HE JOURNAL CHEMISTRY
OF BIOLOGICAL
0 1994 by The American Societyfor Biochemistry and Molecular Biology, Inc.
Protein Kinase C-dependentand -independent Pathways of
Mitogen-activated Protein Kinase Activationin Macrophages by
&*
Stimuli That Activate Phospholipase
(Received for publication, March 25, 1994, and in revised form, May 13, 1994)
Zhi-Hua Qiu and ChristinaC. Leslie$
From the Division of Basic Science, Department of Pediatrics, National Jewish Center for Immunology and Respiratory
Medicine, Denver, Colorado 80206 and the Department of Pathology, University of Colorado School of Medicine,
Denver, Colorado 80262
The activation of mitogen-activated protein kinase
( M A P kinase) in macrophages and the involvement of
protein kinase C (PKC) in MAP kinase activation was
investigated in macrophages exposed to agents that
have previously been shown to activate the 85-kDa cy-
tosolic phospholipaseA, (PLA,)and induce arachidonic
acid release. Phorbol 12-myristate13-acetate(PMA)and
zymosan maximallystimulated MAP kinase activity by 5
and 15 min, respectively,whereas the response to oka-
daic acid was maximal by 60-90 min. MAP kinase acti-
vation correlated with tyrosine phosphorylation of p44
MAP kinase in PMA-stimulated cells and p44 and p42
MAP kinases in zymosan- and okadaic acid-stimulated
cells. MAP kinase activity was not elevated in A23187-
stimulated macrophages. Inhibition of PKC with the in-
hibitor, bisindolylmaleimide (GF109203X), or by pro-
longed exposure to PMA suppressed both arachidonic
acid release and MAP kinase activation in PMA- and
zymosan-stimulated macrophages but not in okadaic
acid or A23187-treated cells. However, prolonged expo-
sure to PMA did not suppress the increased cytosolic
PLA, activity in agonist-treated macrophages. This ap-
proach was complicatedsince initial exposure to PMA to
down-regulate PKC increased cytosolic PLA, activity
which remained elevated for 16 h. In contrast,
GF109203X treatment suppressed the increase in cyto-
solic PLA, activity in response to zymosan and PMA but
not to okadaic acid or A23187. The results demonstrate
that PMAand zymosantrigger PKC activation that leads
to theactivation of MAP kinase and PLA,, whereas these
responses are PKC independent in okadaic acid-treated
cells. In addition, the results are consistent with a role
for MAP kinase activation in regulating the activation of
the 85-kDa PLA, and arachidonic acid release in PMA-,
zymosan-, and okadaic acid-stimulated cells, whereas
these responses in A23187-treated cellsare MAP kinase-
and PKC-independent.
PLA, enzymes play a central role in the production of in-
flammatory lipid mediators, the eicosanoids and platelet acti-
* This work was conducted in the F. L. Bryant Research Laboratory
for the Mechanisms of Lung Disease and supported by National Insti-
tutes of Health GrantHL 34303. The costsof publication of this article
were defrayedin part by the payment of page charges. This article must
thereforebeherebymarked advertisement in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed: Dept. of Pediatrics,
National Jewish Center for Immunology and Respiratory Medicine,
1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1214;Fax: 303-398-
1851.
The abbreviations used are: P q , phospholipase 4;PMA, phorbol
12-myristate13-acetate; MAP kinase,mitogen-activatedprotein ki-
nase; PKC,protein kinase C; ECL,enhancedchemiluminescence;
19480
Vol. 269,No. 30,Issue of July 29,pp. 19480-19487, 1994
Printed in U.S.A.
vating factor (1-3). Mammalian cells contain multiple struc-
turally diverse forms of PLA,, and therehas been considerable
interest in determining the role that specific PLA, enzymes
play in mediating arachidonic acid release. Recent evidence
suggests that depending on the cell type and stimulus involved,
different forms of PLA, including the85-kDa cytosolic PLA, (4),
the 14-kDa secreted group I1 P L 4 (5-7), and a calcium-inde-
pendent cytosolic PLA, (8,9) canplay a role in arachidonic acid
release. We have been interested in understanding themecha-
nisms involved in the regulation of the 85-kDa cytosolic PLA, in
macrophages, which are an important source of lipid mediators
produced during inflammation. Previous studies have shown
that diverse agents that stimulate arachidonic acid release in
macrophages, including zymosan, PMA, A23187, and okadaic
acid, activate the 85-kDa P- by enhancing serine phospho-
rylation of the enzyme (10).In fibroblasts and Chinese hamster
ovary cells, serine phosphorylation of the 85-kDa PLA, has also
been shown to correlate with enhanced cytosolic PLA, activity
and arachidonic acid release (4).
Studies recently have been carried out to identify the kinases
that maybe involved in phosphorylating the85-kDa PLA, and
have implicated a role for MAP kinase. MAP kinase isa proline-
directed serinelthreonine kinase that is activated in many cell
types by diverse stimuli (11-13). The 85-kDa P q can be phos-
phorylated by MAP kinase in vitro, which results in both a n
increase inactivity ofthe PLA, and a decrease in electrophoretic
mobility (14, 15). Evidencehas also been provided that the85-
kDa PLA, is phosphorylated by MAPkinase in vivoand that this
phosphorylation event is importantfor activation of the PI.&
and arachidonic acid release (14). In addition to MAP kinase,
activation of PKC has been shown to play a role in P q acti-
vation. The PKC activator, PMA, can stimulatearachidonic acid
release in macrophages and induce phosphorylation and acti-
vation of the 85-kDa PLA, (10, 16, 17).In addition,down-regu-
lation of PKC, by prolonged exposure to PMA, has been shown
to suppressarachidonic acid release inmacrophages (18).PKC
can phosphorylate the 85-kDa PI& in vitro but has not con-
sistently induced an increase in activity (14,15,19). At this time
there is no evidence that PKC directly phosphorylates the PLA,
in vivo.Since PKC activation can lead to activation of MAP ki-
nase in several cell types, it is possible that PKC activation trig-
gers a kinase cascade that leads toP L 4 phosphorylation. This
study was carried out to determine if agents that enhance serine
phosphorylation and activation of the 85-kDa P q in macro-
phages, induce MAP kinase activation, and to determine role the
of PKC activation in mediating activation of M A P kinase.
GF109203X, bisindolylmaleimide; DMEM, Dulbeccosmodified Eagles
medium; FBS,fetal bovine serum; BSA, bovine serum albumin; HBSS,
Hanks balancedsalts solution; EGFR, epidermal growthfactor recep-
tor; Me,SO, dimethyl sulfoxide; A23187, calcium ionophore.
 Role of Kinases in Macrophage
Phospholipase A, Activation 19481
14
A

