(Received for publication, December 1, 1995; and in revised form, January 5, 1996)
From the
The phosphorylation sites on the human, 85-kDa cytosolic
phospholipase A (cPLA
) were identified using
recombinant cPLA
expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of
tryptic digests of
P-labeled recombinant cPLA
showed four major peaks of radiolabeled phosphopeptides. The
phosphorylated residues were identified as Ser-437, Ser-454, Ser-505,
and Ser-727 using mass spectrometry and automated Edman sequencing. Sf9
cells infected with recombinant virus expressing cPLA
exhibited a time-dependent release of arachidonic acid in
response to the calcium ionophore A23187 or the protein phosphatase
inhibitor okadaic acid, which was not observed in Sf9 cells infected
with wild-type virus. Stimulation of Sf9 cells with A23187 and okadaic
acid also increased the level of phosphorylation of cPLA
.
Okadaic acid, but not A23187, induced a gel shift of cPLA
and increased the level of phosphorylation of Ser-727 by 4.5-fold,
whereas the level of phosphorylation of the other sites increased by
60% or less in response to both agonists. To determine whether the same
sites on cPLA
were phosphorylated in mammalian cells, human
monocytes were studied. Okadaic acid stimulation of monocytes induced a
gel shift of cPLA
, increased the release of arachidonic
acid, and increased the level of phosphorylation of cPLA
on
serine residues. Comparison of two-dimensional peptide maps of tryptic
digests of
P-labeled recombinant cPLA
and
human monocyte cPLA
demonstrated that the same peptides on
cPLA
were phosphorylated in mammalian cells as in insect
cells. These results show that the Sf9-baculovirus expression system is
useful for investigation of the phosphorylation sites on
cPLA
. The results also suggest that phosphorylation of the
cPLA
by protein kinases other than mitogen-activated
protein kinase may be important for the regulation of arachidonic acid
release.
Arachidonic acid is the precursor for a variety of
proinflammatory lipid mediators including the leukotrienes and
prostaglandins. The production of these potent agents is largely
controlled by the availability of free arachidonic acid. The release of
arachidonic acid from membrane phospholipid is a regulated process that
is catalyzed by phospholipase A and occurs in many cell
types in response to a variety of physiological and pharmacological
agonists. Current evidence implicates the 85-kDa, cytosolic PLA
(cPLA
) (
)as an important enzyme in
mediating agonist-induced arachidonic acid release and eicosanoid
production(1, 2) . cPLA
, which shows
specificity for arachidonic acid-containing
substrates(3, 4, 5) , is posttranslationally
regulated both by phosphorylation and by the level of intracellular
calcium. Calcium is not required for catalytic activity of cPLA
but is required for binding of the cPLA
to
phospholipid vesicles(6, 7, 8) .
Calcium-dependent membrane binding of cPLA
occurs at
calcium concentrations of 300 nM or greater and is mediated by
a calcium-dependent lipid binding domain present at the amino terminus
of cPLA
(6, 9, 10) . Stimulation
of mast cells by calcium ionophore or by IgE/antigen has recently been
shown to induce translocation of the cPLA
from the cytosol
to the nuclear membrane(11) .
Treatment of a variety of cell
types with diverse physiological agonists such as growth factors,
thrombin, colony-stimulating factor-1, interleukin-1,
lipopolysaccharide (LPS), bacteria, vasopressin, and zymosan induce
phosphorylation of cPLA resulting in an increase in its
activity(1, 12, 13, 14, 15, 16, 17) .
In stimulated macrophages, platelets, fibroblasts, and Chinese hamster
ovary cells, phosphorylation of cPLA
occurs exclusively on
serine residues(1, 13, 18) . cPLA
is a substrate for protein kinase C, cyclic AMP dependent kinase
(protein kinase A), and mitogen-activated protein (MAP) kinase in
vitro, but only phosphorylation by MAP kinase induces a consistent
increase in cPLA
activity and decreases its electrophoretic
mobility (gel shift)(19, 20, 21) . cPLA
has one consensus site for MAP kinase at Ser-505(21) .
