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INTRODUCTION |
Mammalian tissues contain a soluble phospholipase A1 that can
catalyze the preferential hydrolysis of
PA1 in assays using mixed
micelles (1) or unilamellar vesicles (2). The enzyme has been purified
to homogeneity from bovine testes (3) and shown to have a molecular
mass of 97.6 kDa, as determined by matrix-assisted laser
desorption/ionization (4). Its cDNA has been cloned and
sequenced and shown to encode an 875-amino acid protein that resembles
other phospholipases only in so far as it contains a five-amino acid
lipase consensus domain that includes a central serine residue (serine
540) required for catalysis (4). Moreover, analyses of the distribution
of the human enzyme's mRNA have provided evidence that this enzyme
and one of its splice variants are expressed selectively in human tissues.2 However, the
enzyme's biological role remains to be determined, and little is known
about the regulation of its activity inside cells.
The aim of the present investigation was to explore the possibility
that protein kinases and phosphatases might affect the behavior of the
first identified (bovine) splice variant of the enzyme, which we now
call PA-PLA1
. We expressed an affinity-tagged, recombinant form of
this enzyme in Sf9 cells, purified it, and examined the ability
of several protein kinases and phosphatases to phosphorylate or
dephosphorylate it in vitro. But only CK2 and ERK2
phosphorylated the phospholipase with significant stoichiometry, and
only PP2A could catalyze the hydrolysis of the phosphate esters. We
used mass spectrometry to identify the amino acids that were phosphorylated or dephosphorylated and used several other approaches, including immunoprecipitation, quantitative densitometry, size exclusion chromatography, and enzyme activity analysis to characterize complexes of the enzyme that were formed in vitro or were
identified in homogenates of the macaque testis and cerebral cortex.
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EXPERIMENTAL PROCEDURES |
Materials--
Oligonucleotides for expression of epitope-tagged
PA-PLA1 in Sf9 cells (see below) were synthesized by Life
Technologies, Inc. Phosphatidylcholine and
sn-1-alkyl-2-oleoyl phosphatidic acid were purchased from
Avanti Polar Lipids. FLAG peptide and FLAGM2 affinity beads (FLAGM2
antibody bound to Protein A-Sepharose beads) were from Sigma.
[32P]ATP Easytide (specific activity 6000 Ci/mol) was
from PerkinElmer Life Sciences. Recombinant forms of human
CK22
2, 
protein phosphatase, and rabbit protein phosphatase 1 were from New England Biolabs. Constructs for bacterial expression of human
GST-22, GST-CK2, GST-CK2', and
GST-CK2 were gifts from Dr. Dongxia Li. MAP kinase kinase-activated
preparations of rat ERK2, rat c-Jun N-terminal kinase, mouse p38 S6
kinase, and starfish oocyte p34cdc2 kinase were from
Calbiochem. MAP kinase-activated ERK1 (rat) was from Upstate
Biotechnology, Inc. (Lake Placid, NY). PP2A (catalytic subunit), sequencing grade modified trypsin, and AspN were from Promega. Protein standards for size exclusion chromatography were
from Bio-Rad. Sephadex G50, ATP
S, AMP-PNP, and the mixture of
protease inhibitors used ("TM Complete Protease Inhibitor Mixture," which contained antipain, bestatin, chymostatin, E-64, leupeptin, pepstatin, phosphoramidon, pefabloc, EDTA, and aprotinin) were from
Roche Molecular Biochemicals. Thesit was from ICN Biochemicals. C18
microcolumn packing was from Michrom Bioresources, Inc. High purity
acetonitrile for high pressure liquid chromatography was from Burdick
and Jackson. Glutathione-Sepharose 4B beads, HiLoad Superdex 200 HR
10/30 columns, and HiLoad Superdex 200 26/60 columns were from Amersham
Pharmacia Biotech. PVDF membranes were from Millipore Corp. All other
reagents were from Sigma or J.T. Baker Inc. unless mentioned otherwise.
Antibodies--
Antibodies to CK2, CK2', and CK2, which
had been prepared by David Litchfield, were gifts from Dr. Dongxia Li.
The antibody to ERK2 was from Calbiochem. Polyclonal antibodies against
two peptides from PA-PLA1, TKRRLREIEERLHGLKASS (corresponding to a
putative coiled-coil-forming region,
Thr589-Ser607) and KHEHDNNVKPSLDPV
(corresponding to the C-terminal region, Lys861-Val875) were prepared by Research
Genetics Inc. and subsequently affinity-purified on peptide columns, as
described (4). Horseradish peroxidase-coupled anti-rabbit IgG antibody
was from Amersham Pharmacia Biotech.
Expression and Purification of PA-PLA1 from Sf9
Cells--
The open reading frame of PA-PLA1, attached at its 5'
end to a sequence of nucleotides that corresponded to the FLAG peptide (DYKDDDDK) followed by hexahistidine (HHHHHH), was cloned into pFASTBAC-HTc vector (Life Technologies), and recombinant virus was
prepared according to the manufacturer's instructions. Sf9 cells growing in TNM-FH medium (Grace's insect medium, Life
Technologies) containing 10% fetal calf serum, 100 units/ml
penicillin, 100 µg/ml streptomycin were infected for 66 h with
the recombinant virus at a multiplicity of infection of 5 and a density
of 2.5 × 106 cells/ml. The cells were then harvested
and homogenized in 3 volumes of homogenization buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, protease
inhibitors) at 4 °C using a Dounce homogenizer. The homogenate was
centrifuged for 15 min at 600 × g. The resulting low
speed supernatant was centrifuged for 1 h at 235,000 × g. The recombinant enzyme in the final, high speed
supernatant was adsorbed on a column of FLAGM2 affinity beads. The
column was washed three times with 5 column volumes of homogenization
buffer, and the enzyme was eluted from the column by competitive
replacement with FLAG peptide according to the manufacturer's
instructions. Typically, 1 liter of Sf9 culture medium yielded
2-5 mg of purified protein, which appeared as a single band of about
110 kDa when analyzed by SDS-PAGE and had a specific activity of
300-360 pmol/min/µg of protein when analyzed by the mixed micelle
enzyme assay described below.