11

1c Okadaic Acid

8 PU
p42
-
-
6
"".. H
- 32.5
15' 30 60' 5' 15' 30' 30' 60' 90'
"
U
4 Unstimulated Zymosan Okadaic Acid

Unstimulated
2 B
e
-f
E
p44
p42 -
-
- l "

10 20 30 40 50 60 70 80 90 100 110 120

15' 30' 60' 15'
5' 30' 30' 60' 90'
Time (min) "
U
Fro. 1.Time course of MAP kinase activation in macrophages. Unstimulated Zymosan Okadaic Acid
Macrophages were incubated with zymosan (30 particles/cell, W , PMA C
(32 nM, A), A23187 (0.5 pg/ml, O), okadaic acid (1 PM, A), or Me,SO
(0.1%, unstimulated) for the indicated times a t 37 "C. MAP kinase
activity incell lysates was determined as described under "Experimen-
tal Procedures." Results are presented as the mean of two to four inde-
pendent experiments assayed in triplicate. 2' 5' 15' 2' 5' 15' 2' 5' 15'

Unstimulated PMA A23187

EXPERIMENTAL PROCEDURES FIG.2. Time course of stimulated tyrosine phosphorylation of
MAP kinase in macrophages. Macrophages were incubated with
Materials-[5,6,8,9,11,12,14,15-3H]Arachidonic acid (100 CVmmol),
zymosan (30 particles/cell), PMA (32 nM), A23187 (0.5 pg/ml), okadaic
and [y32PlATP(3000 Ci/mmol) were from DuPont NEN. Pathogen-free acid (1p ~ ) or , Me,SO (O.l%, unstimulated) for the indicated times. Cell
female ICRmice (8 weeks old) were from Harlan Sprague-Dawley. Poly- lysates (75 pg proteidane) were fractionated aon10% SDS-polyacryl-
clonal anti-MAP kinase (erkl-CT) antibody R2 and monoclonal anti- amide gel followed by immunoblotting with anti-phosphotyrosine anti-
phosphotyrosine antibody (4G10) werefrom Upstate Biochemicals,Inc. body (A and c)and anti-MAP kinase antibody( B )a s described under
(Lake Placid,NY).Monoclonal antibody to PKC isozyme type 111 (a)was "Experimental Procedures."
from Seikagaku America, Inc. (Rockville, MD). Anti-mouse IgG and
anti-rabbit IgG horseradish peroxidase-linked F(ab')2 fragment and the
ECL detection kit for immunoblotting were from Amersham Corp. Zy- PMA were added as a Me,SO solution, all cultures including zymosan
mosan, A23187, phenylmethylsulfonylfluoride,leupeptin,aprotinin, and unstimulated cells received Me,SO to a final concentrationof 0.1%.
histone (type 111-S), potato acid phosphatase (typeIII), 1,2-dioleoyl-sn- For experiments with the protein kinase inhibitor, the ["Hlarachidonic
glycerol, and FBS were from Sigma. Prior to use as a stimulus, the acid-labeled cells were washed and then preincubated for 30 min with
zymosan was suspended in phosphate-buffered saline and boiled for 10 an appropriate concentration of inhibitor before addition of stimulus.
min, followed by centrifugation. After resuspension in phosphate-buff- After stimulation, the media was removed and centrifuged a t 1400 x g,
ered saline, the boiling procedure was repeated two more times. One at 4mg
"C for 10 min, and the cells were scraped 1.0 into
ml of 0.1% Triton
of the zymosan was equivalent to approximately 1.3 x lo7particles. The X-100. The amountof radioactivity in the cells and media was measured
zymosan preparation itself did not exhibit any PLA, activity when by liquid scintillation spectrometry.
assayed under the conditions described below. Phosphatidylserine was PLA, Assay-Macrophages that were plated at 10 x 10fi/35-mmdish
obtained from Avanti (Birmingham, AL). Okadaic acid and PMA were were stimulated with zymosan (30 particles/cell), PMA (32 nM), A23187
from LC Services Co. (Woburn, MA). GF109203X (bisindolylmaleimide) (0.5 pg/ml), or okadaic acid (1 PM)in serum-free DMEM. After stimu-
was from Calbiochem-Novabiochem Co. (La Jolla,CA). EGFRfifiHR, pep- lation, the macrophages were scraped in10 mM HEPES, pH 7.4, 2 mM
tide and IP-20 were synthesized by Macromolecular Resources, Colo- EGTA, 0.34 M sucrose, 10%glycerol, 10 pg/ml leupeptin, 10 pg/ml apro-
rado State University, Fort Collins, CO. DMEM and HBSS were from tinin, and 1 mM phenylmethylsulfonyl fluoride, and then sonicated at
Whittaker Bioproducts (Walkersville, MD). Protein concentrations were 0 "C for 10s. The homogenate was centrifuged a t 100,000x g for 60 min,
determined using the BCA reagent from Pierce ChemicalCo. and PLA, activity in the supernatant was assayed using sonicated
Cell Culture-Residentmouseperitonealmacrophageswere ob- dispersions of l-O-hexadecyl-2["H]arachidonoyl-phosphatidylcholine
tained by peritoneal lavage with 4 of mlDMEM containing 10% FBS, 10 prepared as previously described (20). The reaction mixture contained
unitdm1 heparin, 100 pg/ml streptomycin sulfate, 100 units/ml penicil- 30 p~ phospholipid substrate (80,000 disintegrationdmin),5 mM CaCI,,
lin G, and 0.29 mg/ml glutamine. Cells were plated at the indicated 150 mM NaCI, 9 PM dioleoylglycerol (cosonicated with the phospholipid
density and incubated for 2-4ath37 "C in a humidified atmosphere of substrate), 1mg/ml BSA, and 50mM Tris, pH 7.4, in a final volume of 50
5% CO, in air. Each well was then washed twice with calcium- and p1. The reaction was started by the addition of 25 pgof the macrophage
magnesium-free HBSS to remove non-adherent cellsand incubated for cytosolic protein (within the linear protein range of P& activity) and
1618 h in DMEM containing 10% FBS. Approximately 50% of the incubated at 37 "C in a shaking water bath for 10 min. The reaction
peritoneal cells initially seeded adhere to the culture dish. mixture was extracted according toDole and Meinartz (21), and radio-
fH]Arachidonic Acid Release-Cells that had been plated at 1 x labeled fatty acids were isolatedby silicic acid chromatography as pre-
10'12 cm2 well (24-well plate) were labeled with ["Hlarachidonic acid viously described (20).To determine theeffect of dephosphorylation of
(0.25 pCi/ml/well) for 16 h at 37 "C in DMEM containing 10% FBS. For the PLA, on enzyme activity, the 100,000 x g supernatant fraction (100
the PKC down-regulation experiments,PMA was included in the label- pg of protein) was incubated with 10 of pgacid phosphatase in 100mM
ing medium. After labeling, the cells were washed three times with HEPES, pH 6, containing1mM dithiothreitol and 2mM MgCI, at 30 "C
calcium- and magnesium-free HBSS and then stimulated with zymosan for 60 min, as previously described (10).
(30 particles/cell), PMA (32 nM), okadaic acid (1 PM), or A23187 (0.5 MAP Kinase Assay-Macrophages that had been plated a t 6 x lofi
pg/ml) in 1 ml of serum-free DMEM. Since A23187, okadaic acid, and cells/4-cm2well (12-well plate) were either left untreated or treated with
19482 Role of Kinases in Macrophage
Phospholipase A, Activation
A
10000 400
t
Unstimulated
A PMA
A Okadaic Acid
Zymosan