Evidence for a role for MAP kinase in cPLA
phosphorylation
and activation has been provided in transfection studies showing that
agonist-stimulated arachidonic acid release is diminished in Chinese
hamster ovary cells overexpressing cPLA
that has been
mutated at Ser-505 compared with arachidonic acid release that is seen
in cells overexpressing the wild-type enzyme(21) . Although a
role for MAP kinase phosphorylation of cPLA
is implicated,
analysis of two-dimensional phosphopeptide maps of
P-labeled cPLA
from stimulated macrophages
suggests that this enzyme is phosphorylated on multiple sites and that
several of these sites show increased phosphorylation after cell
stimulation(13) . Recent studies have also provided evidence
for MAP kinase-independent pathways leading to cPLA
phosphorylation and
activation(18, 22, 23) .
Identification of
the phosphorylation sites on cPLA is important for
understanding its mechanisms of regulation. To identify the
phosphorylation sites, we have taken advantage of the baculovirus
expression system in which we have previously demonstrated that
cPLA
is expressed as a phosphoprotein and from which large
amounts of cPLA
can be obtained(24) . It has been
observed that the sites that are phosphorylated on proteins expressed
in baculovirus-infected insect cells and mammalian cells are often the
same(25, 26) . A recent report has demonstrated that
cPLA
is phosphorylated on Ser-505 when expressed in insect
cells(27) . In this report we demonstrate that Spodoptera
frugiperda (Sf9) cells expressing cPLA
can be induced
to release arachidonic acid in response to the agonists calcium
ionophore A23187 and the protein phosphatase inhibitor okadaic acid and
that this functional response in okadaic acid-treated cells correlates
with increased phosphorylation of a novel site on cPLA
.
Evidence is also presented demonstrating that the sites phosphorylated
on cPLA
in infected Sf9 cells also occur on cPLA
in human monocytes.
Human monocytes were allowed to
adhere for 4 h and then labeled with
[H]arachidonic acid (0.25 µCi/ml) in RPMI
1640 containing 10% FBS, 100 µg/ml streptomycin sulfate, 100
units/ml penicillin G, and 0.29 mg/ml glutamine. After 16 h the cells
were rinsed three times with RPMI 1640 containing 0.25% HSA. Cells were
stimulated in 1 ml of RPMI 1640 containing 0.25% HSA with PMA (500
nM), A23187 (0.5 µg/ml), okadaic acid (1 µM),
opsonized zymosan (60 particles/cell) or LPS (1 µg/ml). After
stimulation, the medium was removed and centrifuged at 1,400
g for 10 min. The cells were scraped into 1 ml of calcium- and
magnesium-free PBS containing 0.1% Triton X-100. The amount of
radioactivity in the cells and media was measured by liquid
scintillation spectrophotometry.
For P labeling the mononuclear cells were plated
at 70
10
cells/100-mm dish, as above, allowed to
adhere for 4 h, and then the monolayer was washed three times with
phosphate-free RPMI 1640. The cells were labeled overnight with
[
P]orthophosphoric acid (1 mCi/dish) in 5 ml of
phosphate-free RPMI 1640 containing 10% dialyzed FBS, 100 µg/ml
streptomycin sulfate, 100 units/ml penicillin G, and 0.29 mg/ml
glutamine. Stimulants were added to the labeling medium for 2 h. The
cells were rinsed in Hanks' balanced salt solution and scraped
into lysis buffer. The lysates were centrifuged, and the
P-labeled cPLA
was immunoprecipitated from the
monocyte cell lysates, as described below.
Figure 4:
HPLC of tryptic peptides derived from
cPLA from okadaic acid-stimulated Sf9 cells. After
trypsinization of trichloroacetic acid-precipitated cPLA
,
the digest was applied to a C
reverse-phase HPLC column
(Vydac 218TP52). Other details are given under ``Experimental
Procedures.'' Eluted cpm were determined by Cerenkov counting (dotted line), and the peptide elution was monitored by
absorbance at 210 nm.
