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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 930-939
The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase- Are
Substrates, In Vitro, for a Constitutively Associated Protein
Tyrosine Kinase in Platelets
By
Norman R. Geltz and
James A. Augustine
From Blood Research Institute, Milwaukee, WI.
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ABSTRACT |
Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase
consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent
protein serine kinase activity capable of phosphorylating the 85-kD
subunit. Here, we examine the Mn2+-dependent protein
kinase activity of PI3K immunoprecipitated from normal
resting or thrombin-stimulated platelets, and characterize p85/p110
phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated
PI3K showed p85 and p110 were phosphorylated on serine, but in
contrast to previous results, were also phosphorylated on threonine and
tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation;
however, p110 phosphorylation was also inhibited suggesting p110
autophosphorylation on serine/threonine. The protein tyrosine kinase
inhibitor, erbstatin analog, partially inhibited p85 and p110
phosphorylation but did not appear to affect PI3K lipid kinase
activity. The in vitro phosphorylation of p85 or p110 derived
from thrombin-stimulated platelets was no different than that of
resting platelets, but we confirm that in thrombin receptor-stimulated
platelets enhanced levels of p85 and PI3K lipid kinase activity were
recovered in antiphosphotyrosine antibody immunoprecipitates. These
results suggest PI3K can autophosphorylate on serine and threonine,
and both p85 and p110 are substrates for a
constitutively-associated protein tyrosine kinase in platelets.
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INTRODUCTION |
PHOSPHATIDYLINOSITOL 3-kinase (PI3K) is a
heterodimer phospholipid kinase composed of an 85 kD (p85) regulatory
subunit and a 110 kD (p110) catalytic subunit which specifically
phosphorylates the D-3 hydroxyl position of membrane
phosphoinositides.1,2 Multiple isoforms of each subunit
have been discovered, as well as a family of proteins homologous to the
p110 subunit.3-8 Numerous studies have shown PI3K activity
is induced after extracellular ligation of a variety of cell-surface
growth-factor or hormone receptors with their respective ligands. In
many cases, receptor-induced tyrosine phosphorylation of intracellular
proteins appears to play a requisite role in the transmembrane
signaling of PI3K activity.9-11 The physical association of
PI3K with either autophosphorylated receptor protein tyrosine kinases
(PTK)9,12 or with certain members of the nonreceptor
src-family of PTKs has been a recurring theme in the study of
receptor-induced PI3K signaling. Interactions with receptor PTKs occur
as a result of an affinity of specific regions in p85, termed
src-homology-2 (SH2) domains, for specific phosphotyrosine-containing
amino acid sequences (pYMXM, pYXXM, X = any amino
acid).9,11,13 Interactions of p85 with the nonreceptor
src-family tyrosine kinases have been reported to occur through binding
of p85 polyproline motifs to SH3 domains contained in
src-kinases.14,15 In T lymphocytes, interleukin-2 (IL-2)
receptor ligation with the T-cell growth factor, IL-2, induces enhanced
PI3K activity in antiphosphotyrosine immunoprecipitates as well as
association of PI3K with the p59fyn src-family
kinase.16 Thrombin receptor activation of platelets results
in intracellular 3-phosphoinositide synthesis17 and the
association of PI3K with the p60scr or
p59fyn kinases.18 Based on these
studies, it is evident protein tyrosine kinases serve as important
intermediaries in PI3K signaling.
It is still unclear what role phosphorylation of PI3K itself serves in
regulating postreceptor PI3K activity. The p85 subunit of PI3K was
shown to be a substrate for a serine/threonine kinase.19 Recently, p85 was found to be phosphorylated, in vitro, on
serine/threonine residues by serine/threonine kinase activity
copurifying with PI3K from rat liver.20 Dhand et
al,21 have shown with baculoviral-expressed p85 and p110 in
insect cells the p110 subunit is actually a dual specificity enzyme
with intrinsic, Mn2+-dependent protein serine kinase
activity capable of phosphorylating the p85 subunit.21
Their study indicated when p85 and p110 were coexpressed in insect
cells, the p85 subunit was phosphorylated on both threonine and serine,
in vivo, but was phosphorylated only on serine, in vitro.21
In addition, the p110 subunit also became phosphorylated on serine, in
vivo, but was not phosphorylated in vitro. Serine phosphorylation of
p85 by p110 results in a decrease of in vitro PI3K lipid kinase
activity,21 indicating the intrinsic serine kinase activity
of PI3K may be important in regulating its lipid kinase activity.
Tyrosine phosphorylation of PI3K has been a more controversial area of
investigation. Several groups have reported tyrosine phosphorylation of
p85,10,19,22,23 and one study has shown p85 and p110
phosphorylation on tyrosine presumably by the activated
platelet-derived growth factor receptor (PDGFR) in PDGFR
immunoprecipitates of platelet-derived growth factor (PDGF)-stimulated
fibroblasts.24 Recently, the tyrosine phosphorylation of
p85 in response to granulocyte-macrophage colony-stimulating factor
(GM-CSF) stimulation of neutrophils has been shown.25 Despite these findings, a functional role for tyrosine phosphorylation of either p85 or p110 is still unclear.
In previous studies showing the intrinsic protein serine kinase
activity of PI3K, experiments were done with either highly purified
PI3K from rat liver,20 or with baculoviral-expressed p85
and p110 in insect cells.20,21 Under these experimental conditions, PI3K was relatively free of associated proteins. Because platelets are a rich source of PI3K, which is rapidly activated on
stimulation of the thrombin receptor,17,26-28 we chose to
investigate the Mn2+-dependent, intrinsic protein kinase
activity of PI3K immunoprecipitated from lysates of resting or
thrombin-stimulated platelets, and characterize the phosphorylation
profile of the p85 and p110 subunits. In this study, we confirm p85
is phosphorylated on serine, in vitro, as previously
shown,20,21 but we also show it is phosphorylated on
tyrosine and threonine. P85 serine/threonine phosphorylation was
inhibitable by LY294002, a specific inhibitor of PI3K. We show p110
was phosphorylated on serine, threonine, and tyrosine, part of which
was inhibitable by LY294002. Threonine phosphorylation of p110 has
not been previously observed. These in vitro studies suggest that
p110 is capable of autophosphorylation on serine and threonine, that
p110 can phosphorylate p85 on serine and threonine, and both
p85 and p110 are substrates for a constitutively-associated protein tyrosine kinase in platelets. The tyrosine phosphorylation of
p85 or p110, however, did not appear to affect the lipid kinase activity of PI3K.