Analysis of PA-PLA1 Activity--
The enzyme's activity was
analyzed with the use of the mixed micelle assay or unilamellar vesicle
assay described by Lin et al. (2). For the mixed micelle
assays, 40 ng of enzyme in a volume of 10 µl were incubated for 20 min at 37 °C with mixed micelles (90 µl) that contained Triton
X-100/Triton X-114 (1:1), 0.5 mol % [3H]16:0-18:1 PA,
10 mol % sn-1-alkyl-2-oleoyl phosphatidic acid (16 mM total micellar lipid), and the amounts of
[3H]16:0 that were generated were measured as described
(2). For the unilamellar vesicle assays, 40 ng of PA-PLA1 were
incubated for 15 min at 37 °C in 100 µl of MOPS-KCl buffer that
contained 30 µM fatty acid-poor bovine serum albumin plus
unilamellar vesicles (1 mM total vesicle
phosphoglycerides), which had been prepared from a mixture of
3H-labeled 16:0-18:1 PA plus 16:0-18:1 molecular species
of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
and diacylglycerol plus cholesterol (molar ratio = 1:1.5:4.5:2:1:10). Then the reaction was stopped, the lipids were
extracted, and the amount of 3H-labeled 16:0 that had been
released was measured as described (2).
Dephosphorylation of the Purified Recombinant Enzyme--
Before
studying the phosphorylation of recombinant PA-PLA1 (in most
experiments), we removed esterified phosphate groups that Sf9
cells had introduced into it by attaching the enzyme (~1 mg of
protein) to FLAGM2 affinity beads and incubating it for 30 min at
30 °C with 20,000 units of protein phosphatase in the presence
of 0.1 mM Thesit (to stabilize the PA-PLA1). After the
incubations, the enzyme-containing beads were washed three times with 5 column volumes of homogenization buffer, and the enzyme was eluted as
described above.
Measurements of Phosphorylation Reaction Stoichiometry after
Incubations Involving Soluble Enzymes--
Recombinant PA-PLA1 (0.5 µg) was incubated for 10-60 min at 30 °C in 25 µl of
phosphorylation buffer (20 mM HEPES, pH 7.4, 20 mM MgCl2, 0.55 mM MgATP, 100 µM [-32P]ATP) that contained 500 units
of CK222, CK2, or CK2' or the
following amounts of activated MAP kinases: ERK2 (120 units), ERK1 (50 units), c-Jun N-terminal kinase (30 units), p38 S6 kinase (1 µg), and
p34cdc2 (0.5 µg). The reactions were stopped by the addition
of SDS sample buffer (5), the mixtures were boiled for 5 min, and the
phosphorylated enzymes were separated by SDS-PAGE and stained with
Coomassie Blue R-250. The gels were then dried and examined by
autoradiography using Eastman Kodak Co. BIOMAX MS film. The identified
bands were excised and soaked in 5 ml of Ecolume (ICN Biochemicals).
The radioactivity was measured with a Beckman scintillation counter. The molar ratio of incorporated phosphate/PA-PLA1 was calculated on
the basis of the combined specific radioactivity of the ATP and the
total amount of phospholipase that had been used in the assays. This
amount was determined by analysis using SDS-PAGE, Western blotting with
the antibody to the coiled-coil-forming region, and quantitative
densitometry (see below). Control incubation experiments using casein or myelin basic protein as a substrate demonstrated that each of
the kinases used in the above incubations was active.
Identification of Phosphorylated Sites by ESI-LC-MS/MS
Analysis--
After phosphorylating PA-PLA1 with CK2 or ERK2, we
used SDS-PAGE to purify the 32P-labeled enzyme and then
digested aliquots of the purified enzyme separately with trypsin and
AspN as described by Shevchenko et al. (6). Briefly, the gel
band containing the phosphorylated phospholipase was excised,
dehydrated for 10 min in CH3CN, and dried in a Speedvac
(Savant). Pieces of dried gel were incubated for 45 min at 4 °C in
100 µl of 50 mM NH4HCO3 that
contained trypsin (12.5 µg/ml) or AspN (12.5 µg/ml). Then the
temperature was increased to 37 °C, and the incubation was continued
overnight. After the incubations, the digested PA-PLA1 was extracted
from the gel pieces, first with the use of 100 µl of 20 mM NH4HCO3 and then with 100 µl
of 50% CH3CN, 5% formic acid, 45% water. The extracted peptides were pooled and dried and then dissolved in solvent A, which
contained 5% CH3CN, 0.4% acetic acid, 0.005%
heptafluorobutyric acid in water. Approximately 200 fmol of the
digested protein sample were loaded onto a homemade, 75-µm inner
diameter microcolumn of C18, which had been prepared as described (7).
Capillary LC was performed with the use of Applied Biosystems 149B dual syringe pumps at a flow rate of 100 µl/min and a precolumn flow splitting ratio of 50:1, which resulted in a final flow rate through the column of 200 nl/min. After the sample was loaded, the column was
washed for 5 min with 100% solvent A. Then the peptides were eluted
over a 60-min time period with a linear (0-80%) gradient of solvent B
(80% CH3CN, 0.4% acetic acid, 0.005% heptafluorobutyric acid). The eluted peptides were analyzed by on-line ESI-MS/MS using a
Finnigan LCQ ion trap mass spectrometer (Finnigan MAT LCQ, San Jose,
CA) (8). ESI was performed using a needle voltage set at 1.8 kV. The
heated capillary temperature was set at 170 °C. The scan range was
400-1800 m/z. The computer algorithm SEQUEST (9)
was used to compare tandem mass spectra directly with amino acid
sequence data bases and the PA-PLA1 sequence. Peptides that contained phosphorylated serine or threonine residues were identified by searching protein sequences that contained the serine or threonine residues corrected for the presence of phosphate ester groups (m +80). Results obtained from automated sequence data base
searching were manually confirmed.
