From the Institut für Biochemie und
Molekularbiologie II, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany, § Institut für
Pharmakologie, ** Institut für Biochemie, and
Institut für Klinische Pharmakologie
und Toxikologie, Freie Universität Berlin, 14195 Berlin,
Germany,
Max-Delbrück-Zentrum für Molekulare
Medizin, Charité, Humboldt Universität zu
Berlin, 13092 Berlin, Germany, and
§§ Forschungsinstitut für Molekulare
Pharmakologie, 13125 Berlin, Germany
Received for publication, October 9, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Class I phosphoinositide 3-kinases (PI3Ks)
are bifunctional enzymes possessing lipid kinase activity and the
capacity to phosphorylate their catalytic and/or regulatory subunits.
In this study, in vitro autophosphorylation of the G
protein-sensitive p85-coupled class IA PI3K Class I phosphoinositide 3-kinases
(PI3Ks)1 are lipid kinases
that are activated in response to a variety of extracellular stimuli
including hormones, neurotransmitters, and growth factors, which act
via G protein-coupled receptors or receptor tyrosine kinases. These
lipid kinases phosphorylate the D3 position of the inositol ring of
phosphoinositides, thus generating intracellular lipid second
messengers (1, 2). PtdIns, PtdIns-4-P, and PtdIns-4,5-P2
are in vitro substrates of class I PI3Ks, although these
enzymes predominantly produce PtdIns-3,4,5-P3 in
vivo. Class I PI3K lipid products transmit signals by recruiting
intracellular effector molecules to the membrane, which contain
particular pleckstrin homology domain modules. Effectors include
serine/threonine kinases like Akt/protein kinase B, Tec family tyrosine
kinases, and guanine nucleotide exchange factors for monomeric
GTP-binding proteins like Grp1 and Vav (3). Consequently, class I PI3Ks
are involved in the regulation of a wide variety of cellular functions
such as differentiation, proliferation, survival, migration, and
metabolism (4, 5).
All class I PI3Ks are heterodimers consisting of a p110 catalytic and a
p85 or p101 type regulatory subunit. According to the type of their
associated regulatory subunit, class I PI3Ks can be further
distinguished. The class IA catalytic p110 In addition to their lipid kinase activity, all class I PI3Ks
possess an intrinsic protein kinase activity in vitro (13). This enzymatic quality was first characterized as autophosphorylation of catalytic and regulatory subunits. PI3K Autophosphorylation of both catalytic and regulatory subunits of
PI3K Recombinant PI3Ks--
Construction and characterization of
recombinant baculoviruses for expression of GST-p110
Oligonucleotides for generation of p110
Oligonucleotides used to create the p110
Recombinant viruses expressing hexahistidine-tagged p110 G Cell Culture, Transfection, and Preparation of Cell
Lysates--
HEK293 cells (American Type Culture Collection, Manassas,
VA) were grown in minimal essential medium with Earle's salts
supplemented with 10% fetal calf serum and antibiotics. Subconfluent
cells were transfected in 3-cm dishes with pcDNA3-fMLP receptor
(0.2 µg), pcDNA3-p101 (0.4 µg), and pcDNA3-p110 Gel Electrophoresis, Immunoblotting, and
Antibodies--
Characterization of the monoclonal antibody against
p110 Lipid Kinase Assay--
In vitro lipid kinase
activity was determined basically as described previously (11). In
brief, assays were conducted in a final volume of 50 µl, containing
0.1% bovine serum albumin, 1 mM EGTA, 0.2 mM
EDTA, 7 mM MgCl2, 100 mM NaCl, 40 mM HEPES, pH 7.4, 1 mM dithiothreitol, 1 mM Protein Kinase Assay--
In vitro protein kinase
activity was determined as described for the lipid kinase activity with
some modifications. The assay volume was 25 µl (2 µCi of
[ Phosphoamino Acid Analysis--
Autophosphorylated PI3K Determination of p110 In Vitro Autophosphorylation of Class IA
PI3Ks--
Class IA PI3K p110
Both phosphorylation of the p85 adaptor by p110
Next we created a peptide corresponding to the C terminus of p110 Regulation of p110
These observations suggest a high level of basal p110 Autophosphorylation of p110 Autophosphorylation of p110 Identification of the p110 Lipid Kinase Activity of Mutant PI3K Mechanism of p110 The present study describes the autophosphorylation sites of the G
protein-sensitive class I PI3K Recently, Williams and co-workers published the crystal structure of
p110 In contrast to the predominant autophosphorylation of p110 The data presented in this study as well as in other reports provide
evidence that the C terminus of p110 is a common site of
autophosphorylation for three out of four class I PI3K isoforms. Despite this conformity, PI3K We also addressed the control of autophosphorylation by upstream
regulators. Under basal conditions, PI3K Data obtained from aspartate and glutamate mutants of p110 We found that PI3K and
p101-coupled class IB PI3K
was examined.
Autophosphorylation sites of both PI3K isoforms were mapped to
C-terminal serine residues of the catalytic p110 subunit
(i.e. serine 1070 of p110
and serine 1101 of p110
).
Like other class IA PI3K isoforms, autophosphorylation of
p110
resulted in down-regulated PI3K
lipid kinase activity. However, no inhibitory effect of p110
autophosphorylation on PI3K
lipid kinase activity was observed. Moreover, PI3K
and PI3K
differed in the regulation of their autophosphorylation. Whereas
p110
autophosphorylation was stimulated neither by G
complexes
nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110
was
significantly enhanced by G
in a time- and
concentration-dependent manner. In summary, we show that
autophosphorylation of both PI3K
and PI3K
occurs in a C-terminal
region of the catalytic p110 subunit but differs in its regulation and
possible functional consequences, suggesting distinct roles of
autophosphorylation of PI3K
and PI3K
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
, and
-
subunits complex with adaptor molecules containing two Src
homology 2 domains, of which p85 is the prototype. Through interaction
of the adaptor Src homology 2 domains with phosphotyrosines, class
IA PI3Ks are activated by receptor tyrosine kinases. In contrast, the only class IB member, p110
, associates
with a p101 regulatory subunit and is not recruited to
tyrosine-phosphorylated receptor tyrosine kinases. PI3K
is regulated
by G protein-coupled receptors through direct interaction with G
complexes of heterotrimeric G proteins (6-9). Furthermore, class
IA PI3K
is also sensitive to G
and thus may
function as a coincidence detector integrating tyrosine kinase- and G
protein-dependent signals (10-12).