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MATERIALS AND METHODS |
Reagents and antibodies.
The murine monoclonal anti-PI3K (p85) antibody, AB6,29
against human p85 was the generous gift of Dr Shinya Tanaka (Hokkaido University, Sapporo, Japan), and was also obtained from Upstate Biotechnology Inc (Lake Placid, NY). Polyclonal antibody (C-17) to the
p110 subunit of PI3K was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine antibody, 4G10, was obtained from
Upstate Biotechnology Inc. -Actinin monoclonal antibody was obtained
from ICN Biomed (Costa Mesa, CA). IgG1 (MOPC-21) antibody
was from Cappel (West Chester, PA). The thrombin
receptor- activating peptide (TRAP),
SFLLRNPNDKY,30 was synthesized on a Milligen (Framingham,
MA) model 9050 PepSynthesizer. Phosphatidylinositol (PtdIns) and phosphatidylserine were from Avanti Polar Lipids (Alabaster, AL). Erbstatin analog (methyl 2,5-dihydroxycinnamate), PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 were from Calbiochem
(San Diego, CA). Wortmannin and thrombin were from Sigma Chemical Co (St Louis, MO). LY294002 was from BIOMOL (Plymouth Meeting, PA). All
other reagents were obtained from Sigma Chemical Co, unless specified
otherwise.
Platelet isolation and lysis.
Human platelets were isolated from platelet-rich plasma (PRP) from
healthy male volunteer donors (The Blood Center of Southeastern Wisconsin, Milwaukee, WI). PRP was supplemented with 25 ng/mL Prostaglandin E1 and 1 mmol/L acetylsalicylic acid, and
subjected to centrifugation at 120g for 20 minutes to sediment
contaminating blood cells. Platelets were then procured from the
remaining PRP by centrifugation at 500g for 20 minutes. The
platelet pellet was resuspended in "platelet buffer" containing 5 mmol/L piperazine-N,N'-bis-[2-ethanesulfonic acid] (PIPES) [pH
6.8], 145 mmol/L NaCl, 4 mmol/L KCl, 0.5 mmol/L Na2HPO4, 1 mmol/L MgCl2, 0.1%
glucose, 0.1% bovine serum albumin (BSA), and 1 U/mL apyrase,
immediately loaded onto a Sepharose CL-2B (Sigma, St Louis, MO) gel
column, and eluted with platelet buffer. Gel-filtered
platelets were then counted with a Coulter Counter (Hialeah, FL) and
adjusted to the appropriate concentration with platelet buffer.
Equivalent amounts of platelets (1 to 2 × 109) were
used per sample in all assays. Gel-filtered platelets were treated at
25°C with the appropriate stimulus concentration and solubilized in
lysis buffer (137 mmol/L NaCl, 1 mmol/L CaCl2, 1 mmol/L
MgCl2, 20 mmol/L Tris [pH 8], 10% glycerol, 1% Triton X-100) supplemented with 20 µg/mL aprotinin, 20 µg/mL leupeptin, 1 mmol/L Na3VO4, and 1 mmol/L
phenylmethylsulfonyl fluoride (PMSF). The Triton-insoluble fraction was
pelleted by centrifugation at 15,000g for 4 minutes at 4°C,
or for cytosol preparation, at 130,000 g for 45 minutes at
4°C. The supernatants were isolated and used where indicated.
Immunoprecipitation.
Platelet lysates (equivalent to 1 or 2 × 109
platelets) were incubated with equal amounts of the appropriate
antibodies for 2 hours at 4°C, and the antibody immunoprecipitates
were collected on Protein G Agarose for an additional hour. Immune
complexes were washed twice with lysis buffer, twice with 100 mmol/L
Tris (pH 7.6)/500 mmol/L LiCl, and twice with kinase buffer (50 mmol/L HEPES [pH 7.4], 20 mmol/L MnCl2, 5 mmol/L EDTA, 150 mmol/L NaCl, 10% glycerol, and 0.02% Triton X-100). All washes were
supplemented with 20 µg/mL aprotinin, 20 µg/mL leupeptin, 1 mmol/L
Na3VO4, and 1 mmol/L PMSF. Immune complexes
were solubilized in 2×-concentrated sodium dodecyl sulfate
(SDS)-containing sample buffer and frozen until examined by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described below.
Phosphatidylinositol 3-kinase assay.
Phosphatidylinositol 3-kinase activity was determined as described
elsewhere.16 Briefly, washed immunoprecipitates were incubated on ice for 10 minutes in 20 µL of a sonicated substrate mixture containing either PtdIns or PtdIns(4,5)P2, and
phosphatidylserine (1:1) at a concentration of 0.5 mg/mL in 20 mmol/L
HEPES (pH 7.4). Reactions were initiated by addition of 20 µL of
kinase buffer containing 20 mmol/L HEPES (pH 7.4), 50 mmol/L
MgCl2, 50 µmol/L adenosine triphosphate (ATP), 20 µCi
of  -32P-ATP (specific activity, 6,000 Ci/mmole;
Dupont-NEN Research Products, Boston, MA). After 10 minutes at
37°C, the reactions were terminated with 200 µL of 1 N HCl. Four
hundred microliters of CHCl3/CH3OH (1:1) was
added and the phospholipids were extracted. The aqueous layer was
aspirated and the CHCl3 layer was washed once with 160 µL
of CH3OH/1 N HCl (1:1). The resulting CHCl3
layer was dried with nitrogen gas and the phospholipid residues were solubilized in CHCl3/CH3OH (2:1). Radiolabeled
PtdIns(3,4,5)P3 was separated from
PtdIns(4,5)P2, PtdIns(4)P, and PtdIns standards by
thin-layer chromatography (TLC). Briefly, samples were spotted onto
diaminocyclohexane tetra-acetic acid (CDTA)-treated aluminum-backed Silica Gel 60 plates (250 µm, Merck, Darmstadt,
Germany)31 and developed in a mobile phase composed of
n-propanol:2 N acetic acid (65:35).32 Radiolabeled
PtdIns(3)P was separated from PtdIns(4)P as previously
described.31 32P-radiolabeled phosphoinositides
were visualized by autoradiography. 32Pi incorporated into PtdIns(3)P or
PtdIns(3,4,5)P3 was quantitated directly on the TLC plates
with an automated microbiological imaging system (AMBIS) computerized
imaging/radioscanning system (CSP Inc, Billerica, MA). Authentic
PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 standards were
chromatographed in parallel lanes and visualized by spraying with 10%
H2SO4 and heating to 100°C.