A
portion of this two-dimensional peptide map-purified phosphopeptide
(400 cpm) was submitted to Edman sequencing. The peptide was coupled to
a Sequelon-AA membrane (MilliGen) according to the procedure provided
by the manufacturer. The membrane disc was washed with two 1-ml
portions of methanol, and 200 cpm of peptide remained on the disc,
which was stored at -20 °C for a few days. The disc was
placed in the reaction vessel of a 477A Sequencer (Applied Biosystems).
Sequencing reaction cycles were carried out with standard reagents and
conditions except that the S3 wash, which is usually n-butyl
chloride, consisted of 2 mM NaHPO
in
methanol:water (9:1), pH 7 (not adjusted). The entire S3 wash from each
cycle was diverted into glass vials.
Mass spectra were acquired as peptides eluted from the LC by
scanning Q3 at a rate of 500 u/s over the range 400-1,500 m/z. Peak widths ranged from 1.5 to 2.0 u. Signal was
detected with a conversion dynode/electron multiplier. Sequence
analysis of peptides was performed during a second HPLC analysis by
selecting the precursor ion with a 2-3-u (full width at half
height) wide window in the first mass analyzer and passing the ions
into a collision cell filled with argon to a pressure of 3-4
millitorr. Collision energies were on the order of 10-50 eV
(E). The fragment ions produced in Q2 were transmitted to
Q3, which was scanned at 500 u/s over a mass range of 50 u to the
molecular weight of the precursor ion to record the fragment ions. Peak
widths in the second mass analyzer ranged from 1.5 to 2.0 u. The
electron multiplier setting was 200-400 V higher than that used
to record the molecular weight.
Figure 1:
[H]Arachidonic
acid release from Sf9 cells. Sf9 cells were infected with either
wild-type virus (panel A) or recombinant virus containing the
gene for cPLA
(panel B). Cells were labeled with
[
H]arachidonic acid 18 h prior to the addition of
stimuli. At the indicated times postinfection
[
H]arachidonic acid-labeled Sf9 cells were
treated with A23187 (0.5 µg/ml, solid bars), okadaic acid
(1 µM, striped bars), or vehicle (0.1%
Me
SO, open bars) for 2 h.
H label
released into the medium is expressed as a percent of the total
radioactivity (cell associated plus media). The results are expressed
as mean ± S.D. of triplicate values from a representative
experiment.
Figure 2:
Time course of
[H]arachidonic acid release from Sf9 cells.
[
H]Arachidonic acid-labeled Sf9 cells infected
with recombinant virus were stimulated at 67 h postinfection for the
times indicated with A23187 (0.5 µg/ml,
), okadaic acid (1
µM,
), or vehicle (0.1% Me
SO,
).
H label released into the media is expressed as a percent
of the total radioactivity (cell associated plus media). The results
are expressed as the mean ± S.D. of triplicate values from a
representative experiment.
Figure 3:
Effect of stimulation of Sf9 cells on
mobility of recombinant cPLA on a SDS-polyacrylamide gel.
Sf9 cells infected with recombinant virus were stimulated at 67 h
postinfection for 2 h with Me
SO (0.1%), A23187 (0.5
µg/ml), or okadaic acid (1 µM). Cell lysates (400
ng/lane) were separated on a 10% SDS-polyacrylamide gel prepared as
described previously(24) . Immunoblots were incubated with a
polyclonal antibody against the recombinant cPLA
, and the
Amersham ECL system was used for detection.