phosphorylates its p85
adaptor subunit at serine 608, whereas autophosphorylation of PI3K
occurs at serine 1039 of the catalytic p110
subunit. Both
phosphorylations result in down-regulation of the lipid kinase activity
(14-16). Moreover, in Jurkat T cells, p110
phosphorylation was
stimulated by CD28 in vivo (15). There is some in
vitro evidence that class IA PI3Ks can phosphorylate
other substrates such as the insulin receptor substrate-1 adaptor
protein and PDE3B, but the physiological significance of these
phosphorylations remains unknown (17-21). Recently it was reported
that a "protein kinase-only" mutant of the G-protein-regulated
PI3K
still activated mitogen-activated protein kinase pathways in
cells, whereas no activation of Akt/protein kinase B by this mutant
occurred (22).
and PI3K
has been proposed, but many questions regarding the
autophosphorylation sites and the functional relevance of these
autophosphorylation events remain unanswered. Therefore, in the present
study, we examined the in vitro autophosphorylation of the G
protein-sensitive class I PI3K
and PI3K
. Recombinant heterodimeric PI3Ks were purified and analyzed for their
autophosphorylation. Phosphorylated amino acids were identified, and
the effect of autophosphorylation on PI3K lipid kinase activity was
studied. Interestingly, we observed significant differences in the
regulation and functional consequences of the autophosphorylation of
PI3K
and PI3K
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p110
,
GST-p110
, GST-p110
K833R, GST-p101, p101, and p85
were
described previously (6-8). A pFastBacHTa baculovirus transfer vector
(Invitrogen) was used to generate His-p110
and His-p110
full-length constructs using NcoI/SalI and
NcoI/BamHI cloning sites, respectively. Point
mutations were introduced using the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA).
S1070 mutants (mutated
residues are underlined) were as follows: 5'-GGA AAG ACT ACA GAG CTT AAG CTG CAG TCG-3' and 5'-CGA CTG CAG CTT
AAGCTC TGT AGT CTT TCC-3' (for S1070A), 5'-GGA AAG ACT ACA
GAG ATT AAG CTG CAG TCG-3' and 5'-CGA CTG CAG
CTT AAT CTC TGT AGT CTT TCC-3' (for S1070D),
and 5'-GGA AAG ACT ACA GAG AGT AAG
CTG CAG TCG-3' and 5'-CGA CTG CAG CTT ACT CTC
TGT AGT CTT TCC-3' (for S1070E).
S1101 mutants were as
follows: 5'-GGC ATC AAA CAA GGA GAG AAA CAT GCA GCC TAA TAC TTT AGG CTA GAA TC-3' and 5'-GAT TCT AGC CTA AAG TAT TAG GCT
GCA TGT TTC TCT CCT TGT TTG ATG CC-3' (for S1101A), 5'-GGC
ATC AAA CAA GGA GAG AAA CAT GAC GCC TAA TAC TTT AGG CTA GAA
TC-3' and 5'-GAT TCT AGC CTA AAG TAT TAG GCG
TCA TGT TTC TCT CCT TGT TTG ATG CC-3' (for S1101D), and
5'-GGC ATC AAA CAA GGA GAG AAA CAT GAA GCC TAA TAC TTT AGG
CTA GAA TC-3' and 5'-GAT TCT AGC CTA AAG TAT TAG GCT TCA
TGT TTC TCT CCT TGT TTG ATG CC-3' (for S1101E).
, p110
,
and mutants thereof were generated using the Bac-to-Bac Expression
System (Invitrogen) following the manufacturer's instructions. Expression and purification of PI3K isoforms were carried out according
to published protocols (11) with the exception that the partially
purified proteins were subjected to an additional chromatographic step
on a 1-ml Resource Q fast protein liquid chromatography column
(Amersham Biosciences). For that purpose, proteins were diluted in
buffer A (20 mM Tris/HCl, pH 8.0, 10 mM
-mercaptoethanol) and loaded onto the column. The column was subsequently washed with buffer A, and proteins were eluted with a
linear gradient of 0-500 mM NaCl in buffer A.
Complexes and Peptides--
Expression and purification
of recombinant G
1
2-His complexes was
carried out as published (11). Purified proteins were quantified by
Coomassie Blue staining following SDS-PAGE with bovine serum albumin as
the standard (23). The tyrosine-phosphorylated peptide used in this
study, CGGpYMDMSKDESVDpYVPMLDM (where pY represents phosphotyrosine),
was derived from the human platelet-derived growth factor receptor (24)
and kindly donated by Dr. Andreas Steinmeyer (Schering AG, Berlin,
Germany). A nonphosphorylated peptide served as a control and had no
effect on PI3K enzymatic activity. The peptides derived from the C
termini of p110
and p110
(WMAHTVRKDYRS and WFLHLVLGIKQGEKHSA,
respectively) were kindly provided by Dr. Michael Beyermann
(Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany).
(0.4 µg) variants, using the FuGene 6 transfection reagent (Roche
Molecular Biochemicals) following the manufacturer's instructions. For
preparation of whole cell lysates, cells were directly lysed
in sample buffer according to Laemmli (39).
has been described elsewhere (7). The polyclonal
anti-extracellular signal-regulated kinase and anti-phospho-Akt
antibodies were purchased from New England Biolabs. Whole cell lysates
were fractionated by SDS-PAGE and blotted onto nitrocellulose membranes
(Amersham Biosciences). Visualization of specific antisera was
performed using the ECL chemiluminescence system (Amersham Biosciences) according to the manufacturer's instructions.
-glycerophosphate (vesicle buffer) with some
modifications. Lipid vesicles (30 µl containing 320 µM
phosphatidylethanolamine, 300 µM phosphatidylserine, 140 µM phosphatidylcholine, 30 µM
sphingomyelin, and 40 µM PtdIns-4,5-P2 in
vesicle buffer) were mixed with stimuli as indicated and incubated on
ice for 10 min. It should be noted that we ensured that the effects of
G
on PI3K activity were not affected by their
detergent-containing vehicles. Thereafter, the enzyme (20-100 ng of
PI3K
or 2-10 ng of PI3K
) was added, and the mixture was
incubated for further 10 min at 4 °C in a final volume of 40 µl.
The assay was started by adding 40 µM ATP (1 µCi of
[
-32P]ATP; PerkinElmer Life Sciences) in 10 µl of
vesicle buffer. After an incubation period of 15 min (unless otherwise
stated) at 36 °C, the reaction was stopped by adding 150 µl of
ice-cold 1 N HCl, and lipids were extracted with 450 µl
of chloroform/methanol (1:1). Following centrifugation, the organic
phase was washed twice with 150 µl of 1 N HCl.
Subsequently, 40 µl of the organic phase were resolved on potassium
oxalate-pretreated TLC plates (Whatman, Maidstone, UK) using a mixture
of 35 ml of 2 N acetic acid and 65 ml of
n-propyl alcohol as the mobile phase. Dried TLC plates were
exposed to Fuji imaging plates, and autoradiographic signals were
quantitated with a BAS 1500 Fuji-Imager (Raytest, Straubenhardt, Germany).