One- and two-dimensional gel electrophoresis and immunoblotting.
Proteins were separated by SDS-PAGE as previously
described.16 For immunoblotting, proteins were
electrophoretically transferred to Immobilon-P membrane (Millipore
Corp, Bedford, MA) in 25 mmol/L Tris/192 mmol/L glycine at 300 mA for
30 minutes, then stepped to 800 mA for 1 hour. The membrane was
incubated for 1 hour in blocking solution (Tris-buffered saline [TBS]
in 3% BSA) and incubated overnight with the appropriate antibody in
TBS/1%BSA/0.05% Tween-20. The blots were washed with TBS/0.2%
Tween-20/0.5% BSA, incubated for 1 hour in 1 µg/mL horseradish
peroxidase-conjugated secondary antibody (Pierce, Rockford, IL), and
washed extensively. Immunoreactive proteins were detected by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL) according to
the manufacturer's protocol. For two-dimensional electrophoresis,
proteins were solubilized in urea sample buffer (9.5 mol/L urea, 4%
Triton X-100, 2% ampholines [pH 3-10], 5% 2-mercaptoethanol) and
loaded onto 1.5 mm, prefocused (200 V, 1 hour), isoelectric-focusing
tube gels containing ampholines (pH 3-10). Proteins were focused to
equilibrium at 400 V for 15 hours. Resolution of the proteins in the
second dimension was carried out by SDS-PAGE in 8.75% gels as
described above.
In vitro kinase assay.
Kinase assays were performed on washed immune complexes and initiated
in 20 µL of kinase buffer (50 mmol/L HEPES [pH 7.4], 20 mmol/L
MnCl2, 5 mmol/L EDTA, 150 mmol/L NaCl, 10% glycerol, and
0.02% Triton X-100) containing 50 µmol/L ATP supplemented with 20 µCi -32P-ATP. Wortmannin stocks were in dimethyl
sulfoxide (DMSO) and erbstatin analog and LY294002 stocks were in
ethanol; each were diluted with reaction buffers to desired
concentrations for use in experiments. After 15 minutes at 25°C,
the beads were washed twice with phosphate-buffered saline (PBS)
containing 0.5 mmol/L EDTA and the immunoprecipitated proteins were
solubilized in boiling SDS-containing sample buffer and separated by
SDS-PAGE.
Phosphoamino acid analysis.
32P-radiolabeled proteins were separated by SDS-PAGE and
identified by autoradiography. Bands of interest were excised,
rehydrated in 0.05 mol/L NH4HCO3 containing
0.1% SDS and 30%  -mercaptoethanol, and extracted from the gel. The
proteins were precipitated with trichloroacetic acid using BSA as
carrier. The labeled proteins were hydrolyzed at 110°C in 6 N HCl
and nitrogen gas for 1 hour. The hydrolysate was lyophylized and the
residue was reconstituted in water and spotted onto thin-layer
cellulose plates. 32P-labeled phosphoamino acids were
resolved electrophoretically in the first dimension in pH 1.9 buffer
containing 88% HCOOH, glacial acetic acid, H2O
(50:156:1,794) at 1,000 V for 25 minutes. Resolution in the second
dimension was performed in pH 3.5 buffer containing pyridine, 100 mmol/L EDTA, acetic acid, H2O (10:10:100:1,880) at 500 V
for 25 minutes. Phosphotyrosine, phosphoserine, and phosphothreonine standards were run concurrently and detected with 0.2% ninhydrin in
acetone. Radiolabeled amino acids were detected by autoradiography.
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RESULTS |
Manganese-dependent phosphorylation of the p85 and p110 subunits of
PI3K, in vitro.
The protein kinase activity of PI3K has largely been studied in systems
using either purified PI3K, or baculoviral-expressed PI3K from insect
cells.20,21 Here, we examine the
Mn2+-dependent, in vitro protein kinase activity of PI3K
immunopurified from platelets. PI3K was immunoprecipitated from the
Triton X-100-soluble portion of lysates prepared from resting or
thrombin-treated platelets using an antibody to the p85 subunit of
PI3K. It should be noted, under our lysis conditions p110 remained
bound to p85 during immunoprecipitation as evidenced by coprecipitation
of both subunits (see Fig 2B) as well as PI3K lipid kinase activity
(see Fig 7 and Fig 8B). p110 protein kinase activity was then examined
by in vitro kinase assay in the presence of Mn2+ and
-32P-ATP as previously described.21 Labeled
proteins were separated by SDS-PAGE and subjected to autoradiography.
An 85-kD band immunoprecipitated from either resting or
thrombin-aggregated platelet lysates was heavily phosphorylated
(Fig 1, lanes 2 and 4). A 110-kD band was also very heavily phosphorylated in this reaction. The p85 and p110 in
vitro phosphorylation was Mn2+-dependent (occurring at 50 µmol/L Mn2+) and was not simply due to a nonspecific
divalent cation effect because it did not occur in the presence of
Ca2+ alone, and was only slightly observable in the
presence of Mg2+ alone (unpublished data). Similar results
were obtained when PI3K was immunoprecipitated from
post-130,000g supernatants of platelet lysates
(see Fig 2A).
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| Fig 2.