The P-labeled tryptic
phosphopeptides were also analyzed on two-dimensional phosphopeptide
maps. A representative phosphopeptide map of
P-labeled
cPLA
from okadaic acid-treated Sf9 cells is shown in Fig. 5. Five major spots were observed. To determine the
correspondence between the HPLC peaks and the spots on the
two-dimensional peptide maps, each of the HPLC-purified phosphopeptides
derived from cPLA
expressed in Sf9 cells was individually
analyzed by two-dimensional peptide mapping. Since phosphopeptide spots
1, 2, and 3 run close together, pairs of HPLC peaks (1 + 2, 1
+ 3, and 2 + 3) were also analyzed by two-dimensional
phosphopeptide mapping to make the assignments unequivocal. In this way
phosphopeptide spots 1-4 were assigned. There still remained a
significant phosphopeptide spot near spot 1 (labeled 5 in Fig. 5) which had no corresponding peak in the HPLC. Since HPLC
peaks 1-4 account for virtually all of the eluted cpm, it was
suspected that phosphopeptide spot 5 was the result of decomposition of
one or more phosphopeptides during analysis. This was indeed the case.
The intensity of spot 5 relative to its neighboring spot 1 was highly
variable in independent analyses. When spot 5 was eluted from the
cellulose plate and then injected onto the HPLC column, all of the cpm
eluted in the void volume, and as described above, virtually no cpm was
detected in the void volume of trypsin-digested cPLA
that
was injected onto HPLC without prior submission to two-dimensional
peptide mapping. Further evidence indicated that phosphopeptide spot 5
comes completely from phosphopeptide 1. When the phosphopeptide spot 1
was eluted from the cellulose plate and reanalyzed by two-dimensional
peptide mapping, both spot 1 and 5 were seen. The nature of this
decomposition was not investigated further.
Figure 5:
Two-dimensional tryptic phosphopeptide
maps of P-labeled recombinant cPLA
from Sf9
cells. A tryptic digest of immunoprecipitated, gel-purified recombinant
cPLA
from okadaic acid-stimulated Sf9 cells was separated
by two-dimensional phosphopeptide mapping as described under
``Experimental Procedures.'' Phosphopeptides were detected by
autoradiography. The sample was spotted at the position marked by
. Electrophoresis was run in the horizontal dimension, with the
anode on the left, and chromatography was run in the vertical
dimension. Phosphopeptides are labeled 1, HPLC-purified tryptic peptide
1; 2, peptide 2; 3, peptide 3; 4, peptide 4 and were identified by
comigration with HPLC-purified tryptic peptides (see
``Results'' for a description of spot
5).
Figure 6:
Collision-induced dissociation mass
spectra recorded on ions generated from cPLA tryptic
peptides. Fragments of types b and y having the general formulas
H(NHCHRCO)
and
H
(NHCHRCO)
OH
,
respectively, are shown above and below the amino acid sequence at the top of the figure. In addition, double-charged y ions are also
shown. All ions correspond to average masses. Ions observed in the mass
spectrum are underlined, and ions resulting from neutral loss
of phosphoric acid are shown in boldface type. The serine in boldface type was determined to be phosphorylated. Leu and Ile
were assigned by correspondence to the known sequence. In the spectrum,
ions resulting from neutral loss of phosphoric acid are denoted by -P. Panel A, collision-induced dissociation
mass spectrum recorded on the (M+3H)
ions at m/z 759 of a tryptic fragment, residues
428-445, in the ethylester form. Panel B,
collision-induced dissociation mass spectrum recorded on the
(M+3H)
ions at m/z 854 of a
tryptic fragment, residues 446-467. Only residues 446-463
are shown at the top of the figure. Panel C,
collision-induced dissociation mass spectrum recorded on the
(M+2H)
ions at m/z 939 of a
tryptic fragment, residues 721-736. In the spectrum, asterisks are used to denote single or multiple neutral losses
of water (or ammonia) from an ion.
Analysis of HPLC peak 3 by
MALDI-TOF gave two major peaks of molecular weights 2,483 and 2,562
(not shown). These differ in mass by 79, suggesting that the lighter
peptide is the nonphosphorylated version of the heavier peptide. The
mass corresponds to the tryptic peptide 446-467. Analysis of this
material by micro-LC/MS also showed the presence of these two peptides.
Only a very weak neutral loss ion was seen for the phosphopeptide. The
micro-LC/MS/MS spectrum of the putative phosphopeptide is shown in Fig. 6B. A series of y ions,
y-y
, as well as a series of
double-charged y ions, y
-y
, was seen,
indicating that Ser-454 is phosphorylated. Loss of phosphoric acid from
double-charged y ions, y
-y
, was
observed.