-32P]ATP/tube), the vesicle buffer contained 0-10
mM MgCl2 and/or 0-10 mM
MnCl2 as indicated, and lipid vesicles lacked
PtdIns-4,5-P2. The reaction was stopped after an incubation
period of 30 min (unless otherwise stated) at 36 °C by adding 10 µl of 4× sample buffer according to Laemmli (39). Following
separation on SDS-polyacrylamide gels, proteins were transferred to
nitrocellulose membranes. Dried membranes were exposed to Fuji imaging
plates, and autoradiographic signals were quantitated. For the
determination of PI3K autophosphorylation sites, 20-50 µg of PI3K
or PI3K
were phosphorylated in a final volume of 1,500 µl. Samples
were subjected to SDS-PAGE, and gels were stained with Coomassie Blue.
Calculation of the stoichiometry of p110 autophosphorylation was based
on the specific activity of [
-32P]ATP incorporated
into p110, and counts were determined by Cerenkov counting. The amount
of p110 was estimated from the Coomassie-stained gel by comparison with
stained bovine serum albumin standards.
was
subjected to SDS-PAGE and blotted onto a polyvinylidene difluoride
membrane (Millipore Corp.), and the phosphorylated p110
band was
excised. The protein was hydrolyzed in 6 N HCl for 1 h
at 110 °C. The sample was vacuum-dried, and amino acids were
resuspended in 5 µl of pH 1.9 buffer (0.078% (v/v) acetic acid,
0.025% (v/v) formic acid) containing 2 µg each of phosphoserine,
phosphothreonine, and phosphotyrosine as internal standards. The sample
was applied to a cellulose thin layer plate, and electrophoresis in the
first dimension was carried out in pH 1.9 buffer at 550 V for 1 h.
After drying the plate, an electrophoretic separation in the second
dimension was carried out in pH 3.5 buffer (0.05% (v/v) acetic acid,
0.005% (v/v) pyridine, 0.5 mM EDTA) at 500 V for 50 min.
The unlabeled phosphoamino acids were visualized by spraying the plate
with 0.2% (w/v) ninhydrin in acetone, and the radiolabeled
phosphoamino acids were detected by autoradiography.
and p110
Autophosphorylation
Sites--
Gel-excised autophosphorylated p110 spots (100-150 pmol)
were washed with 50% (v/v) acetonitrile in 25 mM ammonium
bicarbonate, shrunk by dehydration in acetonitrile, and dried in a
vacuum centrifuge. Disulfide bonds were reduced by incubation with 10 mM dithiothreitol in 100 mM ammonium
bicarbonate for 45 min at 55 °C. Alkylation was performed by
replacing the dithiothreitol solution with 55 mM
iodoacetamide in 100 mM ammonium bicarbonate. Following a
20-min incubation period at 25 °C in the dark, the gel pieces were
washed with 50% (v/v) acetonitrile in 25 mM ammonium
bicarbonate, shrunk by dehydration in acetonitrile, and dried in a
vacuum centrifuge. The gel pieces were incubated overnight at 37 °C
in 5 mM ammonium bicarbonate, containing 1 µg of
chymotrypsin (sequencing grade; Roche Molecular Biochemicals), for
p110
or at room temperature in 50% (v/v) trifluoroacetic acid,
containing 10 mg/ml cyanogen bromide, for p110
. To extract the
peptides, 0.5% (v/v) trifluoroacetic acid in acetonitrile was added,
and the separated liquid was dried under vacuum, redissolved in 5 µl
of buffer B (0.1% (v/v) formic acid), and loaded onto a Vydac C18
column (150 × 1 mm, 5 µm, type 218 TP 5115) for micro-liquid
chromatography separation. Elution was performed using a linear
gradient of 5-80% buffer C in 60 min at an eluent flow rate of 30 µl/min. Buffer C was 0.1% (v/v) formic acid in acetonitrile/water
(8:2, v/v), containing 0.1% (v/v) formic acid. Fractions were
collected, their radioactivity was determined by Cerenkov counting, and
phosphopeptides were identified by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS
measurements were performed on a Voyager-DE STR BioSpectrometry work
station MALDI-TOF mass spectrometer (Perseptive Biosystems, Inc.,
Framingham, MA) using
-cyano-4-hydroxycinnamic acid as the matrix.
The program FindMod (available on the World Wide Web at
expasy.ch/tools/findmod) was used to interpret the MS spectra of
protein digests. Amino acid sequences of the phosphopeptides were
determined by nanoelectrospray tandem mass spectrometry
(nanoESI-MS/MS). The liquid chromatography fractions were lyophilized
and redissolved in 5 µl of 1% (v/v) formic acid in methanol/water
(1:1, v/v). The MS/MS measurements were performed with a
nanoelectrospray hybrid quadrupole mass spectrometer Q-TOF (Micromass,
Manchester, UK). The collision gas was argon at a pressure of 6.0 × 10
5 millibar in the collision cell.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PI3K
isoforms
phosphorylate either their p85 adaptor subunit and/or the catalytic
p110 subunit itself (14, 15). Autophosphorylation of p110
occurs on
serine 1039 within the C terminus. Alignment of the C termini of class
IA catalytic subunits shows that p110
, which does not
autophosphorylate, contains no C-terminal serine, whereas p110
does
have one (serine 1070) (Fig.
1A). Recent reports have
described autophosphorylation of both subunits of PI3K
(i.e. p85 and p110
) (25-27). In order to analyze
autophosphorylation of PI3K isoforms in vitro, we expressed recombinant heterodimeric PI3K
and PI3K
in insect cells and measured their protein kinase activities (Fig. 1B). As
anticipated, the p85 adaptor of PI3K
was phosphorylated in the
presence of Mn2+ only (see Fig. 1B, left
panel). In contrast to p110
, p110
autophosphorylated its
catalytic subunit. This autophosphorylation of p110
was also largely
Mn2+-dependent, since in vitro
phosphorylation levels in the presence of Mg2+ reached a
maximum of only 5-10% compared with the level observed in the
presence of Mn2+ (see Fig. 1B, right
panel). Furthermore, in the presence of Mn2+, a small
but significant phosphorylation of the p85 subunit of PI3K
was
evident. These data indicate that both subunits are phosphorylated,
with p110
being the main substrate of PI3K
autophosphorylation.
View larger version (29K):
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Fig. 1.
Autophosphorylation of class IA
PI3Ks. A, alignment of the C-terminal amino acid
sequences of class IA PI3K catalytic subunits. The
arrowhead indicates the p110 autophosphorylation site.