(A) In vitro phosphorylation of PI3K from immunodepleted
lysates. Post-130,000g cytosolic fractions of lysates from
resting platelets were serially immunodepleted (preclear) four times
with either Protein G beads alone (lane 1), isotype-matched
IgG1 antibody (lane 2), p85 antibody (lane 3), or an
irrelevant antibody (lane 4). All of the precleared lysates were then
re-immunoprecipitated (IP) with equal amounts of PI3K antibody and the
immune complexes subjected to an in vitro kinase assay with
-32P-ATP and Mn2+ as described in
Materials and Methods. Immune complex proteins were eluted, separated
by SDS-PAGE and visualized by autoradiography. Molecular mass markers
in kilodaltons are indicated at the left. (B) The immune complexes
immunoprecipitated from each lane in (A) were eluted from the Protein G
beads, separated by SDS-PAGE and immunoblotted for p110 content with a
p110-specific antibody. Numbers below the lanes indicate the particular
preclearing step. Molecular mass markers in kilodaltons are indicated
at the left.
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| Fig 7.
Effect of Erb-A on PI3K lipid kinase activity. (A)
Triton-soluble fractions of resting platelet lysate were
immunoprecipitated with equal amounts of either PI3K antibody (lanes 5 through 10) or an isotype-matched antibody (lane 4). The washed immune
complexes were subjected to a "cold" in vitro kinase assay in the
presence of ATP/Mn2+, and in the absence or presence of
the indicated concentrations of Erb-A. Control reactions containing no
Erb-A were incubated with either buffer alone (lanes 4 and 5) or
ethanol/buffer vehicle (lane 6). The phosphorylated immune complexes
were washed and incubated for 10 minutes in the presence of
-32P-ATP, Mg2+, and PI(4,5)P2
substrate. Radiolabeled phosphoinositides were extracted and separated
by TLC as described in Materials and Methods. Migrations of authentic
PI(4,5)P2, PI(4)P, or PI standards are indicated by
outlined circles. PI; phosphatidylinositol. (B) Direct quantitation of
radiolabeled spots from lanes 6 through 10 in (A). Each bar represents
the fold-increase of PI(3,4,5)P3 formed (net counts per
minute) relative to the buffer-only control (A, lane 5). Results are
presented as the mean of three experiments ± standard error.
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| Fig 8.
Recovery of p85 and PI3K lipid kinase activity in
anti-Ptyr immune complexes from thrombin receptor-activated platelets.
Platelets were treated for 1 minute with either PBS vehicle or the
indicated concentrations of SFLLRNPNDKY (SFLL) and lysed in 1%
Triton-containing buffer. (A) Triton-solubilized protein was
immunoprecipitated with either no antibody (lane 1) or with anti-Ptyr
antibody (lanes 2 through 9). Immune complexes were washed and
solubilized in SDS-PAGE sample buffer. Proteins were separated by
SDS-PAGE and immunoblotted with anti-p85 monoclonal antibody. Molecular
mass markers in kilodaltons are indicated at the left. IP;
immunoprecipitation. (B) Triton-solubilized protein was
immunoprecipitated with either no antibody (lane 1) or with anti-Ptyr
antibody (lanes 2 through 9). Anti-Ptyr immune complexes were incubated
with phosphatidylinositol (PI) and assayed for PI3K lipid kinase
activity as described in Materials and Methods. Phosphorylated products
were separated by TLC and visualized by autoradiography. The relative
migration of PI4P standard (dotted oval) and PI3P are indicated at the
right. The radioactivity (net counts) present in each spot was
quantitated directly on the TLC plate and was as follows: lane 1, 9,713; lane 2, 11,216; lane 3, 13,226; lane 4, 13,324; lane 5, 7,995;
lane 6, 4,830; lane 7, 18,036; lane 8, 24,355; lane 9, 29,064. Results are representative of several independent
experiments. (C) Platelets were treated with the indicated
concentrations of SFLLRNPNDKY (SFLL) and lysed. The Triton-solubilized
lysates were immunoprecipitated with anti-Ptyr antibody in the absence
( ) or presence (+) of excess (5 mmol/L) phosphotyrosine (Ptyr).
The anti-Ptyr immune complexes were examined for PI3K activity as in
(B).
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| Fig 1.
In vitro phosphorylation of PI3K from resting or
thrombin-stimulated platelets. Control (CO) or thrombin-treated (THR)
platelets were lysed in 1% Triton-containing lysis buffer and
Triton-soluble fractions were immunoprecipitated with equal amounts of
either p85 antibody (lanes 2 and 4) or an isotype-matched control
antibody (lanes 1 and 3). The washed immune complexes were incubated in the presence of -32P-ATP and Mn2+ in an in
vitro kinase assay as described in Materials and Methods. Phosphorylated proteins were eluted, separated by SDS-PAGE and detected
by autoradiography. Molecular mass markers in kilodaltons are indicated
at the left.
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To show the p85 and p110 bands corresponded to the PI3K heterodimer,
equivalent amounts of post-130,000g, cytosolic fractions were
serially precleared four times with either Protein G beads alone, an
isotype-matched antibody, p85 antibody, or an irrelevant antibody. All
of the precleared lysates were then reimmunoprecipitated with p85
antibody and the washed immune complexes subjected to an in vitro
kinase assay with -32P-ATP and Mn2+. Lysates
from resting platelets precleared four times with either Protein-G
beads alone, IgG1, or antibody to the cytoskeletal protein, -actinin, followed by immunoprecipitation with p85 antibody showed extensive p85 and p110 phosphorylation in the in vitro kinase assay
(Fig 2A, lanes 1, 2, and 4). Lysates precleared four times with p85
antibody were completely depleted of p85 and p110 resulting in the
absence of phosphorylated p85 and p110 in the in vitro kinase assay
(lane 3). Figure 2B confirms the PI3K heterodimer was specifically
precleared by the p85 antibody. The four immune complexes precleared
from each lane in Fig 2A were eluted from the Protein G beads,
separated by SDS-PAGE, and immunoblotted for p110 content with a
p110-specific antibody. As is clearly shown, proteins eluted from
beads of the first anti-p85 preclearing step show a considerable amount
of p110 (lane 3), followed by a diminishing amount with each successive
preclearing step. p110 did not appear when the preclearing was done
with beads alone, anti-IgG1, or with antiactinin
antibodies. These results, clearly show the p110 band coprecipitating
with p85 is the p110 subunit of PI3K.