Analysis of HPLC peak 2 by micro-LC/MS was problematic in
that this HPLC fraction contained multiple peptides, and no major peak
was accompanied by a fragment resulting from the neutral loss of
phosphoric acid. Furthermore, no pair of peaks that differed by the
mass of the phosphate group was found. However, phosphoamino acid
analysis of P-labeled peak 2 material confirmed that
phosphoserine was present (Fig. 7, lane 2).
Consequently, HPLC peak 2 material was purified further by HPLC and
two-dimensional peptide mapping (see ``Experimental
Procedures''). Even after further purification, a candidate
phosphopeptide could not be identified. As a result, the peptide was
submitted to automated Edman sequencing. This analysis revealed a major
component that was identified as the tryptic peptide starting at
residue 446, which suggests that it is the tryptic peptide
446-467. Analysis of the original micro-LC/MS data set revealed
the presence of this peptide in its nonphosphorylated form. Thus,
remarkably, HPLC peak 2 material is contaminated with HPLC peak 3
material even after extensive purification. The sequencing also
revealed a minor component with the sequence
QNPSXXXVSLSNVEAX, where X designates amino
acids that could not be identified in the sequencing cycle. The
sequencing data suggested that this peptide(721-736) is the
result of incomplete trypsin digestion. The original micro-LC/MS data
set was analyzed for the presence of this peptide and its
phosphorylated derivative. Although ions corresponding to this peptide
were not observed, a major peptide in the mixture had a mass
corresponding to the 721-736 peptide with one phosphate and three
additional oxygens (calculated 1,876.0 from the observed m/z values of the +2 and +3 ions of 626 and
939, respectively). It should be stressed that no loss of phosphoric
acid was seen from this peptide. The mass data suggested that the
cysteine residue of the 721-736 peptide had become oxidized
during isolation. A major peak of mass 1,876.2 was also seen in the
MALDI-TOF analysis of this phosphopeptide (not shown). Fig. 6C shows the micro-LC/MS/MS spectrum of the
+3 ion. The y ions y
-y
were
observed, and the series indicates that Ser-727 is phosphorylated. An
incomplete series of b ions was observed, as well as fragments that are
the result of multiple losses of water and ammonia. This b ion series
is also consistent with Ser-727 as the phosphorylation site since the
high negative charge near the internal trypsin cleavage site is
probably the reason that trypsin did not cleave at this site. Finally,
an authentic sample of the Ser-727 phosphopeptide was prepared by
oxidation (29) of a machine-synthesized phosphopeptide and was
found to have the same HPLC retention time as cPLA
-derived
peak 2.
Figure 7:
Phosphoamino acid analysis. HPLC-purified P-labeled tryptic peptides derived from cPLA
from okadaic acid-stimulated Sf9 cells (lane 1, peak 4; lane 2, peak 2) or
P-labeled tryptic digests
derived from cPLA
from human monocytes (lane 3,
unstimulated monocytes; lane 4, okadaic acid-stimulated
monocytes) were hydrolyzed with HCl and separated along with
phosphoserine (PS), phosphothreonine (PT), and
phosphotyrosine (PY) standards on cellulose thin layer plates
as described under ``Experimental Procedures.'' Phosphoamino
acid standards were visualized with ninhydrin, and the
P-labeled phosphoamino acids were detected by
autoradiography.
Figure 8:
Effect of stimulation of human monocytes
on [H]arachidonic acid release and on the
mobility of cPLA
on a SDS-polyacrylamide gel. Panel
A, [
H]arachidonic acid-labeled monocytes
were incubated with Me
SO (0.1%,
), okadaic acid (1
µM,
), LPS (1 µg/ml,
), PMA (500
nM,
), opsonized zymosan (60 particles/cell,
), or
A23187 (0.5 µg/ml,
) for the indicated times.