Analysis of Cross-antagonism between
CK222 and ERK2--
protein
phosphatase-pretreated PA-PLA1 (0.5 µg) was incubated for 30 min
at 30 °C with CK222 (500 units) in 25 µl of phosphorylation buffer that contained unlabeled MgATP (0.55 mM). Then ERK2 (120 units) and [32P]ATP (100 µM) were added in 10 µl of phosphorylation buffer and the incubation was continued for an additional 60 min. Alternatively, the phospholipase was incubated for 30 min with ERK2 plus unlabeled ATP. Then CK222 and radioactive ATP were
added, and the incubation was continued for an additional 60 min. In
either case, the reaction was stopped by the addition of SDS sample
buffer, the mixture was boiled for 5 min, radioactive PA-PLA1 was
isolated by SDS-PAGE, the gel was stained with Coomassie Blue R-250,
the band of radioactive phospholipase was excised and counted in a
scintillation counter, and the number of moles of phosphorus that had
been incorporated per mole of phospholipase in the incubations with
radioactive ATP was calculated on the basis of the combined specific
radioactivity of ATP in the incubations.
Dephosphorylation of PA-PLA1 by PP2A--
PA-PLA1 that had
been phosphorylated by either CK2 or ERK2, as mentioned above, was
brought up to a volume of 100 µl by the addition of 50 mM
Tris-HCl, pH 8.5, 20 mM MgCl2, 1 mM
DTT, 0.01% -mercaptoethanol, 0.1 mg/ml BSA; passed through a column
of Sephadex G50 to remove free nucleotides; and incubated for 30 min at
30 °C with PP2A. The reaction mixture was then boiled in SDS sample buffer, PA-PLA1 from the reaction mixture was purified by SDS-PAGE, and the amount of radioactive phosphate that remained associated with
the phospholipase was calculated as described above.
Binding of PA-PLA1 to CK222 or
Its Subunits--
Recombinant, GST-tagged preparations of CK2
22 (4 µg), CK2
'22 (4 µg), CK2 (2 µg), CK2'(2
µg), or CK2 (2 µg) were bound separately to beads of
glutathione-Sepharose 4B and then incubated for 30 min at 30 °C with
dephosphorylated, recombinant PA-PLA1 (5 µg) in 500 µl of
phosphorylation buffer. After the incubations, the beads were washed
three times with 1-ml portions of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and then extracted with SDS sample buffer. The
PA-PLA1 in the extracts was purified by SDS-PAGE and transferred to
PVDF membranes; then the enzyme protein on the membranes was identified
by Western blotting using the antibody to the putative
coiled-coil-forming region of PA-PLA1.
Relation between Phosphorylation of PA-PLA1 by CK2 and
Complex Formation by the Two Enzymes--
protein
phosphatase-pretreated, recombinant PA-PLA1 (0.2 pmol) was incubated
for 30 min at 4 °C with FLAGM2 affinity beads (40 µl of a 50%
slurry) in homogenization buffer (500 µl) that contained 0.1 M Thesit. After the incubation, aliquots of the beads were
washed three times with 1-ml portions of homogenization buffer (to
remove unbound PA-PLA1) and then incubated separately for 5-120-min
periods at 30 °C with 5 mol of GST-CK2 in phosphorylation buffer
(25 µl) containing 32P-labeled ATP (for phosphorylation
studies) or unlabeled MgATP (for studies of complex formation). After
each incubation, the beads were washed three times with 1 ml of
homogenization buffer, the reactions were stopped by the addition of
SDS sample buffer, the mixtures were boiled for 5 min, and the enzymes
they contained were purified by SDS-PAGE and stained with Coomassie
Blue R-250. After this, phosphate incorporation into the enzymes was
measured as described under "Measurements of Phosphorylation Reaction
Stoichiometry after Incubations Involving Soluble Enzymes," or the
time course and stoichiometry of complex formation by PA-PLA1 and
CK2 was determined by transferring the enzymes to PVDF membranes and
probing them with the antibody to CK2 or the coiled-coil-forming
region of PA-PLA1 followed by the horseradish peroxidase-coupled
antibody to rabbit IgG. The response of each enzyme was visualized by
enhanced chemiluminescence (2), quantitated with the use of a Bio-Rad model GS-700 imaging densitometer (2), and converted into a molar
concentration by comparison with signals from standards containing
recombinant GST-CK2. Different amounts of sample were analyzed to
ensure that the amount of GST-CK2 measured would fall within the
linear range of the standard curve.
Stability of the Complex between PA-PLA1 and
CK2--
PA-PLA1 that had been immobilized on FLAGM2 beads was
incubated for 30 min at 30 °C with CK2 and MgATP in
phosphorylation buffer. Then the beads were washed with homogenization
buffer to remove unbound CK2 and either extracted directly with SDS sample buffer or incubated separately for 30 min at 30 °C in control buffer (50 mM Tris-HCl, pH 8.5, 20 mM
MgCl2, 1 mM DTT, 0.01% -mercaptoethanol, 0.1 mg/ml BSA) or in this buffer plus PP2A (5 units), 1% Triton X-100,
150 mM NaCl, or 350 mM KCl. After the
incubation, the amounts of CK2 that had dissociated from the beads
were determined by SDS-PAGE, Western blotting, and quantitative densitometry.
Molecular Basis of Complex Formation between PA-PLA1 and
CK2--
Samples of kinase-free, recombinant PA-PLA1 that had
been phosphorylated by CK222 were
prepared by immobilizing protein phosphatase-treated, recombinant
PA-PLA1 (0.5 µg) to FLAGM2 beads, as described above, and treating
the bound phospholipase successively for 30 min at 30 °C with 1)
CK222 (400 units) plus MgATP in phosphorylation buffer that contained 0.1 M Thesit; 2)
buffer alone (50 mM Tris-HCl, pH 8.5, 20 mM
MgCl2, 1 mM DTT, 0.01% -mercaptoethanol, 0.1 mg/ml BSA, 0.1 mM Thesit) or buffer plus PP2A (5 units); 3) 350 mM KCl plus 0.1 mM Thesit; and
4) 50 mM Tris-HCl, pH 7.5, 150 mM NaCl (three
times). These treatments yielded kinase-free preparations of PA-PLA1
that contained phosphate groups that were esterified either to serines
93, 105, and 716 or only to serines 93 and 105. The ability of these
preparations to bind CK2 in the presence or absence of MgATP,
nonhydrolyzable analogs of ATP, or other nucleotides was then
determined as described under "Binding of PA-PLA1 to
CK222 or Its Subunits". Control samples of bound, protein phosphatase-pretreated PA-PLA1 were incubated successively with CK222, buffer
or PP2A, 350 mM KCl, and CK2 but no MgATP.