Wortmannin is a potent, irreversible inhibitor of PI3K catalytic
activity. Because p110 has been shown to phosphorylate p85 on serine
608,21 we would expect to see inhibition of p85
phosphorylation in the presence of wortmannin, but we were also
interested in whether p110 phosphorylation would be inhibited.
Anti-PI3K immune complexes from, post-130,000g, cytosolic
fractions of resting platelet lysates were preincubated in the absence
or presence of various concentrations of wortmannin and then subjected
to an in vitro kinase assay. Figure 3A
shows heavy phosphorylation of p85 and p110 occurred in anti-p85 immune
complexes incubated with buffer only (lane 2) or with buffer plus DMSO
vehicle only (lane 3). Anti-p85 immune complexes preincubated in the
presence of 5 or 10 nmol/L wortmannin showed a significant decrease in phosphorylation of p85 showing the protein kinase activity of p110 was
indeed inhibited (lanes 7 and 8). Wortmannin, also inhibited the
phosphorylation of p110 (lanes 7 and 8). The same experiment conducted
with the specific PI3K inhibitor, LY29400233 (1 to 400 µmol/L), gave similar results but even at the higher concentrations
some residual phosphorylation of p85 and p110 remained (Fig 3B). These
data provide additional evidence the p85 and p110 bands phosphorylated
in the in vitro kinase assay are PI3K, and indicate a good portion of
p110 phosphorylation, in vitro, may be due to autophosphorylation.
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| Fig 3.
Effect of wortmannin or LY294002 on protein kinase
activity of PI3K. (A) PI3K was immunoprecipitated from,
post-130,000g, cytosolic fractions of platelet lysates (lanes 2 through 8). Lane 1 consisted of Protein G beads alone incubated with
lysate. The washed immune complexes were incubated for 20 min with
either kinase buffer alone (lanes 1 to 2), buffer/dimethylsulfoxide
vehicle (lane 3), or the indicated wortmannin (Wort) concentrations
(lanes 4 through 8). (B) Same as in (A) except washed immune complexes were incubated with either kinase buffer alone (lanes 1 and 2), buffer/ethanol vehicle (lane 3), or the indicated LY294002 (LY) concentrations (lanes 4 through 8), and lane 1 represents an
IgG1 control antibody immunoprecipitate. Incubations were
then subjected to an in vitro kinase assay as described in Fig 1.
32P-labeled proteins were eluted, separated by SDS-PAGE and
detected by autoradiography. Molecular mass markers in kilodaltons are indicated at the left.
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To analyze for the possibility of multiple bands migrating at 85 and
110 kD, in vitro-phosphorylated PI3K immune complexes from resting
platelet cytosol were analyzed by two-dimensional electrophoresis
involving isoelectric focusing (pH 3-10) in the first dimension
followed by SDS-PAGE in the second dimension. Figure 4, upper panel, illustrates
two-dimensional electrophoresis of in vitro phosphorylated PI3K immune
complexes from resting (CO) platelet cytosol showing heavily
phosphorylated, discrete spots at 85 and 110 kD localizing at acidic
isoelectric points. There was some streaking of p110, which did not
always occur. Two-dimensional analysis of PI3K immune complexes
phosphorylated in vitro in the presence of the specific PI3K inhibitor
LY294002 showed greatly diminished phosphorylation (Fig 4, lower
panel). P85 and p110 phosphorylation was inhibited by 88% and 87%,
respectively, as determined by AMBIS scan quantitation of
radioactivity in each spot. This was a specific decrease in p85 and
p110 because the decrease in intensity of three consistently seen
unidentified spots in the lower panel was only 28%, 25%, and 33%
respectively, relative to the same spots labeled 1, 2, and 3 in the
upper panel. From these results it appears that multiple proteins were
not present in the 85-kD and 110-kD bands and the majority, if not all,
of the bands consisted of the PI3K heterodimer.
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| Fig 4.
Two-dimensional electrophoresis of PI3K immune complexes
phosphorylated, in vitro. PI3K was immunoprecipitated from,
post-130,000g, cytosolic fractions of resting platelet lysates
and the immune complexes subjected to an in vitro protein kinase assay
in the presence of Mn2+ and -32P-ATP in
the absence (CO) or presence of 30 µmol/L LY294002. Radiolabeled proteins were eluted with urea sample buffer and analyzed by
two-dimensional electrophoresis as described in Materials and Methods.
Spots 1, 2, and 3 are unidentified reference points used for relative
quantitation (see Results). Molecular mass markers in kilodaltons are
indicated at the left. IEF; isoelectric focusing.
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Both p85 and p110 are phosphorylated in vitro on serine, threonine,
and tyrosine.
To determine the nature of p85 and p110 phosphorylation, phosphoamino
acid analysis was performed on each phosphoprotein. PI3K was
immunoprecipitated from the cytosolic fraction of resting platelet
lysates and subjected to an in vitro kinase assay with -32P-ATP. The p85 and p110 bands were separated by
SDS-PAGE, localized by autoradiography, extracted from the gel, and
subjected to two-dimensional phosphoamino acid analysis as described in
Materials and Methods. Figure 5A and B,
left panels, clearly show that amino acid hydrolysates of in vitro
phosphorylated p85 and p110 consisted of primarily phosphoserine (pS),
but also significant phosphothreonine (pT) and phosphotyrosine (pY).
Threonine phosphorylation of p110 has not been previously observed.
Figure 5A, right panels, show that addition of LY294002 to the in vitro
kinase assay caused a significant decrease in p85 pS (41%) and pT
(82%) and a slight decrease in pY (10%). For p110, a significant
decrease in pS (55%) and pT(71%) occurred, but a modest decrease in
pY (29%) also occurred. These results show that LY294002 primarily
inhibited serine and threonine phosphorylation of p85 and p110, which
can be expected, with only a modest effect on tyrosine phosphorylation,
indicating again, that the serine/threonine phosphorylation of p85 and
p110 was due to p110 catalytic activity. Erbstatin analog (Erb-A), a
stable analog of erbstatin, is a protein tyrosine kinase inhibitor
capable of inhibiting tyrosine phosphorylation of the epidermal
growth-factor receptor (EGFR) tyrosine kinase (Ki = 3.3 µmol/L).34,35 Erb-A has also been shown to inhibit
autophosphorylation of the p60src tyrosine
kinase.36 Figure 5B, right panels, shows that the presence
of 5 mmol/L Erb-A caused a 27% decrease in p85 pS, an 18% decrease in
pT, and a modest 21% decrease in pY. For p110, a 36% decrease in pS,
a 56% decrease in pT, and a 24% decrease in pY was noted. Although
both p85 and p110 tyrosine phosphorylation was inhibited by Erb-A, it
appears that serine and threonine phosphorylation, primarily of p110,
was also sensitive to the effects of Erb-A.