H
label released into the media is expressed as a percent of the total
radioactivity (cell associated plus media). The results are the average
of duplicate values from a representative experiment. Panel B,
monocytes were stimulated for 2 h with Me
SO (0.1%), okadaic
acid (1 µM), LPS (1 µg/ml), PMA (500 nM),
opsonized zymosan (60 particles/cell), or A23187 (0.5 µg/ml). Cell
lysates (20 µg/lane) were separated on a 7.5% SDS-polyacrylamide
gel. Immunoblots were incubated with a polyclonal antibody against the
recombinant cPLA
, and the Amersham ECL system was used for
detection.
Monocytes were P
labeled and stimulated with okadaic acid and opsonized zymosan to
determine if agonist treatment induced an increase in phosphorylation
of the cPLA
. Incubation with okadaic acid for 2 h induced a
2.5 ± 0.97-fold (mean ± S.E., three experiments) increase
in phosphorylation of the cPLA
, whereas in one experiment
opsonized zymosan increased phosphorylation by 2.7-fold. Phosphoamino
acid analysis showed that phosphorylation occurred exclusively on
serine residues in unstimulated and okadaic acid-stimulated monocytes (Fig. 7, lanes 3 and 4). Initial attempts to
obtain enough
P-labeled monocyte cPLA
to
prepare tryptic digests for HPLC to compare with cPLA
phosphorylated in Sf9 cells were unsuccessful because of
inefficient labeling of the monocyte cPLA
. Consequently,
tryptic phosphopeptides of the
P-labeled cPLA
from monocytes and Sf9 cells were compared by two-dimensional
phosphopeptide mapping. The migration pattern of the phosphopeptides
derived from monocyte cPLA
was similar to that of
phosphopeptides derived from recombinant cPLA
except for
novel phosphopeptides that appeared in monocyte samples in the spot 7
region. Two principal phosphopeptides, 4 and 7, were observed on
peptide maps of tryptic digests of cPLA
derived from
unstimulated monocytes (Fig. 9A and Fig. 10A). After stimulation with okadaic acid
phosphopeptide 2 became very prominent, and new phosphopeptides, 3 and
6, appeared (Fig. 9B). After zymosan stimulation
phosphopeptides 4 and 7 were most prominent, and also evident were
phosphopeptides 2 and 6 (Fig. 10B). The yields of
phosphopeptides transferred from the tubes to the cellulose plates as
gauged by Cerenkov counting were variable, and thus the increases in
the phosphorylation of the phosphopeptides on the maps could only be
considered in a qualitative manner. The yields were nearly quantitative
and consistent in the HPLC separations, and only these data were used
for obtaining quantitative data after stimulation. To confirm the
identity of the monocyte phosphopeptides, samples from stimulated
monocytes were cospotted with samples from okadaic acid-stimulated Sf9
cells (Fig. 9D and Fig. 10D).
Comparison of the maps with the cospotted samples with the maps of the
samples run alone showed that the phosphopeptides 2, 3, and 4 derived
from monocyte cPLA
comigrated with those derived from
cPLA
expressed in Sf9 cells. In addition, weak spots in the
1 and 5 positions were seen in the sample from zymosan-stimulated
monocytes. Spot 7 is probably an oxidized derivative of the MAP kinase
peptide (spot 4). This spot was seen only on some of the phosphopeptide
maps. In addition, oxidation of pure phosphopeptide 4, which was eluted
from the cellulose plate, with performic acid gave rise to material
that migrated in the spot 7 region (not shown). In the analysis of some
samples of trypsin-digested cPLA
from Sf9 cells by HPLC, a
small peak of cpm eluted just prior to peak 4. Two-dimensional
phosphopeptide mapping analysis of this pre-peak 4 revealed a spot in
the 7 region, showing that it is the oxidized phosphopeptide 4 (not
shown). Spot 6, seen in Fig. 9and Fig. 10, requires
additional comment. As seen in these figures, spot 6 is seen with
cPLA
derived from monocytes and Sf9 cells. However, as
shown in Fig. 5, in this map spot 6 (not labeled, between 2 and
4) represents a small percentage of the total phosphopeptides from
cPLA
expressed in Sf9 cells. Spot 6 intensity was highly
variable in comparison with the other phosphopeptides and was seen in
only 5 out of 25 independent tryptic digests of cPLA
from
Sf9 cells. This suggests that spot 6 is either a partial tryptic
fragment or a derivative of one of the other phosphopeptides.