Size Exclusion Chromatography of the Complex between PA-PLA1
and CK2--
Recombinant forms of PA-PLA1 (50 µg), GST-tagged
CK2 (50 µg), and a complex of PA-PLA1 and GST-tagged CK2
(which had been prepared by incubating 50 µg of the phospholipase
with 31 µg of the kinase for 30 min at 30 °C in phosphorylation
buffer that contained 0.55 mM MgATP plus 0.1 mM
Thesit) were chromatographed separately at 4 °C on a column of
HiLoad Superdex 200 HR 10/30 that was connected to a BioLogic HR
Chromatography System (Bio-Rad). The column had been preequilibrated
with a "cytosolic" buffer that contained 1) 10 mM
PIPES, pH 7.2, 150 mM potassium glutamate, 5 mM
nitrilotriacetic acid, 0.5 mM EGTA, 2 mM MgATP,
1 mM DTT (a mixture of ingredients reported to support the
metabolism of permeabilized mammalian cells (10)) and 2) the following
mixture of protease inhibitors: 1 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, 2 µg/ml each of
leupeptin, pepstatin, and aprotinin. A similar buffer was used for the
size exclusion chromatography at a flow rate of 0.4 ml/min. An aliquot
(50 µl) of each fraction (0.5 ml) from the column was blotted onto an
Immobilon-P PVDF membrane (Millipore) using a Bio-Dot apparatus
(Bio-Rad) and probed with either an antibody to the predicted coiled
coil-forming region of PA-PLA1 as described (2) or an antibody to
CK2. The chemiluminescent response also was measured, as described
(2). Then molecular masses of the analyzed proteins and complex were
determined on the basis of comparisons of their elution volumes with
those of the Bio-Rad size exclusion standards, blue dextran 2000 (2000 kDa), bovine thyroglobulin (670 kDa), bovine -globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B12 (1.3 kDa).
Analysis of Complexes Containing PA-PLA1 in Homogenates of
Macaque Testis or Cerebral Cortex--
Testes or brain prefrontal
cortical regions were removed from adult male macaques shortly before
death and minced by hand. The minced tissues were washed three times
with ice-cold cytosolic buffer and then homogenized in 3 volumes of
this buffer using a Potter-Elvehjem homogenizer. The homogenate was
centrifuged for 10 min at 800 × g, and the low speed
supernatant was collected and centrifuged for 1 h at 235,000 × g in a Beckman Ti 45 rotor. The resulting high speed
supernatant was flash-frozen in 10-ml aliquots. Subsequently, the
aliquots were thawed separately and loaded at a flow rate of 2.6 ml/min
onto a column of HighLoad Superdex 200 26/60 that had been
preequilibrated with cytosolic buffer as described above for the size
exclusion chromatography of recombinant proteins. Fractions were
collected at 1.8-ml intervals beginning at 107 ml, aliquots of the
fractions were analyzed by Western blotting and quantitative
densitometry using antibody to the C-terminal region of PAPLA1, and
peaks containing the enzyme were pooled and concentrated to a final
volume of about 1 ml using a Centricon concentrator (Millipore).
Finally, the concentrated peaks containing PA-PLA1 from the testes
or brains were precleared by treatment for 1 h at 4 °C with 2 µg of nonspecific rabbit IgG and 40 µl of protein A-Sepharose beads
(50% slurry). The remaining, unabsorbed material was incubated for
3 h at 4 °C with antibody to the C-terminal region of
PA-PLA1 (2 µg) plus protein A-Sepharose beads (40 µl). The beads
were then washed three times with 1 ml of cytosolic buffer, extracted
with SDS sample buffer, boiled for 5 min, and analyzed by Western
blotting using antibodies to PA-PLA1, CK2, or CK2.
Analysis of Complex Formation between PA-PLA1 and
ERK2--
Recombinant PA-PLA1 (0.5 µg) that had been pretreated
with protein phosphatase was immobilized on FLAGM2 beads and then incubated for 30 min at 30 °C with recombinant ERK2 (2 µg) in 500 µl of phosphorylation buffer in the presence or absence of 0.55 mM MgATP. After the incubations, the beads were washed
three times with 1-ml portions of 50 mM Tris-HCl, pH 7.5, plus 150 mM NaCl and then extracted with SDS sample buffer.
The extract was analyzed by SDS-PAGE and transferred to PVDF membranes.
Then an antibody to ERK2 was used to probe the membranes.
Other Methods--
Proteins were measured by the micro-BCA
method (Bio-Rad). Phosphorylation sites were predicted with the use of
the protein data base search software program PROSITE (available on the
World Wide Web) for protein functional regions and
post-translational modifications. The molecular masses of recombinant,
epitope-tagged PA-PLA1 and GST-CK2 were determined with the
software program PeptideMass (available on the World Wide Web).
Statistical analyses were done with Microsoft Excel. Molecular modeling
studies of the predicted coiled-coil-forming region were done with Rasmol.
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RESULTS |
CK222, CK2, CK2', and a
Putative Sf9 Cell MAP Kinase Phosphorylate PA-PLA1--
The
sequence of PA-PLA1 contains predicted phosphorylation sites for
several protein kinases including CK222
(not shown). To investigate the possibility that
CK222 might catalyze the phosphorylation
of the phospholipase in vitro, we incubated purified recombinant preparations of the two enzymes together for 60 min in the
presence of radioactive ATP and then measured the stoichiometry of
PA-PLA1 phosphorylation (see "Experimental Procedures"). The results of six experiments demonstrated that 2 mol of phosphorus were
incorporated per mole of the phospholipase (Table
I). To identify the sites that were
phosphorylated, we analyzed digests of the phospholipase by
ESI-LC-MS/MS (see "Experimental Procedures"). Unexpectedly, the
analysis identified four phosphorylated peptides, not two,
as would have been predicted from the measurements of phosphorylation
stoichiometry that were made after the in vitro incubations.