L200
FIG.3. Anion-exchange chromatog-
raphy of a c t i v a t e d MAP kinase. A,
macrophages were stimulated with zymo-
san (30 particles/cell,W) for 15 min, PMA
(32 nM, A) for 5 min, okadaic acid(1PM,A)
for 60 min,or unstimulated (0.1% Me,SO,
0) for 15 min. Cell lysates were chromato-
graphed on a Mono-Q column and MAP
kinase assayed as described under "Ex- 0 0
perimental Procedures." Results are rep- 0 5 10 15 20 25 30
resentative of three independent experi-
ments. B , immunoblotting of Mono-Q Fraction Number
fractions (12-23) withanti-phosphoty-
rosineantibody (left) and anti-MAP ki-
nase antibody (right).
B
Anti-Phosphotyrosine Anti-MAP Kinase

Unstimulated 7 1 I =zo- - p44p42

- - 1
Zymosan "" 4"
"" - p42
PMA [
Okadaic Acid
1213
"

14 15 16 17 18 19 20 21 22 23
zymosan (30 particles/cell), PMA(32 nM), okadaic acid(1PM), orA23187
(0.5 pg/ml) for the indicated times. After stimulation, the cells were
rinsed once with calcium-and magnesium-free HBSS and then lysed on
ice with 100 pl of 50 mM 6-glycerophosphate, pH 7.2, containing 100 PM
sodium orthovanadate, 2mM MgCI,, 1mM EGTA, 0.5%Triton X-100,lO
pg/ml leupeptin, 2 pg/ml aprotinin, and1mM dithiothreitol (BufferA).
After centrifugation a t 16,000 x g for 10 min, the MAP kinase activity
in the supernatant was assayed by measuring the incorporation of
r32Plphosphate intoEGFR,,, peptide in a reaction mixture contain-
ing 50 mM 6-glycerophosphate, pH 7.2, 100 p~ sodium orthovanadate,
10 mM MgCI,, 100 p~ ATP (1 pCi of [y-"2PlATP/sample), 25 pg/ml IP-20,
1mM EGTA, and 200 p~ EGFR,,,,, M MAP kinase reaction mixture) in
a final volumeof 40 p1. The reaction was startedby the addition of 20
pl of the cell lysate (20 pgof protein) and incubated for 10 min a t 30 "C.
The incubation was stopped with 10 pl of 25% trichloroacetic acid and
then spotted on Whatman phosphocellulose filters (P81, 2 cm). The
filters were washed three times in m75 M phosphoric acid for 5 min and
then rinsed once in acetone followed by Cerenkov counting. The MAP
kinase activity in the fractions from the Mono-Q column was assayedby
mixing 40 pl of each fraction with 20 p1 of a three times concentrated
MAP kinase reaction mixture. The reaction was stopped by adding 15 pl
of a 37.5% trichloroacetic acid solution.
Mono-& Chromatography-Macrophage lysates (1.2 mg of protein)
prepared in Buffer A from stimulated and unstimulated cells were
loaded onto a Mono-Q (HR 5/5)anion-exchange column equilibrated in
50 mM 6-glycerophosphate, pH7.2, containing 1mM dithiothreitol, 1mM
EGTA, and 100 p~ sodium orthovanadate. The column was eluted with
a 30-ml linear gradient of 0-0.35 M NaCl a t 1 mllmin, and 1.0 ml
fractions were collected.
PKC Assay-Macrophages were treated with different concentra-
tions of the PKC inhibitor GF109203X for 30 min in serum-free DMEM.
The cells were scraped in 20 mM Tris, pH 7.4, containing 2 mM EGTA,
IC 1213 14 15 16 17 18 19 20 21 22 23
I.$
200 p~ sodium orthovanadate, 100mM sodium fluoride, 10 mM sodium
pyrophosphate, 300 nMp-nitrophenyl phosphate, 10 pg/ml aprotinin, 10
pg/mlleupeptin,and 1 mM phenylmethylsulfonylfluoride and then
sonicated a t 0 "C for 10 s. The homogenate was centrifugeda t 100,000
x g for 60 min, and the PKC activity assayed in the supernatant by
measuring the incorporation of 1"2P]phosphate into histone in the pres-
ence or absence of phosphatidylserine (40 pg/ml) and 1,2-dioleoyl-sn-
glycerol (4 pg/ml) in a final volume of 50 p1. The reaction mixture
contained 20 mM Tris, pH 7.4, 1mg/ml histone, 12.5 mM MgCl,, 0.4 mM
EGTA, 1.0 mM CaCI,, 20 PMATP (1pCi of [y-"P]ATP/sample), with or
without lipids (dispersed by sonication). The reaction was started by
5 min at
adding 5 p1 of cell lysate (5 pg of protein). After incubating for
30 "C, the reaction was stoppedby adding 10 pl of 2 5 8 trichloroacetic
acid and the mixture was spotted on Whatman phosphocellulose filters
(P81, 2 cm). The papers were washed twice for 5 min with 75 mM
phosphoric acid, once with 75mM Na,HPO, for 5 min, and then rinsed
oncewithacetone. The filters were air dried and Cerenkov counts
determined. PKC activity was determined by subtracting the activity
obtained in the absence of phosphatidylserine/dioleoylglycerolfrom the
activity obtained in the presence of these lipids.
Immunoblotting-Macrophage lysates were prepared in Buffer A and
centrifuged for 10 min ina microcentrifuge at full speed. The superna-
tant was boiled for 5 min in Laemmli buffer (22), and proteins were
resolved on a 10% SDS-polyacrylamide gel and then transferred to a
nitrocellulose membrane. After blocking for 1 h at room temperature
with 5% BSA in TTBS (20mM 'his, 134 mM NaCl, 0.05% Tween 20, pH
7.6), the nitrocellulose membrane was incubated overnight a t 4 "C in
TTBS containing 5% BSA and a 1:2000 dilution of 4G10 anti-phospho-
tyrosine or anti-PKC-cr monoclonal antibody. After washing with TTBS
(two washes for15 min followed by three washes for 10 rnin), the mem-
brane was incubated with sheep anti-mouse IgG-horseradish peroxidase
antibody (1:5000 dilution in TTBS) for1h at 25 "C, and proteins were
 Role of Kinases in Macrophage Phospholipase A, Activation