Altogether, these results suggest that the major phosphorylation sites
on cPLA
from Sf9 cells and monocytes are the same.
Figure 9:
Two-dimensional tryptic phosphopeptide
maps of P-labeled cPLA
from human monocytes.
Tryptic digests of immunoprecipitated, gel-purified cPLA
from unstimulated monocytes (panel A), from okadaic
acid-stimulated monocytes (panel B), from okadaic-stimulated
Sf9 cells (panel C), or from okadaic acid-stimulated monocytes
cospotted with okadaic acid-stimulated Sf9 cells (panel D)
were separated by two-dimensional phosphopeptide mapping as described
under ``Experimental Procedures.'' Phosphopeptides were
detected by autoradiography and are labeled as described under
``Results.'' The samples were applied at the spots marked by arrows. Electrophoresis was run in the horizontal dimension,
with the anode on the left, and chromatography was run in the vertical
dimension.
Figure 10:
Two-dimensional tryptic phosphopeptide
maps of P-labeled cPLA
from human monocytes.
Tryptic digests of immunoprecipitated, gel-purified cPLA
from unstimulated (panel A), from zymosan-stimulated
monocytes (panel B), from okadaic-stimulated Sf9 cells (panel C), or from okadaic-stimulated Sf9 cells cospotted with
zymosan-stimulated monocytes (panel D) were separated by
two-dimensional phosphopeptide mapping as described under
``Experimental Procedures.'' Phosphopeptides were detected by
autoradiography and are labeled as described under
``Results.'' The samples were applied at the spots marked by arrows. Electrophoresis was run in the horizontal dimension,
with the anode on the left, and chromatography was run in the vertical
dimension.
The Sf9 baculovirus expression system has been used to study
phosphorylation of mammalian proteins using numerous approaches.
Coexpression of protein along with its upstream activating kinase has
resulted in the expression of activated phosphoprotein(39) .
Insect cell kinases activated during viral infection also phosphorylate
expressed proteins(26) . In addition, expressed proteins can
undergo stimulus-dependent phosphorylation in insect
cells(40) . In this study expression of cPLA in
insect cells enabled these cells to release arachidonic acid, but only
after stimulation with either A23187 or okadaic acid, demonstrating
that in this model pathways for the activation of cPLA
appear to exist which are similar to those in mammalian cells.
Previously, it was found that cPLA
is phosphorylated on
Ser-505 during expression in Sf9 cells(27, 37) . In
this study we identified three additional serines that are
phosphorylated on cPLA
during expression in insect cells.
Stimulation of insect cells with okadaic acid resulted in an increase
in phosphorylation of Ser-727 on cPLA
. Phosphorylation of
this site also appeared to be increased in monocytes after stimulation
with okadaic acid and opsonized zymosan, suggesting that this site may
have a regulatory role in activation of cPLA
resulting in
arachidonic acid release in mammalian cells.