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| Fig 5.
Two-dimensional phosphoamino acid analysis of
p85 and p110 phosphorylated, in vitro. PI3K was immunoprecipitated
from, post-130,000g cytosolic fractions of platelet lysates and
the immune complexes subjected to an in vitro protein kinase assay in
the presence of Mn2+ and -32P-ATP in the
absence or presence of either 30 µmol/L LY294002 (A), or 5 µmol/L
erbstatin-A (B). Radiolabeled proteins were eluted and separated by
SDS-PAGE. p85 and p110 bands were subjected to two-dimensional
phosphoamino acid analysis as described in Materials and Methods.
Phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y)
standards were run concurrently with the p85 and p110 amino acid
hydrolysates and stained with ninhydrin. Migration of phosphoamino acid
standards was coincident with the radiolabeled spots.
|
|
Erb-A attenuates PI3K phosphorylation, in vitro.
The p110 subunit of PI3K has not been shown to possess intrinsic
tyrosine kinase activity. Therefore, phosphorylation of p85 and p110 on
tyrosine in the in vitro kinase assay suggests they are substrates for
a tyrosine kinase intimately associated with the PI3K immune complex.
To substantiate this, anti-PI3K immunoprecipitates of platelet lysates
were subjected to in vitro kinase assays in the absence or presence of
various concentrations of Erb-A (Fig 6).
Relative to the "ethanol only" control in lane 3, AMBIS scan quantitation of radioactivity incorporated into p85 and p110 shows p85
phosphorylation was inhibited 27% by 5 µmol/L Erb-A (Fig 6, lane 4),
inhibited 52% by 25 µmol/L Erb-A (lane 5), and inhibited 57% and
65% in the presence of 50 and 100 µmol/L Erb-A, respectively (lanes
6 and 7). Phosphorylation of p110 was inhibited 40% by 5 µmol/L
Erb-A (lane 4), inhibited 78% by 25 µmol/L Erb-A (lane 5), and
inhibited 85% and 90% in the presence of 50 and 100 µmol/L Erb-A,
respectively (lanes 6 and 7). These results show that both p85 and p110
phosphorylation was decreased 27% and 40%, respectively, by 5 µmol/L Erb-A, a concentration around the Ki for EGFR
kinase inhibition. The reason for the significant inhibition at the
higher Erb-A concentrations is not understood, but may reflect
competition of high erbstatin concentrations with ATP.34
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| Fig 6.
Effect of erbstatin analog on p85 and p110
phosphorylation, in vitro. PI3K was immunoprecipitated (IP) from
Triton-soluble fractions of resting platelet lysates. Isotype-matched
IgG1 antibody was used as a control (lane 1). Washed immune
complexes were incubated in the absence or presence of the indicated
concentrations of Erb-A for 15 minutes, and subjected to an in vitro
kinase assay in the presence of -32P-ATP, and
Mn2+ as described in Materials and Methods. In the
absence of erb-A, kinase buffer alone (lanes 1 and 2), or
ethanol/buffer vehicle (lane 3) were used as controls.
32P-labeled proteins were eluted, separated by SDS-PAGE,
and identified by autoradiography. Molecular mass markers in
kilodaltons are indicated at the left.
|
|
Erb-A does not affect PI3K lipid kinase activity.
Reports have shown PI3K lipid kinase activity is attenuated by serine
phosphorylation of p85 by p110.21 It was of interest to
know whether PI3K lipid kinase activity was affected by p110 tyrosine
phosphorylation. To determine this, PI3K was immunoprecipitated from
platelet lysates, using equal amounts of p85 monoclonal antibody, and
subjected to a cold in vitro protein kinase assay with ATP in the
absence or presence of various concentrations of Erb-A. The
phosphorylated immune complexes were washed extensively and subjected
to a PI3K lipid kinase assay using
phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) as
substrate in the presence of -32P-ATP and
Mg2+ as described in Materials and Methods.
PtdIns(4,5)P2 was used as a substrate to ensure that the
product of the reaction, PtdIns(3,4,5)P3, was labeled only
on the 3 position. Labeled phospholipids were extracted and separated
by TLC in a mobile phase, which separates phosphoinositides based on
their level of phosphorylation.32 Figure 7A illustrates the separation of
32P-PtdIns(3,4,5)P3 from
PtdIns(4,5)P2, PtdIns(4)P, and PtdIns standards. PtdIns(3,4,5)P3 was formed only on p85 antibody
immunoprecipitates (Fig 7A, lanes 5 through 10), and not on the control
isotype-matched IgG1 immune complexes (Fig 7A, lane 4)
indicating the specificity of the assay. The same profile of lipid
separation was seen when PtdIns(4,5)P2 was used as a
substrate in a PI3K lipid kinase assay performed on antiphosphotyrosine
immune complexes from PDGF-stimulated fibroblast lysates (data not
shown). Figure 7B illustrates quantitation of the radiolabel in spots
shown in Fig 7A, and shows PI3K subjected to in vitro phosphorylation
in the presence of 5 to 100 µmol/L Erb-A (lanes 7 through 10) was no
different in its ability to convert PtdIns(4,5)P2 to
PtdIns(3,4,5)P3 than in the presence of the vehicle
(buffer/ethanol) alone (lane 6).
Recovery of p85 and PI3K activity in anti-Phosphotyrosine immune
complexes from SFLLRNPNDKY-treated platelets.
