(Received for publication, March 7, 1997)
From the Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland
A G-protein subunit (G
)-responsive
phosphoinositide 3-kinase (PI 3-kinase) was purified approximately
5000-fold from pig platelet cytosol. The enzyme was purified by
polyethylene glycol precipitation of the cytosol followed by column
chromatography on Q-Sepharose fast flow, gel filtration,
heparin-Sepharose, and hydroxyapatite. The major G
-responsive PI
3-kinase is distinct from p85 containing PI 3-kinase as the activities
can be distinguished chromatographically and immunologically and is
related to p110
as it cross-reacts with anti-p110
-specific
antibodies. The p110
-related PI 3-kinase cannot be activated by
G-protein
i/o subunits, and it has an apparent
native molecular mass of 210 kDa. The p110
-related PI 3-kinase
phosphorylates phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The apparent Km values
for ATP were found to be 25 µM with PtdIns, 44 µM with PtdIns4P, and 37 µM with
PtdIns(4,5)P2 as the substrate. G
subunits did not
alter the Km of the enzyme for ATP; however,
Vmax increased 2-fold with PtdIns as substrate,
3.5-fold with PtdIns4P, and 10-fold with PtdIns(4,5)P2.
Under basal conditions the apparent Km values for
lipid substrates were 64, 10, and 15 µM for PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively. In the presence
of G
subunits the dependence of PI 3-kinase activity on the
concentrations of lipid substrates became complex with the highest
level of stimulation occurring at high substrate concentration,
suggesting that the binding of G
and lipid substrate
(particularly PtdIns(4,5)P2) may be mutually cooperative.
Wortmannin and LY294002 inhibit the G
-responsive PI 3-kinase
activity with IC50 values of 10 nM and 2 µM, respectively. Unlike the p85 containing PI 3-kinase in platelets, the p110
-related PI 3-kinase is not associated with a
PtdIns(3,4,5)P3 specific 5-phosphatase.
The p85-associated PI 3-kinase was not activated by G alone but
could be synergistically activated by G
and phosphotyrosyl platelet-derived growth factor receptor peptides. This may represent a
form of coincidence detection through which the effects of tyrosine kinase and G-protein-linked receptors might be coordinated.
Phosphoinositide 3-kinases (PI 3-kinases,1 EC 2.7.1.137) phosphorylate the D-3 position of the inositol ring in inositol phospholipids and were originally identified through their association with viral oncoproteins and activated tyrosine kinases (1, 2). Receptor-regulated forms of PI 3-kinase appear to prefer to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in vivo (3-5). The product of this reaction, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), is a candidate second messenger that regulates a variety of cellular responses to growth factors perhaps through the activation of serine/threonine protein kinases such as Akt/PKB and/or certain protein kinase C isoforms and small GTP-binding proteins such as Rac 1 (6-10).
The first form of PI 3-kinase to be purified and cloned was identified as a heterodimer composed of a 110-kDa catalytic subunit with a tightly bound regulatory subunit of 85 kDa (11-14). The p85 subunit possesses a number of regions with homology to recognized signaling proteins. These include a Bcr homology domain, a Src homology (SH) 3, and two SH2 domains and two proline-rich regions. Binding of the SH2 domains to phosphotyrosine residues within the sequence context, YMXM, which occurs in a wide range of activated growth factor receptors and adaptor proteins, causes the translocation and activation of the catalytic subunit (2, 15-16). More recently, a number of distinct p85 and p110 subunit isoforms have been identified (11, 14, 17), but the functional significance of this heterogeneity is not yet clear (18).
Heterotrimeric G-protein-regulated forms of PI 3-kinase have also been
identified following the observations that activation of
G-protein-coupled receptors in neutrophils and platelets caused a rapid
accumulation of PtdIns(3,4,5)P3 (19-21). Stephens et
al. (22) partially purified a G-protein subunit
(G
)-responsive PI 3-kinase from a myeloid cell line (U937). This
enzyme is immunologically and biochemically distinct from a growth
factor-regulated, p85-containing PI 3-kinase present in the same cells.
Using degenerate oligonucleotide primers based on conserved regions of
known PI kinases, Stoyanov et al. (23) isolated a p110
homologue, designated p110
, from a U937 cell cDNA library.
Recombinant p110
has a predicted molecular mass of 120 kDa and can
be activated in vitro by both G
and the
subunits
of transducin and Gi. p110
differs from the
and
isoforms mainly by its lack of the recognized p85 binding site which is
replaced by a region which has been proposed to resemble a pleckstrin
homology (PH) domain (23).
A G-responsive PI 3-kinase has also been reported to be present
in platelet cytosol (24). Because this activity associated with a PDGF
receptor phosphotyrosyl peptide and was immunoprecipitated with a
monoclonal antibody raised against the p85 subunit of PI 3-kinase, it
was thought to possess a p85-related subunit. However, Zhang et
al. (25) reported a G
-stimulated PI 3-kinase in platelets that was not recognized by p85-directed antibodies. The latter study
established that this platelet G
-stimulated enzyme was immunologically related to p110
.