ac/'
t
- 100
FIG.4. Effect of the protein kinase C
inhibitor, GF109203X on PKC and
MAP kinase activity (A) and tyrosine
phosphorylation ( B ) . Macrophages
were preincubated without (0.1% Me,SO)
or with theindicatedconcentration of
GF109203X for 30 min followed by stimu-
lation with zymosan (30 particles/cell,W)
for 15 min, PMA (32 nM, A) for 5 min,
okadaicacid (1 p ~ 0, ) for 60 min, or
Me,SO (0.1%, unstimulated) for 15 min.
A , MAP kinase (-) and PKC (- - - -1 ac-
tivities incell lysates were determined as
describedunder"ExperimentalProce-
dures." Results are expressed as percent
of control that was not treated with in-
hibitor andrepresents the mean * S.E. of
three to five independentexperiments I I 0
assayed in triplicate for MAP kinase ac- 0 0.01 0.1 1
tivity and mean e S.D. of triplicate
samples of a representative experiment GF109203X (pM)
forPKC activiti B, macrophage lysates
(75 pg proteidane) were separated on a
10% SDS-polyacrylamide gel followed by
phosphoty-
with
immunoblotting B
rosine monoclonal antibody.
p44 -
p42 -
I

GF109203X - + - + - + - + - +
uuuuu

detected using thehersham ECLSystem. To reprobe the nitrocellulose,
the membrane wasstripped by incubationfor 30 min at 37 "C in 62.5 mM
Tris-HC1 buffer, pH 6.7, containing 100 mM 2-mercaptoethanol and 2%
SDS, followed by washing five times (5 min each) with TTBS. The mem-
brane was then incubated with a polyclonal anti-" kinase antibody
(1:5000 dilution in 5% BSAPI"rBS buffer) overnightat 4 "C followed by
incubationwith anti-rabbit IgG-horseradish peroxidase
antibody
(1:15,000 dilution inTTBS) for 1 h, at 25 "C. For anti-phosphotyrosine
and anti-MAP kinase immunoblotting ofMono-Q fractions, 900 p1 of
Mono-Q fractions was mixed with 100 pl of 72% trichloroacetic acid and
0.15%deoxycholic acidand incubated overnight at 0 "C. Themixture was
centrifuged in a microcentrifuge for10 min at 4 "C. Cold acetone (1 ml)
was added to the pellet, followedby vortexing and centrifugation, the and
process was repeated twice. The dried pellet was dissolvedin Laemmli
buffer and boiled for 5 min. TheMAP kinases were separated on a 10%
SDS-polyacrylamide geland immunoblotted with anti-phosphotyrosine
and anti-" kinase antibodies,as described above.
RESULTS
MAP Kinase Activation-The effect of agents that promote
arachidonic acid release and phosphorylation of the 85-kDa
P W , including zymosan, PMA, A23187, and okadaic acid, on
MAP kinase activation was investigated. Macrophages were
incubated with the agonists for the indicated times, andMAP
kinase activity inwhole cell lysates was assayedby measuring
the phosphorylation of a synthetic EGFR,,,,,, peptide which
contains a consensus sequence for MAP kinase. As shown in
Fig. 1, all the stimuli, except A23187, induced a time-depend-
ent increase inMAP kinase activity. In unstimulated cells the
activity of MAP kinase remainedat a constant low level during
the 2-h incubation period. When macrophages were stimulated
with PMA or zymosan, the activityof MAP kinase rapidly in-
creased by &5-fold, reaching a maximum by 5 and 15 min,
respectively. In contrast, MAP kinase activation by okadaic
acid was preceeded by a 30-min lag and then increased up to
5-6-fold by 90 min. No significant increase in MAP kinase
activity wasobserved in A23187-treatedcells. The timecourse
of MAP kinase activation in response to zymosan, PMA, or
okadaic acid was similar to the time course previously observed
for the increase in activityof the 85-kDa P W in macrophage
cytosols induced by these stimuli (10).
MAP kinases area family of serinelthreonine protein kinases
that are dually regulated by phosphorylation on tyrosine and
threonine residues, and enhanced tyrosinephosphorylation of
MAP kinases occurs in manycell types in response to a variety
of agonists (11-13). To provide more evidence that the en-
hanced activity in cell lysates was due toMAP kinase activa-
tion, the effect of cell stimulation on tyrosine phosphorylation
of MAP kinase was determined by immunoblotting (Fig. 2).
Zymosan stimulation induced tyrosine phosphorylation of a
44-kDa protein (p44) and, toa lesser extent, a 42-kDa protein
(p42) that was evident at 5 min, and increaseda t 15 min (Fig.
M).PMA stimulated tyrosine phosphorylation of p44 that was
 Role of Kinases in Macrophage PhospholipaseA, Activation