The equivalent serine residue in p110
is marked in
boldface type. B, heterodimeric
recombinant PI3K
(GST-p110
/p85) and PI3K
(His-p110
/p85)
were purified from Sf9 cells, and the proteins were separated by
SDS-PAGE and analyzed by Coomassie staining. PI3K
and PI3K
were
assayed for the incorporation of 32P into the catalytic and
regulatory subunits in the presence of either Mn2+ (2 mM) or Mg2+ (7 mM) as indicated.
Shown are representative autoradiographs and the corresponding
Coomassie-stained gels as loading controls.
Autophosphorylates a C-terminal Serine
Residue--
Speculating that p110
autophosphorylates its C
terminus, the in vitro phosphorylated and
[32P]phosphate-labeled protein (Fig.
2A) was cleaved with cyanogen bromide in order to generate a C-terminal peptide, which was then analyzed by mass spectrometry. The resulting peptides were separated by
reversed-phase HPLC, and the radioactivity of each fraction was
determined. The main radioactive fraction was examined by MALDI-MS, and
a peptide corresponding to the phosphorylated C terminus of p110
(m/z 1312.65) could be identified (Fig. 2B). Sequencing of this phosphopeptide by nanoESI-MS/MS revealed the 1061AHTVRKDYRpS1070 (where pS represents
phosphoserine) sequence of p110
and serine 1070 as the site of
autophosphorylation (Table I). In order
to verify this finding, a p110
mutant in which serine 1070 was
changed to alanine was created, and autophosphorylation of this mutant was compared with the wild-type enzyme in the presence of
Mn2+. Fig. 2C shows that no significant
phosphate incorporation into the mutant p110
S1070A of PI3K
took
place while this mutant was still catalytically active as a lipid
kinase (see below). Hence, results obtained by mass spectrometric and
mutagenic analysis demonstrate that serine 1070 represents in fact the
main site of autophosphorylation in p110
.
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Fig. 2.
p110
autophosphorylation at a C-terminal serine residue.
A, heterodimeric recombinant PI3K
purified from
Sf9 cells was subjected to SDS-PAGE and visualized by Coomassie
staining. Apparent molecular masses (kDa) of marker proteins are
indicated. B, peptide mass fingerprint analysis of p110
.
Autophosphorylated p110
was digested in gel using cyanogen bromide,
the resulting peptides were separated by reversed-phase HPLC, and the
fractions were analyzed by MALDI-MS. The peak with
m/z 1312.65 (calculated m/z
1312.62) corresponds to the phosphorylated sequence
1061AHTVRKDYRpS1070. C, p110
in
which serine 1070 was mutated to alanine shows a loss of
autophosphorylating activity. Heterodimeric PI3K
either containing
wild-type p110
(WT) or a p110
mutant (S1070A) was
subjected to a protein kinase assay in the presence of
Mn2+. Shown are one representative autoradiograph and the
corresponding Coomassie-stained protein bands as loading control.
D, lipid kinase activity of mutant p110
. Equal amounts of
purified heterodimeric PI3K
(His-p110
/p85) either containing
wild-type p110
(WT) or a p110
mutant (S1070D, S1070E,
or S1070A) were tested for their enzymatic activity in a lipid kinase
assay in the absence (
) and presence (+) of 120 nM
purified G
1
2-His, 100 nM
tyrosine-phosphorylated peptide, and both stimuli. Experiments were
carried out in the presence of Mg2+. Indicated are mean
values ± S.D. of three independent experiments. E,
Mn2+-dependent protein kinase activity of
PI3K
in the presence of increasing amounts of a synthetic peptide
derived from the C terminus of p110
. One representative
autoradiograph of three independent experiments is shown.
MS/MS fragment ions of the p110 C-terminal phosphopeptide
. Since all C-terminal
y" ions show corresponding peaks with loss of 98 mass units, whereas
only the N-terminal b10 ion shows the neutral loss of
H3PO4, the site of phosphorylation could be clearly
assigned to the C-terminal amino acid serine 1070.
and
autophosphorylation of p110
down-regulate the enzymes' lipid kinase activities (14-16, 20). p110
, like p110
, autophosphorylates a
serine residue at the extreme C terminus, which may also affect the
catalytic activity of PI3K
. In order to test this assumption, we
measured lipid kinase activities of p110
variants in which serine
1070 was mutated. Since the lipid kinase activity of PI3K
can be
synergistically stimulated by G
complexes and a
tyrosine-phosphorylated peptide derived from the platelet-derived
growth factor receptor, we measured formation of
PtdIns-3,4,5-P3 under basal conditions and after
stimulation of PI3K
variants with either stimuli in the presence of
Mg2+ (11). Wild-type p110
and the nonphosphorylating
p110
S1070A mutant exhibited the same enzymatic activity under basal
conditions and after stimulation with G
, phosphotyrosyl peptide,
or both stimuli (Fig. 2D). This finding indicates that
serine 1070 is not essential for the catalytic activity of p110
.
However, purified mutant PI3K
heterodimers are less stable than the
wild-type enzyme (data not shown). In order to mimic the effects of
p110
autophosphorylation, mutants of p110
containing the
negatively charged aspartic and glutamic acid instead of serine 1070 were employed. These p110
S1070D/E mutants no longer
autophosphorylated (data not shown). Moreover, as shown in Fig.
2D, the lipid kinase activity of either p110
S1070D/E mutant was reduced by 4-7-fold under basal conditions and following stimulation with G
and phosphotyrosyl peptides. This implies a
regulatory function of the p110
autophosphorylation.
(WMAHTVRKDYRS) as a pseudosubstrate in order to test whether the
protein kinase activity of the autophosphorylated p110
was still
intact. Applying mass spectrometry, no phosphorylation of this peptide
by wild-type PI3K
was observed (data not shown). Moreover, as
indicated in Fig. 2E, increased amounts of the C-terminal peptide did not influence the autophosphorylation of p110
by competition. Therefore, we assume that protein phosphorylation by
PI3K
requires highly specific protein-protein interactions, and due
to the lack of appropriate substrates the effect of p110
autophosphorylation on the protein kinase activity of the enzyme remains unknown so far.
Autophosphorylation--
The finding that
autophosphorylation of p110
on serine 1070 results in
down-regulation of the lipid kinase activity of PI3K
(see Fig.
2D) suggests a regulatory function of this
autophosphorylation. Therefore, one may suppose that the protein kinase
activity like the lipid kinase activity of PI3K
is controlled by
cell surface receptors. In order to address this hypothesis, we
compared both kinase activities after incubation of PI3K
with
increasing concentrations of G
complexes and the
tyrosine-phosphorylated peptide. As indicated, Fig.