Moreover, each peptide had a molecular mass that exceeded the value
predicted from cDNA sequence analysis by m +80 and
therefore contained a single esterified phosphate group.
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Table I
Stoichiometry of PA-PLA1 phosphorylation by CK2
Recombinant PA-PLA1 that had or had not been pretreated with protein phosphatase was incubated for 60 min at 30 °C with
recombinant preparations of CK222, CK2, or
CK2' in the presence of radioactive ATP, and the stoichiometry of
phosphorylation was measured (see "Experimental Procedures").
Values are means ± S.E. from duplicate analysis of six
experiments for the nonpretreated PA-PLA1 sample and three
experiments for the pretreated PA-PLA1 sample.
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Three of the peptides contained serine residues that preceded nearby
glutamates and were predicted CK222
phosphorylation sites. 1) Peptide 91D ...
R102 was phosphorylated on serine 93 (Fig.
1A). 2) Peptide
103Y ... R143, which contained eight
serines, appeared to be phosphorylated only on serine 105 (Fig.
1B). We identified this phosphorylation site by exclusion on
the basis of the combined results of the b and y ion series. We
detected a mass of +80 in the b ion series b1-14
(103Y ... S116) but were unable to assign
the phosphate from this series. On the other hand, we detected no mass
of +80 in y1-37 (143R ...
G107), which contained seven out of the eight serines in
the sequence, serines 109, 114, 115, 116, 117, 128, and 130. 3) Peptide
711D ... R735 was phosphorylated on serine
716 because there was a loss of mass of 80 between
b5 and b7 (Ile715 and
Glu717; Fig. 1C).
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Fig. 1.
Identification of phosphorylated sites after
incubation of recombinant PA-PLA1 with
CK222
and MgATP. Recombinant PA-PLA1 (which had not been pretreated
with protein phosphatase) was incubated for 30 min at 30 °C with
CK222 plus [-32P]ATP and
then purified by SDS-PAGE, subjected to in-gel digestion with trypsin
or AspN, and analyzed by micro-LC/MS/MS (see "Experimental
Procedures"). A-C, CK2 phosphorylation sites;
D, presumptive proline-directed protein kinase
phosphorylation site. Sequences of identified phosphopeptides are shown
above the mass spectra; phosphorylation sites are indicated
by asterisks; numbers in superscript
on the amino and carboxyl termini of each peptide denote the location
of the peptide within the PA-PLA1 sequence; bn
denotes the ion generated by cleavage of the peptide bond after the
nth amino acid from the amino terminus;
yn denotes the ions generated from the carboxyl
terminus; identified b or y ions are shown in boldface
letters; and values of m/z
(mass/charge) for ions are indicated in the mass spectra.
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The fourth phosphorylated peptide, 786D ...
L812, contained an esterified phosphate group on serine
793, which was not a predicted CK222
phosphorylation site (Fig. 1D). Instead, proline residues were present nearby, consistent with the possibility that serine 793 might have been phosphorylated by a proline-directed kinase in
Sf9 cells. Note that others have shown that Sf9 cells
contain a MAP kinase that can phosphorylate a recombinant form of the arachidonoyl-specific phospholipase A2 (11) but also that the phosphorylated peptide 786D ... L812,
which we detected, comprised only a minority of the total
786D ... L812 peptide identified in the
digests of recombinant PA-PLA1.
The results of other experiments using a preparation of the
phospholipase that had been treated with protein phosphatase before
being incubated with radioactive ATP plus either
CK222, CK2, or CK2' showed that
each of the three preparations of CK2 could catalyze the incorporation
of a maximum of 3 mol of phosphorus into the phospholipase (Table I;
also see Fig. 5A). Therefore, it appeared that all three
forms of CK2 could phosphorylate the phosphatase-pretreated PA-PLA1
on serine 93, serine 105, and serine 716.
The fact that incubation with CK2 in vitro caused two
phosphate groups to be incorporated into the untreated
phospholipase but three to be incorporated into the treated
phospholipase suggested that Sf9 cells might have partially
phosphorylated the phospholipase on CK2-dependent sites
during its expression. In support of this possibility, the mobility of
a preparation of untreated phospholipase that had not been incubated
with CK2 in vitro increased when it was incubated with protein phosphatase, as determined by SDS-PAGE (data not shown), and
analysis of the resulting product by mass spectrometry provided
evidence that it contained no residual esterified phosphate (data not
shown). However, we were unable to obtain further proof that Sf9
cells had phosphorylated the enzyme on CK2-dependent sites
by examination of a digest of a phospholipase preparation that had been
exposed to neither the kinase nor the phosphatase. The only
phosphorylated peptide that we could detect was one containing serine
793 (data not shown). The MS analysis was not quantitative, so we may
well have missed peptides that were phosphorylated on CK2 sites in low
stoichiometry. But conclusive proof that Sf9 cells
phosphorylated the phospholipase on CK2 sites remains to be obtained.
ERK2 Phosphorylates PA-PLA1--
In subsequent in
vitro incubation experiments, we attempted to identify a MAP
kinase that could catalyze the phosphorylation of serine 793, as the
putative proline-directed kinase in Sf9 cells did. We incubated
phosphatase-pretreated, recombinant PA-PLA1 with radioactive ATP
plus a constitutively activated form of either ERK1, ERK2, c-Jun
N-terminal kinase 1, p38, or p34cdc2 but found that only ERK2
could phosphorylate the PA-PLA1 with significant stoichiometry. It
catalyzed the incorporation of 1 mol of phosphate/mol of the
phospholipase (Table II), and we
identified only one phosphorylated peptide, 711D ...
R735 upon examining digests of the ERK2-phosphorylated
phospholipase using LC-MS/MS (Fig. 2).