Although previous
studies have suggested that the cPLA is phosphorylated on
multiple sites after cell activation, only one site, Ser-505, which is
phosphorylated by MAP kinase, has been identified (13, 21) . Phosphorylation of cPLA
on
Ser-505 also occurs during expression in Sf9 cells possibly by an
insect MAP kinase homolog resulting in activated enzyme and retardation
in electrophoretic mobility on SDS-polyacrylamide
gels(24, 27) . The recombinant cPLA
mutant, which has an alanine replacing Ser-505, does not exhibit
this gel shift when expressed in Sf9 cells(27) . However,
although cPLA
is partially phosphorylated on Ser-505 in
unstimulated Sf9 cells, we found that this is not sufficient to cause
these cells to release arachidonic acid since stimulation with A23187
or okadaic acid was required to induce arachidonic acid release. Since
neither of these agonists induced a large increase in phosphorylation
at Ser-505, this suggests that other mechanisms are required for full
activation of cPLA
leading to arachidonic acid release. It
has now been established that in some systems in addition to
phosphorylation an increase in calcium is required for full activation (21) . For example, stimulation of macrophages with
colony-stimulating factor-1, which does not increase calcium levels in
these cells, can induce phosphorylation of cPLA
and
activation of MAP kinase without inducing arachidonic acid
release(
)(41) . However, agonists that induce an
increase in calcium act synergistically to affect arachidonic acid
release when added with agonists that fully induce a gel shift, such as
colony-stimulating factor-1. These results have led to the hypothesis
that an increase in intracellular calcium, to promote association of
the cPLA
with the membrane, as well as phosphorylation of
cPLA
is required for full activation (21, 41) . However, an increase in intracellular
calcium does not appear to be required in some cells as some agonists
that can stimulate arachidonic acid release do not raise intracellular
calcium levels. For example, okadaic acid induces arachidonic acid
release in mouse peritoneal macrophages and activates MAP kinase but
does not raise intracellular calcium levels
(23) .
Similarly, okadaic acid did not raise intracellular calcium levels in
Sf9 cells, unlike the calcium ionophore bromo-A23187 (data not shown).
Consequently, the results suggest that phosphorylation of Ser-727 on
cPLA
in okadaic acid-stimulated Sf9 cells allows
association of the cPLA
with the membrane in the absence of
an increase in intracellular calcium levels. Additional experiments are
required to explore this possibility fully.
Many investigators use
the retardation of cPLA on SDS-polyacrylamide gels as an
indicator of phosphorylation of cPLA
on Ser-505. Recently,
MAP kinase (ERK1/ERK2)-independent pathways have been implicated in
phosphorylation of the cPLA
leading to a gel
shift(22) . The okadaic acid-induced gel shift (Fig. 3)
was independent of the phosphorylation of the MAP kinase peptide.
Okadaic acid slightly increased the level of phosphorylation of the MAP
kinase peptide to levels similar to those of A23187, but only okadaic
acid induced a gel shift, suggesting that the okadaic acid-induced gel
shift was caused by the phosphorylation of Ser-727.
The kinase that
phosphorylates the cPLA in insect cells on Ser-727 has not
been identified, but this serine lies within a consensus sequence for a
basotrophic kinase (RXS), such as protein kinase C or protein
kinase A(42, 43) . These kinases can phosphorylate
cPLA
, in vitro, but phosphorylation has not
resulted in a consistent increase in enzyme
activity(20, 21) . Protein kinase C-dependent pathways
have been implicated in the activation of the cPLA
. PMA, an
activator of protein kinase C, can stimulate phosphorylation of
cPLA
in mammalian cells(13, 19) . PMA can
also stimulate phosphorylation of the cPLA
in insect
cells(27) . However, since PMA can also activate MAP kinase in
mammalian cells further studies are needed to determine whether protein
kinase C can directly phosphorylate cPLA
or whether this
kinase produces an indirect effect(23, 44) . The
results of this study suggest that Ser-727 is also phosphorylated in
okadaic acid- and opsonized zymosan-stimulated human monocytes,
indicating that this site may be regulated in monocytes during cell
activation. The conservation of Ser-727 (as is seen with Ser-505), even
in an evolutionary distant species such as zebrafish, suggests that
this serine may have an important regulatory role. Ser-454, on the
other hand, which is phosphorylated only to a small extent in Sf9 cells
and monocytes, is not conserved even in the mouse, whereas Ser-437 is
conserved in mouse and chicken(6) . In conclusion, the Sf9
baculovirus expression system is a useful system for studying
cPLA
phosphorylation and activation and has led to the
identification of multiple novel phosphorylation sites on
cPLA
. Further studies will determine whether these sites
play a functional role in the activation of cPLA
.