Unstimulated Zymosan PMA Okadaic Acid A231 87

T

A23187 1 '1
t I T

0.01 0.1 1
GF109203X - + - + - i - + - +
GF109203X (pM) FIG.6. Effect of the proteinkinase C inhibitor GF109203X on
the activity of the 85-kDa cytosolic P m .Macrophages were pre-
FIG.5. Effect of the protein kinase C inhibitor GF109203X on incubated without (0.1% Me,SO) or with 1 p~ GF109203X for 30 min
[SH]-arachidonicacid release. ['HlArachidonicacid-labeled mac- followed by stimulation with zymosan (30 particledcell) for 30 min,
rophages were preincubated without (0.1% Me,SO) or with the indi- PMA(32 MI) and A23187 (0.5pg/ml) for15 min, okadaic acid (1J~M) for
cated concentration GF109203X for 30 min followed bystimulationwith 60 min, or Me,SO (0.1%,unstimulated)for 30 min. The PLA, activity in
zymosan (30 particledcell, W), PMA (32 m, A),A23187 (0.5 pg/ml, O), 100,000 x g cytosolic fraction was determined as described under "Ex-
okadaic acid (1p ~ A),, or Me,SO (0.1%,unstimulated) for 60 min. The perimental Procedures." Results are expressed as the mean ? S.E. of
amount of 'H-label released intothe media was determined as a percent three to four independent experiments assayed in triplicate using the
of total radioactivity (cell associated plus media). The results are ex- PLA, activityin unstimulatedcells withoutGF109203X treatment as a
pressed as the percent of control not treated with the inhibitor and control.
represent the mean f S.E. of three to four independent experiments.
was evident in fractions 17 and 18 from okadaic acid-stimu-
evident by 2 min, but did not induce tyrosine phosphorylation lated cells, and fractions14-18 from zymosan-stimulated cells,
of p42 (Fig. 2C). Treatment of macrophages with okadaic acid that was not evident in these fractionsfrom PMA-stimulated or
induced tyrosine phosphorylation of both p44 and p42 by 60 unstimulated cells. When the nitrocellulose membrane was
min, which further increased after 90 min of stimulation (Fig. reprobed with antibody to MAP kinase this tyrosine-phospho-
2 A ) . In contrast, stimulation with
A23187 did not induce tyro- rylated protein was verified t o be 42-kDa MAP kinase. Tyrosine
sine phosphorylation of either p44 or p42. When these nitro- phosphorylation of 42-kDa MAP kinase also appeared to result
cellulose membranes were reprobed with antibody specific for in tighter association to the Mono-Q column.
p42 and p44 MAP kinases, bands at 42 and 44 kDa were de- Role of PKC inMAP Kinase and PLA, Activation-In many
tected, which comigrated with the p42 and p44 tyrosine phos- cell types, PKC activation has been shown to play a role in
phoproteins induced by zymosan or okadaicacid(Fig.2B). activation of Pw.PMA can induce serine phosphorylation and
Similarly, the 42- and 44-kDa forms of MAP kinase were de- activation of the 85-kDa PI.& in macrophages and promote
tected in PMA- or A23187-stimulated cells, and the 44-kDa activation of MAP kinase. To investigate whether PKC activa-
form comigratedwith thep44 tyrosine phosphoprotein induced tion was required for activation of MAP kinase, the PKC in-
by PMA (not shown). hibitor, bisindolylmaleimide,GF109203X, was used(23). Bisin-
To provide additional evidence that macrophage stimulation dolylmaleimides have been shown to inhibit PKC-a, PKC-PI,
induced increases in tyrosine phosphorylation and activity of PKC-p,,, PKC-7, and PKC-E but showed slight selectivity for
MAP kinase(s), cell lysateswerechromatographed over a PKC-a (24). To ensure that GF109203X inhibited macrophage
Mono-Q anion-exchange column and assayed for MAP kinase PKC activity, the cells were pretreated for 30 min with the
at 220 mM inhibitor
activity (Fig. 3).A peak of kinase activity was eluted and
the effect on PKC activity determined.
NaCl from cells stimulated with PMA, zymosan, and okadaic GF109203X treatment resulted in a concentration-dependent
acid but not from A23187-stimulated cells (A23187 data not inhibition of PKC activity using histone I11 as substrate (Fig.
shown).When Mono-Q fractions were fractionated by SDS- 4A). The inhibitor was also found to suppress MAP kinase
polyacrylamide gel electrophoresis and immunoblotted using activation in zymosan- and PMA-stimulatedcells but to have
antiphosphotyrosine antibody, a tyrosine phosphoprotein at 44 no effect on M A P kinase activation in cells stimulated with
kDa was present in fractions 19 and 20 from PMA-, okadaic okadaic acid (Fig.4A).GF109203X was slightly less effective at
acid-, and zymosan-stimulatedcells, but not in these fractions inhibiting MAP kinase activation in response t o zymosan than
from unstimulated cells, and correlated with the highest MAP PMA. The lackof effect of GF109203X on okadaic acid-induced
kinase activity. When the nitrocellulose membranes were re- MAP kinase activationprovides evidence that the inhibitor has
probed with antibody to MAP kinase, p44 MAP kinase was no direct effect on MAP kinase activity. When the macrophages
verified to be present in fractions 19 and20. In unstimulated were pretreated with 1J ~ MGF109203X, PMA-induced tyrosine
cell lysates, the p44 MAP kinase eluted in fractions 14-17, phosphorylation of p44 MAP kinase was almost completely
whereas cell stimulation resulted in tyrosine phosphorylation suppressed, whereas tyrosine phosphorylation of p44 and p42
and in tighter bindingof a portion of 44-kDa MAP kinase to the MAP kinase in zymosan-stimulated cells was only partially
Mono-Q column. A 42-kDatyrosine-phosphorylated protein inhibited (Fig. 4B). The inhibitor had no effect on tyrosine
 Role of Kinases in Macrophage Phospholipase A, Activation 19485
30 unstimulated zymosan okadaic
PMA
Acid A23187
1 T
FIG.7. Effect of PKC down-regula-
tion on P3Hlarachidonicacid release. Q)
Macrophageswerelabeledwith['Hlarach- v)
idonic acid in theabsence(0.1% Me,SO)
or presence of differentconcentrations of
2
- 20