3, A and B, shows
that the lipid kinase activity of PI3K
was stimulated in a
concentration-dependent manner by G
(EC50 = 20 nM) or phosphotyrosyl peptide (EC50 = 5 nM). In contrast, neither G
nor phosphotyrosyl
peptide stimulated p110
autophosphorylation (see Fig. 3,
A and B). Moreover, a combination of both stimuli led to a remarkable synergistic activation of PI3K
lipid kinase activity (see Fig. 2B) (11). However, even under these
conditions, autophosphorylation of p110
was not enhanced regardless
of whether Mg2+, Mn2+, or mixtures thereof were
present (data not shown).
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Fig. 3.
Autophosphorylation of
p110 is not stimulated by
G
or a tyrosine-phosphorylated
peptide derived from the platelet-derived growth factor receptor.
A and B, lipid kinase (closed circles)
and protein kinase (open circles) activities of purified
PI3K
were examined in response to increasing concentrations of
purified G
1
2-His (A) and a
tyrosine-phosphorylated peptide (Tyr-P-peptide)
(B). [32P]phosphate-labeled
PtdIns-3,4,5-P3 and p110
were isolated and quantified as
described under "Experimental Procedures." Representative
autoradiographs are shown at the top, whereas mean
values ± S.D. of three independent experiments are shown at the
bottom. Lipid and protein kinase activities of PI3K
were
illustrated as -fold stimulation of basal activities. C,
time course of p110
autophosphorylation. Purified PI3K
was
incubated with phospholipid vesicles and [
-32P]ATP
either in the absence of stimuli (open circles)
or in the presence of 120 nM purified
G
1
2-His (closed
circles), 100 nM tyrosine-phosphorylated peptide
(closed squares), or both stimuli
(closed triangles). At equilibrium, the
stoichiometry of p110
autophosphorylation was ~0.4-0.6 mol of
phosphate/mol of p110
(n = 3). Note that lipid
kinase assays were carried out in the presence of Mg2+,
whereas protein kinase activity was determined in the presence of
Mn2+. Shown is the time course of one representative
experiment of three.
autophosphorylation. Nonetheless, we found that in the presence of Mn2+, the stoichiometry of phosphorylation was maximally
0.5 mol of phosphate/mol of p110
(Fig. 3C), arguing
against a high basal autophosphorylation as the reason for the missing
regulation of PI3K
protein kinase activity in vitro.
Moreover, no differences in the time course of p110
autophosphorylation occurred, regardless of whether G
or
tyrosine-phosphorylated peptide were present. It is interesting that
autophosphorylation peaked after more than 30 min under in
vitro conditions. Taken together, the presented data do not
exclude the possibility that p110
autophosphorylation may be
involved in receptor-independent regulation of PI3K
enzymatic activity.
Is Stimulated by G
--
Major
characteristics of PI3K
autophosphorylation such as Mn2+
dependence and inhibition of lipid kinase activity resemble those of
class IA PI3K
and -
autophosphorylation. However, in
contrast to class IA kinases, a significant
autophosphorylation of p110
occurs in the presence of
Mg2+. Furthermore, autophosphorylation does not change the
lipid kinase activity of PI3K
(11, 28). These findings suggest a
role for PI3K
autophosphorylation different from the class
IA PI3K isoforms. Our observation that G
stimulates
p110
autophosphorylation further supports this assumption (11, 12).
Since others have reported an inhibitory effect of G
on PI3K
protein kinase activity (29), we reexamined G
-induced p110
autophosphorylation using recombinant purified protein (Fig.
4). In the absence of lipid vesicles,
G
did not increase autophosphorylation of p110
. In contrast,
the addition of lipid vesicles led to a significant G
-dependent stimulation of p110
autophosphorylation regardless of whether the lipid vesicles contained
PtdIns-4,5-P2 (see Fig. 4A). These data may
indicate that the orientation of proteins on the lipid bilayer surface
facilitates the interaction of G
with PI3K
. Interestingly, we
observed phosphorylation of p101, which could not be stimulated by
G
even in the presence of lipid vesicles. Since in these
experiments, a GST-p101/p110
heterodimer was analyzed (see Fig.
4B), we also used a His-p110
/p101 heterodimer (Fig.
5A) in order to exclude the
possibility that a bulky GST tag may influence the phosphorylation of
PI3K
subunits. As indicated in the upper panel of Fig.
5B, phosphorylation of a p101 variant without a GST tag was
not visible. Moreover, both G
-stimulated p110
autophosphorylation of the His-p110
/p101 heterodimer
(EC50 = 30 nM) and lipid kinase activity
(EC50 = 10 nM) were comparable with the data
obtained with the GST-p101/p110
heterodimer (see Fig. 5B)
(11, 12). Taken together, these results clearly demonstrate that, in
contrast to class IA PI3K
, autophosphorylation of
PI3K
is sensitive to G
. Whereas under basal conditions
autophosphorylation of p110
increased linearly for a period of more
than 90 min, the presence of G
significantly accelerated this
phosphorylation (Fig. 5C). In particular,
autophosphorylation of p110
reached its maximum after 20 min with a
stoichiometry of about 0.95 mol of incorporated phosphate/mol of
p110
. The latter observation suggests the presence of one
autophosphorylation site in p110
.
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Fig. 4.
Phospholipid vesicles are required for
G stimulation of
PI3K
autophosphorylation. A,
heterodimeric recombinant PI3K
(GST-p101/p110
) purified from
Sf9 cells was assayed for autophosphorylation in response to
increasing concentrations of recombinant
G
1
2-His in the absence (
) and presence
(+) of phospholipid vesicles lacking PtdIns-4,5-P2. Shown
are autoradiographs of one typical experiment (upper
panel) and graph bars with mean values ± S.D. of three
independent experiments (lower panel).
B, heterodimeric recombinant PI3K
(GST-p101/p110
) was
purified from Sf9 cells, subjected to SDS-PAGE, and analyzed by
Coomassie staining. Apparent molecular masses of marker proteins are
indicated.
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Fig. 5.
G
stimulates p110
autophosphorylation. A, heterodimeric recombinant
PI3K
(His-p110
/p101) was purified from Sf9 cells,
subjected to SDS-PAGE, and analyzed by Coomassie staining. Apparent
molecular masses of marker proteins are indicated. B,
purified PI3K
(His-p110
/p101) was assayed for lipid kinase
activity (closed circles) and protein kinase
activity (open circles) in the presence of
increasing concentrations of recombinant
G
1
2-His.