To determine whether PI3K levels and PI3K enzymatic activity were
recoverable in anti-Ptyr antibody complexes from agonist-activated platelets, platelets were treated with either PBS or a range of suboptimal (<2 µmol/L) to optimally-activating (2 to 8 µmol/L) concentrations of SFLLRNPNDKY (SFLL) peptide and immediately lysed. Triton-soluble proteins were then immunoprecipitated with the 4G10
anti-Ptyr antibody. The immune complexes were then analyzed for either
p85 levels (Fig 8A), or PI3K lipid kinase
activity (Figs 8B and C). In platelets stimulated with activating
concentrations of peptide (aggregation observed, data not shown),
increased levels of anti-Ptyr-recoverable p85 were found (Fig 8A, lanes
7 through 9) when compared to controls (lanes 1 and 2). Although p85
was observed in anti-Ptyr immune complexes from untreated platelets (lane 2) or from platelets treated with concentrations of peptide that
were suboptimal in producing aggregation (lanes 3 through 6), no p85
was present in the sample in which no anti-Ptyr antibody was present
(lane 1). These results suggest that p85, or a complex containing p85
tightly-associated with a phosphotyrosyl protein, was specifically
immunoprecipitated by anti-Ptyr antibody.
Based on the results above and Fig 2B, it would be predicted that
increased levels of p85 in anti-Ptyr immunoprecipitates would parallel
elevated PI3K enzymatic activity. To confirm this, platelets were
treated as described in Fig 8A and platelet lysates were
immunoprecipitated with anti-Ptyr antibody. The immune complexes were
then examined for PI3K enzymatic activity as described in Materials and
Methods. Figure 8B illustrates a stimulus-dependent increase in
anti-Ptyr-recoverable PI3K lipid kinase activity that correlates well
with that of p85 levels shown in Fig 8A. Figure 8B, lane 1, shows that
in the control immunoprecipitation performed in the absence of
anti-Ptyr antibody there was slight PI3K activity nonspecifically bound
to the beads. In the absence of stimulation (lane 2) or in the presence
of suboptimal agonist concentrations (lanes 3 through 6),
anti-Ptyr-recoverable PI3K activity remained at or below control
levels. This activity was increased fourfold to eightfold in a
concentration-dependent manner by optimal agonist concentrations (Fig
8B, lanes 7 through 9). Identical results were obtained when thrombin
was used as agonist (data not shown). The anti-Ptyr recoverable PI3K
activity was specifically phosphotyrosine dependent because it could be
effectively competed away when phosphotyrosine was present in excess (5 mmol/L) during the antiphoshotyrosine immunoprecipitation (Fig 8C).
| |
DISCUSSION |
The PI3K p110 subunit possesses a second catalytic function as a
Mn2+-dependent protein kinase and can phosphorylate the p85
subunit of PI3K on serine 608 in vitro and in vivo.21 In
vivo, serine and threonine phosphorylation of p85 as well as serine
phosphorylation of p110 has also been shown in
32Pi-labeled insect cells cotransfected
with p85 and p110.21 When expressed in insect cells, p85
and p110 were shown to be unassociated with other proteins or
kinases.21 Others have shown in vitro serine
phosphorylation of p85 and, to a lesser extent, of p110 purified from
rat liver.20 However, the PI3K heterodimer normally binds
to a variety of intracellular signaling molecules, many of which are
tyrosine kinases,12,18,37 which could potentially regulate
the activity or trafficking of PI3K. Our results indicate when PI3K
is immunopurified from platelets, p85 is phosphorylated on serine, in
vitro, in a Mn2+-dependent manner as previously
shown,20,21 but is also phosphorylated heavily on threonine
and tyrosine. More surprisingly, p110 is heavily phosphorylated in
vitro on serine, and also significantly on threonine and tyrosine;
however, the in vitro phosphorylation of the isolated PI3K complex does
not appear to be affected by thrombin activation and aggregation of
platelets.
We provide four lines of evidence showing the p85 and p110 bands
observed to be phosphorylated in vitro are indeed PI3K; (1) these bands
are phosphorylated only in anti-PI3K immune complexes; Protein G beads
alone or IgG1 immunoprecipitation show very little to no
nonspecifically-bound or autophosphorylated proteins and no
phosphorylated PI3K, (2) serial immunodepletion of PI3K from platelet
lysates followed by anti-PI3K immunoprecipitation and in vitro kinase
assay results in a complete loss of phosphorylated 85- and 110-kD
bands; immunoblotting the cleared anti-p85 immune complexes with a
p110 antibody confirms the presence of p110, (3) incubation of
anti-PI3K immune complexes with the irreversible PI3K inhibitor,
wortmannin, or with the reversible but specific inhibitor LY294002,
greatly inhibits the in vitro phosphorylation of p85 and p110
indicating that p110 catalytic activity is responsible for substantial
phosphorylation of both p85 and p110, and (4) two-dimensional
electrophoresis of phosphorylated PI3K immune complexes derived from
post-130,000g cytosolic fractions show discrete spots for both
p85 and p110, the intensities of which are attenuated 88% and 87%,
respectively, in LY294002-treated samples, with no evidence for
multiple proteins comigrating with either p85 or p110.
Phosphoamino acid analysis of in vitro phosphorylated PI3K
immunoprecipitated from platelets confirmed both p85 and p110 are
phosphorylated on serine, threonine and tyrosine. Because p110 has been
shown to be a serine kinase it is also possible that it phosphorylates
threonine residues. The significant inhibition of both p85 and p110
phosphorylation by wortmannin or LY294002 treatment would strongly
argue that a portion of the in vitro phosphorylation of p110 on serine
and threonine is likely due to autophosphorylation. P110 is already
known to phosphorylate p85 on serine, but our results indicate p85
threonine phosphorylation also appears to be due to p110 catalytic
activity. Although this is the most likely explanation, our data do not
rule out the possibility of PI3K association with another
serine/threonine kinase. Our observed in vitro tyrosine phosphorylation
of p85 and p110 from resting platelet cytosol would suggest the
presence of a tyrosine kinase constitutively associated with the PI3K
immune complex. Many reports show the association of PI3K with a
variety of tyrosine kinases, including src-family
kinases.14,15,18 Many of these interactions, especially
those with tyrosine phosphorylated receptor tyrosine kinases such as
the prototypic PDGFR, are activation-induced and occur via
phosphotyrosine binding to SH2 domains of p85 serving to activate PI3K
catalytic activity.12,37,38 Although one study has shown
both p85 and p110 subunits are substrates for the activated
PDGFR,24 the lack of evidence showing a transmembrane receptor tyrosine kinase in platelets capable of docking PI3K, in
conjunction with our evidence for significant tyrosine phosphorylation of p85 and p110, would argue for an intimate association between PI3K and a nonreceptor tyrosine kinase in platelets. It is unclear whether PI3K tyrosine phosphorylation exerts a regulatory effect on
p110 lipid kinase activity as does p85 serine
phosphorylation.20,21 Our data show p110 lipid kinase
activity towards PtdIns(4,5)P2 is not affected after PI3K
immune complexes are subjected to Mn2+-dependent in vitro
phosphorylation either in the absence or presence of Erb-A. This
suggests PI3K tyrosine phosphorylation does not appear to influence
p110 lipid kinase activity.