PMA(1-100 nM) for 16 h and then stimu-

lated
with
zymosan (30 particles/cell),
2
-
PMA (32n ~ )A23187
, (0.5 pg/ml),okadaic Q) l5
acid (1 PM), or Me,SO (O.l%, unstimu- a
lated) for GO min. The amountof 'H-label
released into the media was determined
and expressed a s a percent of total radio-
activity (cell associated plus media). Re-
5
sults are expressed as the mean t S.E. of
four independent experiments assayed in
s
duplicate using the amount of "-label re-
leased from unstimulated cells without
PMA preincubation as a control.
P P

phosphorylation of p42 and p44 in okadaic acid-stimulated

cells. These results suggest that PKC activation is requiredfor -80 kDa
tyrosine phoshorylation and activationof MAP kinase inPMA-
stimulated cells. In zymosan-stimulated cells, both PKC de- PMA (nM) 0 0.1 1.0 10 100
pendent- and -independent mechanisms appear to contribute FIG. to 8. Down-regulationof PKC-a in macrophages. Macrophages
MAP kinase activation, whereas theactivation of MAP kinase were incubated without(0.1%Me,SO) or with the indicated concentra-
in okadaic acid-stimulated cells is independent of PKC. The tions of PMA for 16 h. Cell lysates (50 pg proteidane)were separated
inhibition of MAP kinase activation in PMA- and zymosan- on a 7.5%SDS-polyacrylamide gel followedby immunoblottingwith anti-
stimulated cells also correlated with the abilityof GF109203X PKC-a antibody as described under "Experimental Procedures."
to inhibit ['Hlarachidonic acid release. PMA-induced arachi-
donic acid release was very sensitive to PKC inhibition by ent down-regulation of PKC-a. The effect of PKC down-regula-
GF109203X, whereas zymosan-inducedarachidonicacidre- tion on MAP kinase activation and tyrosine phosphorylation
lease was only partially suppressed (Fig. 5 ) . The release of was also studied. Preincubation of the macrophages with 10 nM
arachidonic acid in okadaic acid- and A23187-stimulated cells PMA for 16 h almost completely blocked MAP kinase activity in
was not inhibitedby GF109203X. We have previously reported response to subsequent stimulationwith PMA for 15 min (Fig.
that stimulationof macrophages with PMA, zymosan, A23187, 9A ). Zymosan-stimulated MAP kinase activity was suppressed
and okadaic acid induces an increase inactivity of the 85-kDa by 70% following long term exposure to PMA, whereas okadaic
P& in thecytosol. The increasedactivity of the PLA, is due to acid-stimulated MAP kinase activity was only inhibited by
phosphorylation since
dephosphorylation by phosphatase 30%. PMA-induced tyrosine phosphorylation of p44 MAP ki-
treatment reduces the activity to control levels (10). As shown nase was almostcompletely inhibited when the cells were pre-
in Fig. 6, the increased PLA, activity induced by okadaic acid treated with 10 nM PMA for 16 h (Fig. 9B 1. Prolonged exposure
and A23187 was unaffected by PKC inhibition with to PMA also substantially inhibitedtyrosine phosphorylation of
GF109203X, whereas zymosan- and PMA-stimulated PLA, ac- p44 MAP kinase inzymosan-stimulated macrophages, whereas
tivity was partially suppressedby 62 and 68%, respectively. partial inhibition of tyrosine phosphorylation of p42 and p44
It has been shown that prolonged exposure of a variety of MAP kinases in okadaic acid-stimulated cells was evident.
cells to PMA, which down-regulates PKC, can suppress arachi- The effect of PKC down-regulation on cytosolic PLA, activity
donic acid release inresponse to certain stimuli. This approachwas also investigated. The macrophages were incubated with
was also used to investigate the role of PKC in activation of or without PMA for 16 h followed by treatment with the ago-
MAP kinase and the85-kDa P&. As shown in Fig. 7, arachi- nists, and thenP& activity was measured in the100,000 x g
donic acid release in response to PMA was partially inhibited cytosolic fraction (Fig. 10). Onepotential complication in using
by prior exposure to as little a s 1 nM PMA for 16 h and com- this approach was that to down-regulate PKC, the cells are first
pletely suppressed tocontrol levels whenpretreated with 10 nM exposed to a concentration of PMA that can initially activate
PMA for 16 h. Similarly,zymosan-inducedarachidonic acid PKC, MAP kinase, and the P&. In fact we did observe that
release was partially inhibited by prior exposure of the mac- the cytosolic P& activity was elevated after the16-h exposure
rophages to 1nM PMA and suppressedby 85% with 10-100 nM to PMA in cells that were not further stimulated and was al-
PMA. Arachidonic acid release triggered by A23187 was only most a s high as the activity in cells treated with PMA for 15
minimally inhibited (20% a t 100 nlq PMA) by down-regulation min (butnot exposed to PMA for 16 h). Analysis of the 85-kDa
of PKC, and the response to okadaic acid was totally unaf- P& by immunoblotting showed that the increased P& ac-
fected. Resident mouse peritoneal macrophages have been re- tivity observed 16 h after PMA treatment was not due to an
ported to contain multiple PKC isoforms including PKC-CY, -p, increase in amount of the P& (not shown). When the cells
-6, - E , and -(, and a role for PKC-CYin mediating zymosan- were further stimulated, the PLA, activity was enhanced in
induced eicosanoid synthesis in thesecells has been suggested cells even if they hadbeen previously exposed to PMA for 16 h.
(18).As shown in Fig. 8, we have found that prolonged exposure This wasnot unexpected for cells stimulated with okadaic acid
of the macrophages to PMAresulted in a concentration-depend- or A23187 since these responses appear to be PKC-independ-
19486 Role of Kinases
in Macrophage
Phospholipase A, Activation
A
22 ,1
Unstimulated Zymosan PMA
Okadaic
Acid A23187 lhstimulated Zymosan PMA Okadaic Acid A23187