[32P]phosphate-labeled PtdIns-3,4,5-P3 and
p110
were isolated and quantified as detailed under "Experimental
Procedures." Representative autoradiographs are depicted at the
top, whereas graph bars with mean values ± S.D. of
three independent experiments are shown at the bottom. Lipid
and protein kinase activities of PI3K
are illustrated as -fold
stimulation of basal activities. C, autophosphorylation of
p110
is accelerated in the presence of G
subunits. Purified
His-p110
/p101 was incubated with phospholipid vesicles and
[
-32P]ATP in the absence (open
circles) or presence (closed circles)
of 120 nM purified G
1
2-His for the
indicated periods of time. At equilibrium, the stoichiometry of
G
-stimulated p110
autophosphorylation was ~0.95 mol of
phosphate/mol of p110
. The time course of one representative
experiment out of three is shown.
Does Not Inhibit PI3K
Lipid
Kinase Activity--
In order to examine the effect of p110
autophosphorylation on PI3K
lipid kinase activity, p110
was
phosphorylated in the presence of G
. The catalytic activity of
this autophosphorylated PI3K
was compared with the nonphosphorylated
counterpart using PtdIns-4,5-P2 as the substrate (Fig.
6). No differences in the production of
PtdIns-3,4,5-P3 were detected, regardless of whether PI3K
was autophosphorylated. Therefore, in contrast to the
C-terminal autophosphorylation of p110
and p110
, which both
down-regulate lipid kinase activity of PI3K, autophosphorylation of
p110
has no obvious inhibitory effects on PI3K
enzymatic
activity. Hence, we assumed that autophosphorylation of p110
occurs
at a site different from a serine residue at its C terminus.
View larger version (13K):
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Fig. 6.
Autophosphorylation of
p110 does not affect its lipid kinase
activity. Heterodimeric recombinant PI3K
was subjected to a
protein kinase assay either with (closed circles)
or without (open circles) ATP for 30 min at
37 °C in the presence of 120 nM purified
G
1
2-His and phospholipid vesicles lacking
PtdIns-4,5-P2. Thereafter,
PtdIns-4,5-P2-containing phospholipid vesicles were added
to the reaction mixture, the ATP concentration was adjusted, and a
lipid kinase assay was performed as described under "Experimental
Procedures." Reactions were stopped at the time points indicated, and
incorporation of [32P]phosphate was determined (mean
values ± S.D. of three independent experiments).
Autophosphorylation
Site--
Phosphoamino acid analysis revealed that p110
autophosphorylates serine but not threonine or tyrosine residues
(Fig. 7A). In order to
identify the phosphorylated serine residue, in vitro [32P]phosphate-labeled p110
protein was cleaved using
different proteases. The resulting peptides were separated by
reversed-phase HPLC, and the radioactivity of each fraction was
determined. After digestion with chymotrypsin, a phosphopeptide
corresponding to the C terminus of p110
was identified by MALDI-MS
(m/z 1134.56) and nanoESI-MS (doubly charged ion
with m/z 567.77), as shown in Fig. 7B.
The site of modification within the C-terminal sequence 1093-1102 was
determined by nanoESI-MS/MS (see Fig. 7B, lower panel). In particular, the C-terminal y" fragment ion series and the loss of neutral H3PO4 (98 mass units)
confirmed the sequence and identified phosphorylation of serine 1101. Thus, the MS data demonstrate that serine 1101 of p110
is the site
of autophosphorylation. To confirm this finding, a p110
mutant in
which serine 1101 was changed to alanine was examined for its
autophosphorylation. As indicated in Fig. 7C, no significant
G
-stimulated phosphate incorporation into the p110
S1101A
mutant took place. Hence, both class IB p110
as well as
class IA p110
and p110
isoforms autophosphorylate on
a serine residue at the extreme C terminus.
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Fig. 7.
Mapping of the
p110 autophosphorylation site.
A, phosphoamino acid analysis. Purified His-p110
/p101 was
phosphorylated in the presence of recombinant
G
1
2-His (400 nM). After
protein separation by SDS-PAGE and transfer to a polyvinylidene
difluoride membrane, the 32P-labeled p110
subunit was
subjected to phosphoamino acid analysis as described under
"Experimental Procedures." The positions of ninhydrin-stained
phosphoamino acid standards are indicated by dashed
circles. B, MS (upper
panel) and MS/MS (lower panel) spectra
of the chymotryptic fragment
1093GIKQGEKHpSA1102 of p110
isolated by
reversed-phase HPLC. The peak with a m/z ratio of
567.74 (upper panel, calculated
m/z 567.77) corresponds to the double-charged ion
of the phosphorylated sequence. The loss of 98 Da corresponding to
neutral H3PO4 from this precursor ion gave rise
to the peak with an m/z ratio of 518.77 (lower panel). Relevant ions are labeled
according to the nomenclature proposed in Ref. 30. The y" ions in the
spectrum that contain the phosphoserine are produced by consecutive
fragmentation reactions breaking the amino bond and losing the
H3PO4, or vice versa. C,
p110
with a serine 1101 to alanine mutation does not show any
G
-stimulated autophosphorylation. Heterodimeric purified PI3K
(His-p110
/p101) either containing wild-type p110
(WT)
or a p110
mutant (S1101A) was subjected to a protein kinase assay in
the absence (
) and presence (+) of 120 nM purified
G
1
2-His. One typical autoradiograph and
the corresponding Coomassie-stained gel as loading control are
shown.
--
Next we examined the
in vitro lipid kinase activity of heterodimeric PI3K
variants containing either wild-type p110
or a mutant p110
in
which serine 1101 was replaced by either alanine (see above) or the
negatively charged aspartic and glutamic acid (Fig.
8A). No differences in the
production of PtdIns-3,4,5-P3 by these PI3K
variants
were observed under both basal conditions and followed by stimulation
with G
. These data underline that autophosphorylation of p110
does not inhibit PI3K
lipid kinase activity. Moreover, HEK293 cells
were transiently transfected with wild-type or mutant PI3K
as well
as with the G protein-coupled fMLP receptor, and Akt phosphorylation
was subsequently determined. In the absence of PI3K
, no fMLP-induced
phosphorylation of Akt was observed (data not shown), whereas in the
presence of any PI3K
variants (i.e. the wild-type
enzyme or the alanine, aspartate, or glutamate mutants), Akt
phosphorylation was significantly stimulated by fMLP to a comparable
extent (Fig. 8B). Hence, results obtained both in an
in vitro assay using recombinant proteins and in a cell-based assay suggest that autophosphorylation of p110
does not
influence PI3K
lipid kinase activity and thus clearly differs from
the autophosphorylation of class IA PI3K isoforms.
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Fig. 8.
Lipid kinase activity of mutant
p110 . A, in vitro lipid kinase
activity of mutant p110
. Equal amounts of heterodimeric purified
PI3K
(His-p110
/p101) containing either wild-type p110
(WT) or a p110
mutant (S1101D, S1101E, or S1101A) were
tested for their enzymatic activity in a lipid kinase assay in the
absence (
) and presence (+) of 120 nM
G
1
2-His. Basal
PtdIns-3,4,5-P3 formation of all PI3K
variants was
similar (0.25 mol/min/mol enzyme). Shown are mean values ± S.D.
of three independent experiments. B, stimulation of Akt
phosphorylation by mutant p110
. HEK293 cells were transiently
transfected with plasmids for the fMLP receptor, p101, and wild-type
(WT) or mutant (S1101D, S1101E, or S1101A) p110
.