Identification of the phosphorylated amino acid in this peptide was
difficult at first because of the peptide's fragmentation pattern and
its content of four prolines, six serines, and three threonines. But we
scanned through a narrow window of m/z 2500-3000
using alternate MS-MS/MS in a second run and were able to identify
serine 730 as the only phosphorylated residue. Thus, ERK2
phosphorylated the enzyme in vitro but on a different site
than the putative proline-directed protein kinase in Sf9 cells
did. Therefore, the identity of this Sf9 cell protein kinase
remains to be determined.
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Table II
Phosphorylation of PA-PLA1 by ERK2 and cross-antagonism between ERK2
and CK222
Recombinant PA-PLA1 that had been pretreated with protein
phosphatase was incubated for 60 min with 32P-labeled ATP plus
ERK2 or CK222 (as a control). Alternatively, the
phospholipase was first incubated for 30 min with
CK222 plus unlabeled ATP and then incubated for
an additional 60 min with ERK2 plus 32P-labeled ATP or
incubated for 30 min with ERK2 plus unlabeled ATP and then incubated
for 60 min with CK2 plus 32P-labeled ATP. Following the
incubations, radioactive phosphorus (P) incorporated into the
phospholipase was determined on the basis of the combined specific
radioactivity of MgATP in the incubations (see "Experimental
Procedures"). Results represent means ± S.E. of duplicate
measurements from two different sets of experiments.
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Fig. 2.
Identification of phosphorylated site after
incubation of phosphatase- pretreated PA-PLA1
with ERK2 and MgATP. The pretreated phospholipase was
incubated for 30 min at 30 °C with ERK2 and MgATP and then purified,
digested, and analyzed by LC/MS/MS (see "Experimental Procedures").
The sequence of the phosphorylated peptide, the phosphorylation site,
the identified b or y ions, and the m/z value are
shown as in Fig. 1.
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Selective, Cross-antagonism of Phosphorylation by CK2 and
ERK2--
The proximity of the CK2 phosphorylation site, serine 716, and the ERK2 phosphorylation site, serine 730, raised the possibility that phosphorylation reactions involving the two sites might influence each other. To examine this possibility, we first incubated a phosphatase-pretreated preparation of recombinant PA-PLA1 for 30 min
at 30 °C with CK222 plus
unlabeled ATP under conditions that could cause the
incorporation of 3 mol of phosphate into each mole of the phospholipase
and then added ERK2 plus radioactive ATP to the mixture and
continued the incubation for an additional 60 min. The results revealed
that less than 0.1 mol of radioactive phosphate became incorporated
into the phospholipase during the incubation with ERK2 (Table II).
Moreover, when we incubated recombinant PA-PLA1 with ERK2 plus
unlabeled ATP in a parallel experiment and then added
CK222 plus radioactive ATP and
continued the incubation, 2 mol of radioactive phosphate, not 3 mol,
were incorporated per mole of the phospholipase. Because incubation
experiments with mixtures of the phospholipase plus radioactive ATP
plus either ERK2 or CK222
without pretreatment with the other kinase showed significantly higher
amounts of incorporated radioactive phosphate (Table II), these results
provided evidence that phosphorylation of the phospholipase by
CK222 inhibited the subsequent
phosphorylation of the phospholipase by ERK2 and vice versa.
Furthermore, analysis by LC/MS/MS showed that the inhibitory effect of
phosphorylation by ERK2 on phosphorylation by
CK222 specifically involved the CK222 phosphorylation site, serine 716. Thus, upon phosphorylating the enzyme successively with ERK2 and
CK222, we could identify only three
phosphorylated amino acid residues: the
CK222 phosphorylation sites, serine 93 and serine 105, and the ERK2 phosphorylation site, serine 730 (scans
not shown).
PP2A Dephosphorylates PA-PLA1 Selectively--
To identify a
physiologically relevant protein phosphatase that could dephosphorylate
PA-PLA1, we first incubated the phospholipase in the presence of
radioactive ATP and CK222 and then
removed the remaining radioactive nucleotides and incubated the
phosphorylated phospholipase with calcineurin, protein phosphatase 1, or the catalytic subunit of PP2A (see "Experimental Procedures").
Upon reisolating the PA-PLA1 and measuring its content of
radioactive phosphorus, we found that only PP2A could dephosphorylate
the phospholipase and that it removed about one-third of the total radioactivity (Table III). Furthermore,
we obtained a similar result when we incubated the phospholipase with
MgATP and CK222, treated the
phosphorylated phospholipase with 350 mM KCl, washed out
the KCl, and then incubated the phospholipase with PP2A (data not shown). This control experiment ruled out the possibility that a
complex formed between CK222 and
PA-PLA1 (see below) might have prevented the PP2A from catalyzing
the hydrolysis of the remaining esterified phosphate groups on the
phospholipase, because complexes between the enzyme and CK2 or
CK222 dissociate when they are treated
with 350 mM KCl (Table IV and
data not shown).
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Table III
PP2A dephosphorylates PA-PLA1 selectively
Samples containing protein phosphatase-pretreated recombinant
PA-PLA1 were incubated for 30 min at 30 °C in the presence of
radioactive 32P-labeled ATP plus either
CK222 or ERK2 and then treated for 30 min with
PP2A at 30 °C (see "Experimental Procedures"). Alternatively,
control samples of the phospholipase were incubated for 30 min with
radioactive 32P-labeled ATP and CK222 or
ERK2 but were not subsequently treated with PP2A. After the
incubations, radioactive phosphorus (P) in PA-PLA1 was measured as
described (see "Experimental Procedures"). Results represent
means ± S.E. of duplicate measurements from two different sets of
experiments.