T T

l
PMA(16h)

p44
p42

p
-

-
-
M
+

A
uuuuu
h
-

- +
+

-
-

+
-
-
-

pr*-

+ - +
+

- +
- +
PMA(16h) - + - + - + -

85-kDa cytosolic P w .Macropha& were incubated without (0.1%,

Me,SO) orwith 10 nhl PMA for 16 h followed by stimulation with
+
FIG.10. Effect of PKC down-regulation 0 11 the activity of the

zymosan (30 particles/cell) for 30 min, PMA (32 nu) and A23187 (0.5
pg/ml) for 15 min, okadaic acid (1 p ~ for
) 60 min, or Me,SO (0.10,
- +

unstimulated) for 30 min. The PJA, activity inthe 100,000 x g cytosolic

6B a h% 0 fraction was determinedas described under Experimental Procedures
% ..,
3 ,
b
+e%.

,.
9*9,
8,
with l-O-hexadecyl-2-l~Hlarachidonoyl-phosphatidylcholine as a sub-
strate. The reaction was started by the addition of 25 pg of cytosolic
protein. Results are expressed as themean S.E. (% of control) of three
FIG.9.Effect of PKC down-regulation on MAP kinase activity independent experiments assayed in triplicate using the PLA, activity
(A) and tyrosine phosphorylation ( B ) .Macrophages were preincu- in unstimulated cells without PMA preincubation a s a control.
bated without(0.1%Me,SO) or with10 nv PMAfor 16 h a t 37 C followed
by stimulation with zymosan (30particledcell) for 15 min, PMA(32 nM) TABLEI
andA23187(0.5pg/ml)for5min,okadaicacid(1p~1)for60min,orMe,SO Effect of phosphatase treatment of macrophage cytosols on
(0.1%. unstimulated) for 15 min. A, MAP kinase activity in cell lysates stimulated-PLA, activity
was determineda s described under Experimental Procedures.Results For PKC down-regulation, macrophages were cultured for 16 h in the
are expressed as the mean = S.E. of three independent experiments presence or absence of 10 nM PMA. The cells were then exposed to PMA
assayed in triplicate. R,macrophage lysates (75 pg proteidane) were (32nM) or left untreated(Me,SO) for 15 min. The 100,000 x g cytosolic
separated on a IO%, SDS-polyacrylamidegel followed by immunoblotting fraction (100 pg of protein) was incubated in 100 mM HEPES, pH 6, 1
with anti-phosphotyrosine monoclonal antibody. mM dithiothreitol, and 2 mM MgCI,, with or without10 pg of potato acid
phosphatase a t 30C for 60 min. The PLA, activity was determinedas
ent. However, the results were unexpected for cells stimulated described under Experimental Procedures. Results are expressed as
with zymosan and especially for cells stimulated with PMA. means k S.D. of one of two representative experiments assayed in
Since PKC was down-regulated, it was surprising thatsubse- triplicate.
quent stimulation withPMA consistently resulted in a further PLA2 activity
Treatment after
enhancement of PLA, activity above the level in cells treated down-regulation h, +Phosphatase
-Phosphatase
for 16 h with PMA (but not further stimulated). To provide
evidence that the enhancedPLA, activity was due tophospho- dPm
rylation of the PLA,, the cytosols were treated with phospha- A. None - 2742 -c 2688
248 = 165
B. None + 4064 k 581 2839 -c 276
tase. We have previously shown that this results in dephospho- C. PMA - 6101 k 293 3227 k 435
rylation of the 85-kDa PLA, and in a reduction of stimulated D. PMA + 5303 k 116 3372 = 490
PLA, activity to levels in unstimulated cells (10). As shown in
Table I, phosphatase treatment of cytosols from unstimulated
cells that had not been down-regulated did not affect PLA, variety of cell types by diverse stimuli. We have previously
activity(Table I, line A), whereas the increased activity in shown that the85-kDa PLA, is serinephosphorylated on mul-
cytosols of cells treated with PMA for 16 h (Table I, line B),as tiple sites inmacrophages and cell stimulation increasesphos-
well as the further increaseobserved after a subsequent PMA phorylation of several of these sites(10).These results suggest
exposure (Table I, line D), was reduced to control levels. These that multiple kinasesmay be involved in phosphorylating the
results suggest that the enhanced activity is due to phospho- 85-kDa PLA, in macrophages although the identity of these
rylation of the P L h . However, it is interesting that
PMA could kinases is not known. Evidence has been provided that the
induce an increase in PLA, phosphorylation in cells in which 85-kDa P& is a substrate for MAP kinase both in vitro and in
certain isoforms of PKC were down-regulated, and p42 and p44 cells (14, 15). The 85-kDa PLA, contains a consensus sequence
MAP kinase activities were suppressed. for MAP kinase, and phosphorylation of this site increases
PLA, activity (14, 15, 25, 26). To gain more insight into the
DISCUSSION kinases that may be activated by agonists that induce arachi-
There is now substantial evidence demonstrating that the donic acid release and activation of the 85-kDa P& in mac-
85-kDa cytosolic PLA, is activated by phosphorylation in a rophages, the effect of these agonistson MAP kinase activation
 Role of Kinases in Macrophage Phospholipase A, Activation
was investigated. A23187 did not activate MAP kinase in the
macrophages demonstrating that the phosphorylation and ac-
tivation of the 85-kDa PLA, induced by A23187 is independent
of MAP kinase. In contrast, zymosan, PMA, and okadaic acid
all induced a time-dependent increase in MAP kinase activity
that correlated with the time course of PLA, activation in mac-
rophage cytosol as previously described (10). PMA, which in-