Serum-starved cells were treated with either vehicle (
) or 1 µM fMLP. Equal amounts of whole cell lysates were
subjected to SDS-PAGE followed by immunoblotting with anti-phospho-Akt,
anti-p110
, and anti-Erk antibodies as loading control.
Autophosphorylation--
In order to examine
the mechanism of p110
autophosphorylation, we used a peptide
corresponding to the C terminus of p110
(WFLHLVLGIKQGEKHSA).
However, this C-terminal peptide was not a substrate for PI3K
protein kinase activity, since a phosphorylation of this peptide by
wild-type PI3K
was not detected using mass spectrometry (data not
shown). Furthermore, the peptide neither influenced p110
autophosphorylation under basal conditions nor in the presence of
G
(Fig. 9A). Moreover, a
kinase-defective p110
K833R mutant did not autophosphorylate,
emphasizing that phosphorylation of purified PI3K
preparations was
not due to the presence of a contaminant kinase activity copurifying
with the lipid kinase (Fig. 9B). Last, co-incubation of
p110
K833R with enzymatically active wild-type heterodimeric PI3K
did not result in phosphorylation of the mutant p110
K833R, whereas
the wild-type enzyme autophosphorylated. Hence, a transphosphorylation mechanism can be excluded.
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Fig. 9.
Mechanism of p110
autophosphorylation. A, protein kinase activity
of purified PI3K
in the presence of increasing amounts of a
synthetic peptide derived from the C terminus of p110
.
Autophosphorylation of p110
was monitored in the absence or presence
of 120 nM purified G
1
2-His.
Shown is one representative autoradiograph out of three independent
experiments. B, the autophosphorylation of p110
is not
mediated by transphosphorylation. Recombinant purified His-p110
/p101
and GST-p110
K833R were obtained from Sf9 cells, and proteins
were subjected to SDS-PAGE and analyzed by Coomassie staining
(left panel). The His-p110
/p101 complex was
incubated alone or in the presence of kinase-inactive GST-p110
K833R
with [
-32P]ATP and 120 nM
G
1
2-His. Phosphorylated proteins were
separated by SDS-PAGE and visualized by autoradiography.
Autophosphorylated GST-p110
served as a control to indicate the size
of a phosphorylated GST-p110
K833R. One representative autoradiograph
is shown (right panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -
isoforms. The experimental approaches include mass spectrometric analysis of the
posttranslationally modified proteins and site-directed mutagenesis,
which are independent and complementary methods. With these strategies,
we identified the C-terminal residues serine 1070 and serine 1101 of
the catalytic p110
and p110
subunits, respectively, as the
modified amino acids. Previously, the C-terminal serine 1039 was
detected as the site of p110
autophosphorylation (15). Hence, class
I PI3K
, -
, and -
isoforms share the extreme C terminus of the
catalytic subunit as a common site of autophosphorylation, whereas
p110
is not significantly modified, probably due to the lack of a
serine residue in this region.
for a fragment comprising amino acid residues 144-1102 (31).
Although serine 1101 was not resolved in this structure it is clear
from the data that it should be just beyond the C-terminal helix
k
12, which lines the PtdIns-4,5-P2
binding pocket. Therefore, from a sterical point of view, a
preferential phosphorylation of this serine appears reasonable.
Moreover, lipid and protein kinase activities may compete with each
other (32, 33). This assumption is supported by recent data from Yart
et al. (27) demonstrating that a "protein kinase-only"
(PKO) mutant of p110
exhibited an even higher protein kinase
activity than the wild-type enzyme. Our own studies did not indicate
any difference in the autophosphorylation activity of p110
,
regardless of whether lipid substrates such as
PtdIns-4,5-P2 were present (data not shown). Other data are
more complex. Bondeva et al. (22) reported that only those
PKO p110
variants showed an increased autophosphorylation activity
that contained a class II or class III donor activation loop but not
the class IV counterpart. In contrast, wild-type p110
and all PKO
p110
variants phosphorylated p85 equally well, whereas PKO p110
variants containing a class II or class III donor activation loop
exhibited autophosphorylation of the p110
subunit (20). The latter
finding was unexpected, since p110
lacks a C-terminal serine. In
this context, our observation of a residual phosphorylation of the
p110
S1070A mutant may be of interest (see Fig. 2C). The
fact that purified kinase-defective mutants of p110
were not
detectably phosphorylated by a contaminating kinase
activity rather indicates the presence of a second, quantitatively less
important phosphorylation site in p110
, presumably at threonine 1063 (see Fig. 1A). Support for this assumption comes from
MALDI-post-source decay data, which revealed a double
phosphorylated C-terminal peptide; unfortunately, the signal was too
weak for sequencing by nanoESI/MS-MS. A corresponding C-terminal
threonine of p110
(see Fig. 1A) may be a candidate target
for significant autophosphorylation by class II and III PKO p110
variants (20).
observed
in this and previous studies (27), others have reported p85
phosphorylation as the major target of PI3K
autophosphorylation (25,
26). In order to explain the apparent discrepancies, one must consider
the experimental conditions used in these studies. Roche et
al. (25) added purified p85 to immunoprecipitated p110
and
detected a phosphorylated p85 band, whereas the p110
band was not
shown. More important, PI3K
autophosphorylation was examined with
enzyme preparations immobilized to beads in those studies (25, 26).
Interestingly, this may affect autophosphorylation, since we noticed an
increased p85 phosphorylation when we examined PI3K
bound to
Ni2+-nitrilotriacetic acid
beads.2 Hence, experimental
conditions significantly influence in vitro autophosphorylation of PI3K
. Likewise, in addition to p110
autophosphorylation (11, 12, 28), p101 phosphorylation by PI3K
has
been reported (29). In fact, using a GST-p101 construct, we also
observed in initial experiments a phosphorylation of p101, which, in
contrast to p110
autophosphorylation, was not stimulated by G
.
However, p101 phosphorylation was not detectable when a purified
hexahistidine-tagged PI3K
heterodimer was used. Therefore, we cannot
exclude the possibility that the artificial bulky GST tag may have
facilitated phosphorylation of p101.
and -
differ in all other
biochemical characteristics of autophosphorylation. For instance,
PI3K
, like the two other class IA isoforms, PI3K
and
-
, autophosphorylates preferentially in the presence of
Mn2+ (see Fig. 1B and Refs. 14-16). In
contrast, PI3K
exhibits a significantly stimulated
autophosphorylation activity in the presence of Mg2+ as
shown previously (11, 28) and in this study. Interestingly, in
vitro most serine/threonine kinases are
Mg2+-dependent, whereas many tyrosine kinases
show a greater activity in the presence of Mn2+ (34).