The SH3 domains of certain src-family kinases can bind the polyproline
motifs of p85,14,15 and because these interactions are not
phosphotyrosine dependent they may not be contingent on an activation
stimulus. We consistently observe a protein in the molecular mass range
of 60 to 63 kD, which becomes phosphorylated in the in vitro kinase
assay performed on anti-PI3K immune complexes (Figs 1, 2, and 6). The
in vitro phosphorylation of this protein was inhibited by Erb-A (see
Fig 6). This may be one of several nonreceptor src-family kinases, such
as p60src, p59fyn, or
p62yes, found in platelets. We also observe another
autophosphorylated band at approximately 70 to 72 kD, which
consistently coprecipitates specifically with PI3K. One possible
candidate for this band is p72syk, a tyrosine
kinase that has been shown to bind to PI3K in platelets.39 If the p110 subunit of PI3K does not possess intrinsic tyrosine kinase
activity, then an associated tyrosine kinase must be responsible for
tyrosine phosphorylation of p85 and p110 in vitro. Data from our
experiments using Erb-A support this argument. At 5 µmol/L Erb-A,
which approximates the Ki (3.3 µmol/L) for inhibition of epidermal growth factor receptor (EGFR) phosphorylation, in vitro p85 phosphorylation was inhibited by 27% and p110
phosphorylation was decreased by 40%. Our attempts to immunoblot
proteins derived from anti-PI3K immune complexes with antibodies to
either p72syk or the src-family kinases
p60src, p59fyn, or
p62yes were negative. It is possible only a small
percentage of PI3K may associate with these kinases, which would make
detection difficult. A constitutively-associated tyrosine kinase might
easily phosphorylate p85 or p110 in vitro after cell lysis, as we have
observed, especially a src-family kinase which may become
dephosphorylated at its C-terminal regulatory phosphotyrosine by a
phosphatase. In vivo, a tyrosine kinase constitutively associated with
PI3K may be "off" until an activation stimulus triggers
translocation of the cytosolic PI3K/tyrosine kinase complex to the
membrane/cytoskeleton where the kinase may become activated to
phosphorylate p85 or p110. Tyrosine phosphorylation may then serve to
recruit SH2-containing proteins which may either regulate PI3K activity
or bind downstream signaling molecules.
Whether PI3K phosphorylation is an activation-dependent, biologically
significant event, in vivo, remains an unanswered question. Recently,
the tyrosine phosphorylation of p85 in response to GM-CSF stimulation
of neutrophils has been shown.25 We have been unable to
detect thrombin-dependent phosphorylation of either p85 or p110
in platelets, either in vitro or in vivo. We are able to immunoprecipitate from Triton-soluble fractions of lysates from TRAP-stimulated platelets, enhanced amounts of PI3K and
PI3K lipid kinase activity with antiphosphotyrosine antibody, as shown
in Fig 8. These results confirm that increased levels of the p85 regulatory subunit of PI3K can be recovered in anti-Ptyr antibody complexes from thrombin receptor-stimulated platelets, and this recovery is dependent on stimulus concentrations that induce
aggregation. Moreover, the relative increase in PI3K enzymatic activity
recovered in the anti-Ptyr immune complexes paralleled the increase in
p85 recovered (compare Fig 8A and B), indicating that the catalytic p110 subunit was also present in the anti-p85 immune complexes. It
is not known, however, whether the recovery of PI3K by anti-Ptyr antibody is due to tyrosine phopshorylation of PI3K itself or of an
intimately associated protein.
Based on our data examining the in vitro phosphorylation of PI3K
immunopreciptiated from post-130,000g cytosolic fractions of
platelet lysates, our results suggest that in resting platelets PI3K is
constitutively associated with a non-receptor protein tyrosine kinase
which can use p85 and p110 as substrates, although a direct
association with a kinase has not been confirmed. We have not ruled out
the possibility that the PI3K heterodimer has intrinsic protein
tyrosine kinase activity, but based on previous results21
this does not appear to be likely. We show that p110 is also
threonine phosphorylated which has not been previously observed, and in
the context of our studies with wortmannin and LY294002, a considerable
portion of p110 serine/threonine phosphorylation appears to be a
result of autophosphorylation. Recently, a newly discovered
leukocyte-specific PI3K designated p110 was shown to
autophosphorylate on serine in a Mn2+-dependent manner, and
was inhibited by wortmannin.8 However, this same group was
unable to show autophosphorylation of p110 alone.8 In
all of our studies p110 was always bound to p85 and our data show
p110 does autophosphorylate, in vitro. This indicates that the
physical binding of p85 to p110 may permit p110
autophosphorylation on serine and threonine.
| |
FOOTNOTES |
Submitted October 3, 1996;
accepted September 24, 1997.
Supported by Public Health Service grant HL-51413 (J.A.A.) from the
National Institutes of Health (Bethesda, MD).
Address reprint requests to James A. Augustine, PhD, Blood Research
Institute, 8727 Watertown Plank Rd, Milwaukee, WI 53226-3548.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Shinya Tanaka for providing p85 monoclonal antibody. We also
thank Peter J. Newman and Robert T. Abraham for critical comments and
suggestions.
| |
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Abstract
Introduction
Abstract