duced activation of p44, but not p42 M A P kinase, induced rapid
activation of MAP kinase and of the 85-kDa P W , both of
which occurredby 2-5 min. PMA has been reported to activate
both p41 and p44 MAP kinases in a transformed macrophage
cell line (27). Zymosan activation of p42 and p44 MAP kinases
was maximal between 10-15 min after stimulation, and this
slightly preceeded zymosan-inducedPLA, activation which in-
creased up to 30 min after stimulation. In okadaic acid-stimu-
lated cells, MAP kinase and PLA, activation, as well as arachi-
donic acid release, were all preceeded by a 30-min lag phase.
The increase in P q activity in response to okadaicacid
reached a maximum by 60 min (not shown), whereas MAP
kinase activation continued to increase up to 90-120 min. A
delayed activation of MAP kinase has also been observed in
okadaic acid-treated B lymphocytes (28).
We have found that MAP kinase activation and arachidonic
acid release in PMA- and zymosan-stimulated macrophages
are PKC dependent, but are PKC independent in response to
okadaic acid andA23187. The mechanisms involved in activat-
ing MAP kinase by okadaic acidare not understood,but it also
has been reported to be independent of PKC in other cell types
(29). MAP kinase activation and arachidonic acid release in
zymosan-stimulated cells were not completely suppressed by
inhibition of PKC suggesting the involvement of PKC-depend-
ent and -independent mechanisms. Prolonged exposure of the
macrophages to PMA resulted in down-regulation of PKC-a
that correlated with suppression of arachidonic acid release
and M A P kinase activation in response to PMAand zymosan. It
has recently been demonstrated in Madin-Darby canine kidney
cells that inhibition of PKC-a by antisense cDNAinhibits PMA-
induced arachidonic acid release, and PKC-a has been impli-
cated in the regulation of zymosan-stimulated eicosanoid syn-
thesis in macrophages (18, 30).Arachidonic acid release in
response to okadaic acid and A23187 was unaffected by PKC
down-regulation, but down-regulation did result in a slight
inhibition of okadaic acid-inducedMAP kinase activation that
was not observedwith GF109203X. It would not be unexpected
that prolonged inhibition of PKC would have more complex ef-
fects on cell function than observed with more immediate in-
hibition of PKC with GF109203X. This is particularly evident
when the effects of PKC inhibition by GF109203X and down-
regulation on agonist-induced increases in cytosolic P W ac-
tivity are compared. GF109203X inhibited the increase in
PLA, activity induced by PMA and zymosan, whichcorrelated
with the effects of this inhibitor on arachidonic acid release
and MAP kinase activation. However, prolonged exposure to
PMA did not result in an inhibition of PLA, activity and re-
sulted in enhanced activity when the cells were then subse-
quently exposed to PMA, okadaic acid, or A23187. Although a
commonly used technique, prolongedexposure to PMA to
dowr-regulate PKC is complicated, sincethe cells are initially
being activated by the PMA treatment. In addition, the pro-
longed down-regulation of PKC is clearly having other effects
on the cell, thus allowing further enhancement of PLA, activ-
ity, above the level in cells treated with PMA for 16 h, when
they are subsequently exposed t o PMA. In addition, this in-
crease in PLA, activity in PMA down-regulated cells is re-
duced by phosphatase treatment, suggesting it isdue to phos-
phorylation of the P W . This increased phosphorylation
19487
occurs despite suppression of certain isoforms ofPKC and
MAP kinase. It is possible that there is an isoform of PKC
that is activated byPMA but not subject to complete down-
regulation by prolonged PMA treatment. Activation of this
isoform could then directly or indirectly increase PLA, phos-
phorylation. This suggests that, although increased phospho-
rylation and activation of the PLA,maybe required for
arachidonic acid release, as inferred from the data using the
GF109203X inhibition, there maybe an additional step re-
quired that is suppressed after prolonged exposure to PMA.
Of interest, however, is the fact that despite this enhanced
PLA, activity in response to PMA and zymosan in PKC down-
regulated cells, [3H]arachidonic acidwas suppressed.
The data are consistent with a role for MAP kinase in PLA,
activation in zymosan- and PMA-stimulatedmacrophages
since inhibition of MAP kinase by the PKC inhibitor,
GF109203X, also resulted in an inhibition of zymosan- and
PMA-induced arachidonicacid release and the increase in
PLA, activity.However, inhibition of PMA-induced arachidonic
acid release by the inhibitor was evident at lower concentra-
tions than inhibited MAP kinase activity, suggesting there may
be another PKC-dependent step in regulating arachidonic acid
release. Although the effect of GF109203X onzymosan-induced
arachidonic acid release and P- and MAP kinase activation
were only partial, similar concentrations inhibited the three
responses. In addition, the datasuggest that theinvolvement of
PKC in PLA, activation may in part be due to triggering a
kinase cascade that leads to MAP kinase activation.
Acknowledgment-We thank Gail Kastner for the excellent technical
assistance.
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