Conversely, for phosphorylase kinase, a metal ion-dependent dual kinase specificity was reported (35). The presence of
Mg2+ causes serine phosphorylation of phosphorylase
b, and Mn2+ activates tyrosine phosphorylation
of angiotensin II (35). The basis for these properties are still
unclear. In this context, it should be remembered that
autophosphorylation, per se, is not a good indicator of
protein kinase activity, since many ATP-binding proteins that are not
protein kinases are known to autophosphorylate in vitro
(32).
slowly autophosphorylated (i.e. 0.1 mol of phosphate was incorporated into 1 mol of
p110
within 30 min). The addition of G
accelerated phosphate
incorporation by 8-10-fold, resulting in an almost stoichiometric
phosphorylation (see Fig. 5, B and C).
Interestingly, this effect was only seen in the presence of lipid
vesicles, which is in contrast to results obtained by Bondev and
co-workers (29). Since the EC50 values for the stimulation
of lipid and protein kinase activities of PI3K
were concordant, we
hypothesized that the molecular mechanisms of stimulation of these
kinase activities are similar. In contrast, neither G
nor
phosphotyrosyl peptide stimulated autophosphorylation of PI3K
(see
Fig. 3) even under low basal autophosphorylation conditions
(i.e. in the presence of Mg2+). Unfortunately,
in vitro data addressing a possible stimulation of
autophosphorylation of PI3K
and -
by upstream regulators are
missing. However, Vanhaesebroeck and co-workers (15) reported a
CD28-mediated stimulation of C-terminal p110
phosphorylation under
in vivo conditions.
suggest
that a phosphorylated PI3K
displays a hampered lipid kinase activity
(see Fig. 2D). Unfortunately, a more direct experimental approach (e.g. assaying the effect of autophosphorylation on
lipid kinase activity) was inconclusive. In particular, we were unable to completely remove Mn2+, which is necessary for
autophosphorylation but disturbed PI3K
lipid kinase activity,
without the use of immobilizing agents before carrying out the lipid
kinase assay. Nevertheless, our results with the p110
1070D/E mutants
are consistent with data obtained from the other class IA
kinases. Autophosphorylation of PI3K
and -
and exchange of serine
1039 of p110
to aspartate or glutamate inhibited lipid kinase
activity (14-16, 20). Possible explanations for this effect include an
induction of structural/conformational changes of the phosphorylated
enzyme or an impact on the phospho-transfer reaction or on the
ATP/PtdIns-4,5-P2 interaction (15). Furthermore, based on
the crystal structure of p110
, Williams and associates (31) have
speculated that a phosphorylated C terminus may be a sterical
impediment for PtdIns-4,5-P2 substrate binding.
Surprisingly, here we provide experimental evidence that
autophosphorylation of the C terminus of p110
does not inhibit lipid
kinase activity as shown under in vitro conditions with a
prephosphorylated wild-type enzyme and p110
1101D/E mutants (see
Figs. 6 and 8A). These mutants showed full activity on
cellular effectors in HEK293 cells in vivo (see Fig.
8B). Hence, we assume that autophosphorylation of p110
,
which is primarily regulated by G
, has functions distinct from
regulating its lipid kinase activity. One possibility may be the
existence of PI3K
binding partners that specifically interact with
the autophosphorylated form of PI3K
. In fact, recent evidence suggests that PI3K
interacts not only with its principal regulators (i.e. G
and Ras) but also with additional components
of signaling cascades such as the
-adrenergic receptor kinase 1 (36).
and -
did not phosphorylate peptides derived
from their respective C terminus, and vice versa the
peptides did not affect the autophosphorylation capacity of the enzyme (see Figs. 2E and 9A). Similar observations were
reported for PI3K
and -
(15), whereas Beeton et al.
(26) described that PI3K
and -
phosphorylated a p85-derived
peptide containing serine 608. Mechanistically, PI3K
did not
transphosphorylate (see Fig. 9B), which may indicate a high
degree of substrate specificity of the protein kinase activity.
Notably, auto- but not transphosphorylation has also been reported for
the p110
monomer and a phosphatidylinositol 4-kinase
(37,
38). Surprisingly, while we were searching for in vivo
substrates of PI3K protein kinase activity, we failed to detect p110
autophosphorylation in HL-60 and Sf9 cells so far, which
emphasizes the need for further investigations into the regulation,
activity, and targets of PI3Ks in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank H. Lerch and J. Malkewitz for
excellent technical assistance. We thank Drs. Bart Vanhaesebroeck and
Michael Waterfield for providing baculoviruses and Dr. Reinhard
Wetzker for the monoclonal anti-p110 antibody. We also
thank Drs. Michael Beyermann and Andreas Steinmeyer for providing
peptides. Valuable discussions with Drs. Reinhard Wetzker, Len
Stephens, Phil Hawkins, Lewis Cantley, and Roland Piekorz are appreciated.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie (DFG Nu53-6/1; SFB518).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ This work is in partial fulfillment of Ph.D. requirements at Friedrich-Schiller-Universität Jena. Present address: Biotec der TU Dresden, c/o Max-Planck-Institut für Molekulare Zellbiologie und Genetik, Dresden.
¶¶ To whom correspondence should be addressed: Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, Universitätsstr. 1, Gebäude 22.03, 40 225 Düsseldorf, Germany. Tel.: 49-211-811-2724; Fax: 49-211-811-2726; E-mail: bernd.nuernberg@uni-duesseldorf.de.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M210351200
2 C. Czupalla and B. Nürnberg, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PI3K, phosphoinositide 3-kinase;
p110, catalytic subunit of PI3Ks;
p101, subunit associated with p110;
p85, regulatory subunit of class
IA PI3Ks;
PtdIns, phosphatidylinositol;
PtdIns-4-P, phosphatidylinositol 4-phosphate;
PtdIns-4, 5-P2,
phosphatidylinositol 4,5-bisphosphate;
PtdIns-3, 4,5-P3,
phosphatidylinositol 3,4,5-trisphosphate;
GST, glutathione
S-transferase;
His, hexahistidine tag;
MS, mass
spectrometry;
MALDI-MS, matrix-assisted laser desorption/ionization MS;
nanoESI-MS/MS, nanoelectrospray ionization tandem MS;
HPLC, high
pressure liquid chromatography;
fMLP, formylmethionylleucylphenylalanine;
PKO, protein kinase-only;
TOF, time
of flight.
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REFERENCES |
---|
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