
systems,
and
by several
Not
recessive’23
geneity
hated
identified
X-hinked
The discrepancy
that
defect in his X-linked
in a
phosphatases,
definitively
mined.”6
This issue may now be reexamined
understanding
of PK-C and with the use
subcellular
fractions.
shows
with
that
from
autosomal
of autosomal
known
PUTATIVE
in the
studied
sources
phosphorylation
or absent
in some
transfer.
both
lack
only
phosphorylamediator)
components,
that
basis
physiological
from
studies
indicating
that a functional
oxidase
arises
from the
reconstituted
PK-C
system,
and (b) the recent
demonstra-
AA
system
is that
an intermediary
of oxidase
come
that
and
been
products.
of evidence
suggested
membrane
neutrophil
phosphorylated
lines
patients
AA and SDS
different
is crucial
tions
by effecting
preparations
phosphorylation
has
important
col-
burst
characterization
PK-C
protein
products
for elec-
first
to
the reported
findings show discrepancies
and inconsistencies,
making a coherent
statement
as to the identification
of PK-C
dependent
phosphorylations
impossible.
The two most
and
cells,
that
using
Badwey
components
properties.’#{176}’ The
the stereo-relationship
ing in effective
or
for oxidase
of the
detergent
correlation
to the
tion of a component
of AA
is based
to oxygen.
respiratory
in the
required
expression
of oxidase
and Pick postulate
oxidase
affects
properties
for oxidase
with
changes
Bromberg
the
arm
of the PK-C-independent
NADPH
in studies
fluidity
or an ancillary
mechanism
biochemical
of NADPH-oxidase
demonstrating
oxidase
and
closely
tissues.’6
been
PK-C-independent
PK-C-stimulated
Activity
The role of PK-C-mediated
or the physico-
activation
NADPH-oxidase
mechanism.
reported.
to correspond
other
by AA.
chemical
recently
appear
phosphoneutrophil
but consistent
as exclusively
image.
found
further
characterized.’3’
to be a major
PK-C
with
degranulation,
has
not
been
An early
study
of a reconstituted
with disrupted
neutrophils
identified
its
further
PK-C
major
phosphorylated
membrane-bound
products
of 38 and 34 kd,
but the relationship
of these products
to oxidase activation
was
not
investigated.#{176}
sively
in the
phils
prestimulated
Babior
particulate
fraction
with
found
a 22-kd
of disrupted
PMA.’32
Romeo’s
protein
human
group,
excluneutrousing
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ALFRED
716
enucleated,
found
not
granule-free
cytoplast
six PK-C-mediated
attempt
function.’33
earlier
guish
to relate
One
of
ghosts
phosphorylated
these
these
Romeo
study
PK-C specifically
targeted
products
of human
labeled
(cytosol-free)
products,
but
proteins
did
protein.
neutrophils
could
proteins
primarily
An
not distinbecause
of
intact
cell
patterns
study.
46-kd
proteins
in a membrane
corresponding
precipitate,
size
of the
missing
was not estabin light of the
proteins
reported
CGD studies
of Segal and Babior.’23”24
Other activators
of PK-C have also been
phosphorylation
products
of
the
human
preloaded
cells stimulated
with OAG, and
gel electrophoresis,
Fujita
and co-workers
prominent
oxidase
PMAunique
images,
but did
not attempt
used
in the
to examine
neutrophil.
any
of these
to
activation.’39
Berkow and Kraft recently
compared
and Bryostatin-stimulated
neutrophils
and found
products
of 93, 88, 66 and 45 kd in a one-dimensional
system47;
Bryostatin
induced
phosphorylation
of
an
(c)
approaches
to
to oxidase
expression
the high
number
of
which
the
not
are
not
complexity
membranes
are
used;
from
and
products
directly
defining
the
(d)
Purified
containing
PK-C
the
the
controlled
same
process
with
selected
inhibitor
offers
minimal
to oxidase
with
phosphorylation
studies
and
expression.
for
identified,
characterized,
system
Although
this
and
is an
membranes
to examine
reactions
and
NADPH-oxidase
may
use a minimal
products
for
purified
this
the
fewest
activation
PK-C system, which can then be confirmed
COD cells and corresponding
studies
with
activation
New
PMA/PK-C-
targets.
Corresponding
kinetics,
specificity
should
identify
those
PK-C
crucial
or
of relating
of
the opportunity
competing
in
animal
necessity
mechanism
incubated
the oxidase
from other
competing,
of phosphorylation
mediated
activation
of NADPH-oxidase
must
system
to reduce
the number
of phosphorylated
products(s)
Using
two-dimensional
observed
four
to link
activities,
source
phils,
the
kinase
if purified
human
phosphorylated
found
crucial
(b)
systems;
the undifferentiated
system of analysis used.’
Okamura
and
colleagues
attempted
to relate this 46-kd species (probably
two proteins
with distinct
pis of 7.4 and 6.4) to NADPHoxidase
activation.’35
This study,
using guinea
pig neutrobut again their relation
to oxidase activation
lished.
We find the claim interesting
only
products
activities;
non-PK-C
to oxidase
is a 47-kd
guish those
stimulated
I. TAUBER
isolated,
the
be dissected
ambitious
the
NADPH-oxidase
and
then
undertaking,
tracing
this receptorligand-coupled
ing of a major
mechanism
of signal
by
with studies
of
intact
cells. Once
reconstituted.
the
reaction
transduction.
reward
for
is understand-
addi-
tional protein of 70 kd. The similar pattern supports
the same
mechanism
of cell activation
by these stimuli.
These previous
studies alerted
workers to the problems
of
(a) using a prelabeled
intact cell system that cannot distin-
ACKNOWLEDGMENT
I gratefully
appreciate
Blumberg
and acknowledge
Happnie.
the useful comments
the expert secretarial
of Dr Peter M.
assistance
of AM.
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NEUTROPHIL
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1987 69: 711-720
Protein kinase C and the activation of the human neutrophil NADPHoxidase
AI Tauber
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