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Table IV
Effects of Triton X-100, PP2A, and KCl on the stability of the
PA-PLA1 plus CK2 complex
PA-PLA1 that had been immobilized on FLAG beads was incubated for 30 min at 30 °C with CK2 plus MgATP in phosphorylation buffer (see
"Experimental Procedures"). Samples containing the immobilized
complex of PA-PLA1 plus CK2 were then washed with homogenization
buffer to remove unbound CK2 and subsequently incubated separately
for 30 min at 30 °C in buffer containing 50 mM Tris-HCl,
pH 8.5, 20 mM MgCl2, 1 mM DTT, 0.01%
-mercaptoethanol, 0.1 mg/ml BSA 1% (control) or buffer plus either
one of the following components: 1% Triton X-100, 150 mM
KCl, PP2A, or 350 mM KCl. At the end of incubation, the
amount of CK2 in the supernatant was analyzed by SDS-PAGE, Western
blotting, and quantitative densitometry. The total CK2 present in
the original complex was determined by extracting CK2 from the
complex with SDS sample buffer and analyzing the CK2 in the extract
using a similar approach (data not shown). The results indicate
means ± S.E. from two different experiments.
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To determine whether PP2A catalyzed the complete removal of
phosphate from a single site on the phospholipase or catalyzed the
partial removal of phosphates from several sites, we used LC-MS/MS to
analyze digests of phospholipase that had been successively phosphorylated by CK222 and then
dephosphorylated by PP2A. The results of the analysis demonstrated that
serine 716 had lost 80 mass units, whereas serine 93 and 105 were fully
phosphorylated (Fig. 3 and data not
shown). Therefore, the action of the phosphatase was site-specific.
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Fig. 3.
PA-PLA1 that has
been phosphorylated by
CK222
can be selectively dephosphorylated by PP2A. PA-PLA1 that had
been pretreated with protein phosphatase was incubated for 30 min
at 30 °C with CK222 and MgATP and then
isolated by size exclusion chromatography and incubated with PP2A as
described under "Experimental Procedures." Afterward, the
PA-PLA1 was purified by SDS-PAGE, and peptides obtained by in-gel
digestion were analyzed by LC/MS/MS (see "Experimental
Procedures"). The sequences of the phosphorus-containing peptides,
identified b or y ions, and m/z values are shown
as in Fig. 1.
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When we did a comparable set of incubation experiments using
PA-PLA1 that had been phosphorylated by ERK2, we found that PP2A
could hydrolyze almost all of the phosphate that was esterified to the
phospholipase, whereas both protein phosphatase 1 and calcineurin were
without effect (Table III and data not shown). Since ERK2 could
phosphorylate only one site, serine 730, this site was clearly the one
that was dephosphorylated. Therefore, PP2A dephosphorylated both serine
716 and serine 730, the same two serines that showed cross-antagonism
of phosphorylation by CK222 and ERK2.
Whether this was coincidental or reflected a special structural feature of the PA-PLA1 remains to be determined (but see
"Discussion").
PA-PLA1 Forms Stable Complexes with
CK222, CK2, and CK2' in
Vitro--
CK2 has been reported to form stable complexes with several
of its substrates through interactions that involve either its subunit or subunit (12, 13). To determine whether
CK222 and its subunits form stable
complexes with PA-PLA1, we incubated protein
phosphatase-pretreated PA-PLA1 with or without MgATP in the presence
of beads of glutathione-Sepharose 4B that contained immobilized
GST-tagged CK222,
CK2'22, CK2, CK2', or CK2 and
then washed the beads and measured the amount of phospholipase that
bound to them (see "Experimental Procedures"). The results demonstrated that PA-PLA1 could bind to GST-tagged
CK222, CK2, or CK2' in the presence
of MgATP but not in its absence. Furthermore, they showed that the
phospholipase could not bind to GST-tagged CK2 or to GST alone in
the presence or absence of MgATP (Fig. 4
and data not shown). Therefore, PA-PLA1 appears to belong to the
group of CK2 substrates that bind directly to CK2 or CK2'.
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Fig. 4.
PA-PLA1 forms stable
complexes with CK2. Samples of GST-linked
CK222, CK2, CK2', CK2, or GST
alone, which had been immobilized on glutathione-Sepharose 4B beads,
were incubated separately with PA-PLA1 plus MgATP for 30 min at
30 °C. The beads were washed with homogenization buffer, extracted
with SDS sample buffer, and analyzed by SDS-PAGE followed by Western
blotting with the antibody to the coiled-coil-forming region of
PA-PLA1. Similar results were obtained in two additional
experiments.
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In further studies, we focused attention on the complex that
contained PA-PLA1 and CK2. To examine the relation between the
phosphorylation of the phospholipase by CK2 and the formation of
this complex, we incubated the two enzymes together for periods of
5-120 min in the presence of unlabeled MgATP or radioactive ATP and
compared the time courses and reaction stoichiometries of these
processes (see "Experimental Procedures"). The results of two
independent experiments demonstrated that 1) the time courses of
PA-PLA1 phosphorylation and complex formation were similar, although
not completely identical, 2) 3 mol of phosphorus were ultimately
incorporated into each 97.6-kDa molecule of PA-PLA1, 3) the complex
ultimately contained 0.8 mol of CK2/mol PA-PLA1, and 4) no
detectable radioactive phosphate was incorporated into CK2 (Fig.
5, A and B). This
provided evidence that the phosphorylation of PA-PLA1 by CK2
promoted the formation of a stable, 1:1 complex between the two
enzymes.
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Fig. 5.
Relation between the phosphorylation of
PA-PLA1 by CK2 and
the formation of a complex between the two enzymes. PA-PLA1
that had been immobilized on FLAGM2 affinity beads was incubated with
GST-CK2 for periods of 5-120 min in phosphorylation buffer that
contained 32P-labeled ATP or unlabeled MgATP. After each
incubation period, the beads were washed with homogenization buffer and
extracted with SDS-sample buffer. Then the two enzymes were purified by
SDS-PAGE, and the time courses of PA-PLA1 phosphorylation
(A) and complex formation (B) were determined as
described under "Experimental Procedures." Note that no
incorporation of radioactive phosphorus into CK2 was detected, that
the results represent duplicate analyses from two different
experiments, and that similar results were obtained when time course
experiments were performed with enzymes in solution.
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We next examined the stability of the complex and found that it
was relatively resistant to extraction with 1% Triton X-100 or 150 mM KCl and remained essentially intact after approximately one-third of the phosphate esterified to PA-PLA1 was removed by
treatment with PP2A (Table IV). This indicated that the stability of
the complex did not depend on the PP2A-sensitive phosphate group that
was esterified to serine 716 (see Table III and Fig. 3). Importantly,
however, the complex dissociated when it was treated with 350 mM KCl (Table IV), which suggested that the stability of
the complex depended on electrostatic interactions involving other
components of the two enzymes.
Molecular Basis of Complex Formation--
To examine the
possibility that these electrostatic interactions might have involved
the phosphate groups that were esterified to serines 93 and 105, we
took advantage of the results of the above studies to prepare
phosphorylated, kinase-free forms of the phospholipase that contained
esterified phosphate groups on serines 93 and 105 or serines 93, 105, and 716 (see "Experimental Procedures"). Upon incubating these
preparations separately with fresh CK2, we were surprised to find
that even the maximally phosphorylated form of PA-PLA1 did not bind
the CK2 except in the presence of MgATP (Fig.
6A). Furthermore, MgGTP could
substitute for the MgATP, but (Mg)ATPS, (Mg)AMP-PNP, MgADP, MgUTP,
and MgCTP were without effect (Fig. 6, A and B).
These results left open the possibility that formation of the complex
might have depended on the MgATP-dependent phosphorylation
of serines 93 and 105 but showed that an additional, highly specific,
MgATP/MgGTP-dependent mechanism was involved. Because
CK2 can accommodate either MgATP or MgGTP in its
Mg-trinucleotide-substrate-binding site (14) and could phosphorylate
PA-PLA1 in the presence of either one (data not shown), it seemed
likely that MgATP or MgGTP contributed to the formation of the complex
by binding to this site (see "Discussion").
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Fig. 6.
Specificity of the nucleotide requirement for
complex formation between PA-PLA1 and
CK2. Aliquots of GST-CK2 were
incubated separately for 30 min at 30 °C in phosphorylation buffer
that contained one of several nucleotides plus one of the following
preparations of recombinant PA-PLA1 that were bound to FLAGM2-beads:
1) a preparation that had been dephosphorylated by phosphatase; 2)
a similar preparation that had first been dephosphorylated by phosphatase and then phosphorylated for 30 min at 30 °C with CK2
and subsequently treated with 350 mM KCl; or 3) a similar
preparation of successively dephosphorylated and phosphosphorylated
PA-PLA1 that had been treated with PP2A before being treated with
350 mM KCl. After the incubations, the beads were washed
with buffer, and the amounts of CK2 that had bound to PA-PLA1
were determined (see "Experimental Procedures"). In A,
the nucleotides used were MgATP, ATPS, or AMP-PNP; in B,
the nucleotides were MgATP, MgGTP, MgADP, MgCTP, or MgUTP. Similar
results were obtained in two experiments.
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Evidence That the Complex of PA-PLA1 and CK2 Contains
Four Molecules of Each Protein--
We used size exclusion
chromatography to estimate the molecular masses of soluble preparations
of recombinant PA-PLA1 and GST-tagged CK2 and to compare them
with that of a complex of the two enzymes that had been formed in the
presence of MgATP (Fig. 7). When the
recombinant PA-PLA1 was chromatographed by itself, it had an
apparent molecular mass of 220 kDa, which provided evidence that it was
a dimer. On the other hand, the GST-tagged CK2 was a monomer because
it had a molecular mass of 62 kDa, as compared with the expected mass
of 68 kDa (26-kDa GST plus 42-kDa CK2). In view of these results, we
expected that a 1:1 complex of the two enzymes that was formed in the
presence of MgATP would have an apparent molecular mass of about 340 kDa. However, upon incubating the enzymes together under the
appropriate conditions and examining the resulting complex by size
exclusion chromatography, we found that the complex had an apparent
molecular mass of about 650 kDa, which suggested that it was a
heterooctamer formed from four molecules of PA-PLA1 and four
molecules of GST-tagged CK2. These results suggested that at least
three types of binding sites contributed to the complex: 1) a binding
site on the phospholipase that promotes the formation of an enzyme
dimer, 2) a binding site for CK2 on each subunit of the dimer, and
3) a binding site that promotes the conversion of a presumptive,
intermediate enzyme heterotetramer into a heterooctamer. Further
structural work will be required to clarify the molecular basis of
these binding phenomena.
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Fig. 7.
Size exclusion chromatography of recombinant
PA-PLA1, GST-tagged
CK2, and a complex of the two enzymes.
PA-PLA1, GST-tagged CK2, and a complex of PA-PLA1 plus
GST-tagged CK2 were chromatographed separately on a 10/30 column of
Superdex G 200, and positions of the proteins emerging in the effluent
were monitored by dot blotting using antibodies to the enzymes followed
by quantitative densitometry (see "Experimental Procedures").
Molecular masses of the proteins shown were calculated on the basis of
a standard curve generated from experiments with bovine thyroglobulin
(670 kDa), bovine -globulin (158 kDa), and chicken ovalbumin (44 kDa), whose elution positions are indicated by arrows.
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The Effects of Phosphorylation and Complex Formation on the
Phospholipase Activity of PA-PLA1--
To examine the effect of
phosphorylation by CK2 on the catalytic properties of PA-PLA1, we
incubated the kinase with the protein phosphatase-pretreated
phospholipase in the presence of unlabeled MgATP under conditions that
could cause complex formation (control experiments using radioactive
ATP confirmed that 3 mol of phosphorus were incorporated into the
phospholipase) and then used a mixed micelle assay or a unilamellar
vesicle assay to measure the activity of the phospholipase (see
"Experimental Procedures"). The results of two independent
experiments with each type of assay indicated that the incubation
caused a 50% loss of phospholipase activity (Table
V). These results differed from those of
corresponding incubation experiments with PA-PLA1 plus ERK2 and
MgATP (see "Experimental Procedures"), which provided no evidence
for complex formation or phosphorylation-dependent loss of
phospholipase activity (data not shown and Table V).
,
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ABSTRACT
INTRODUCTION