The substrate specificities of identified PI 3-kinases vary
substantially (11, 14, 22, 24, 26-28). To define the molecular characteristics and properties of G-stimulated PI 3-kinases in
platelets more clearly, we have now partially purified the major form
of this enzyme from pig platelets. This enzyme is a p110
-related PI
3-kinase that is distinct from p85-associated species and that
phosphorylates all three potential phosphoinositide substrates with a
marked preference for PtdIns(4,5)P2 which is further
enhanced by G
.
As the results appeared to contradict previous detection of a
G-responsive p85-containing PI 3-kinase in human platelets (24),
we also studied the p85-containing PI 3-kinase in pig platelets. We
found that this enzyme can be activated by G
in a manner that is
largely dependent upon the presence of a tyrosine-phosphorylated PDGF
receptor peptide.
Materials
Polyvinylidene difluoride membrane, PtdIns,
phosphatidyl-L-serine, and protein G-Sepharose were
purchased from Sigma; PtdIns(4)P and PtdIns(4,5)P2 were
prepared as described (38). [-32P]ATP (3000Ci/mmol)
and enhanced chemiluminescence were purchased from Amersham Corp.
Partisphere SAX high performance liquid chromatography columns were
from Whatman; Superose 12 HR 10/30, Mono Q, Q-Sepharose fast flow,
heparin-Sepharose CL-6B and PD-10 column were from Pharmacia Biotech
Inc. Centricon-30 was from Amicon. GTP
S was from Boehringer
Mannheim; mouse monoclonal antibodies to PI 3-kinase p85 were from
Upstate Biotechnology Inc. Genapol C-100 detergent was from CAlbiochem;
G
i/o was kindly provided by Dr. P. Casey (Duke
University, North Carolina). Phosphotyrosyl peptide based on the
sequence of PDGF receptor was provided by Dr. S. Cartlidge, Zenaca
Pharmaceutical. p110
anti-peptide antibodies were provided by Dr. R. Wetzker (Friedrich-Schiller-Universitat Jena).
Methods
Preparation of GThe major G-protein subunits
were purified from cholate extracts of bovine brain membranes as
described by Sternweis and Robishaw (39). The G
subunits were
stored in 20 mM Tris, 1 mM EDTA, 0.1% Genaple
C-100 and were more than 95% pure as determined by SDS-PAGE. The
G
preparation was flash-frozen in 10-µl aliquots and stored at
80 °C until use.
Platelet cytosol was derived from 12 liters of freshly
drawn pig blood. The detailed procedure for preparation of platelet cytosol was essentially the same as described (24). Platelets were
sonicated in 120 ml of lysis buffer (10 mM HEPES, pH 7.4, 1 mM EGTA, 0.2 mM EDTA, 3 mM
MgCl2, 10 mg/ml each of antipain and pepstatin, 1 mM each of DTT, sodium orthovanadate, phenylmethylsulfonyl fluoride, and benzamidine). The platelet lysate was centrifuged at
35,000 rpm for 1 h, and the resulting supernatant (platelet cytosol, 1049 mg of protein) was kept. G-responsive PI 3-kinase was precipitated by 5-15% PEG in buffer A (20 mM HEPES,
pH 7.4, 0.2 mM EDTA, 3 mM MgCl2, 10 mg/ml each of antipain and pepstatin, 1 mM each of DTT,
sodium orthovanadate, phenylmethylsulfonyl fluoride, and benzamidine).
The pellet was resuspended in 36 ml of buffer A. The PEG sample (36 mg)
was loaded onto a Q-Sepharose Fast Flow column (150 × 15 mm),
pre-equilibrated with buffer A, and eluted with a gradient of 0-0.5
M NaCl (150 ml). G
-responsive PI 3-kinase fractions
(12 mg) were pooled and loaded onto a gel filtration column (Sepharose
CL-4B, 100 × 2.6 cm) pre-equilibrated with buffer B (buffer A
plus 100 mM NaCl and 10% sucrose). The G
-responsive PI 3-kinase activity fractions (1.35 mg protein) were pooled and loaded
onto a heparin-Sepharose column (50 × 10 mm) pre-equilibrated with buffer B. The column was washed with 30 ml of buffer B and eluted
with a linear gradient of 100-500 mM NaCl (60 ml).
Fractions (0.36 mg) containing G
-responsive PI 3-kinase were
pooled and concentrated to 6 ml with Prepcentricon 30 (Amicon, Inc.
Beverly, MA). Half of the sample was loaded onto a hydroxyapatite
column (100 × 10) pre-equilibrated with buffer C (20 mM K2HPO4, pH 7.0, 5 mM
DTT, 0.1 mM each of phenylmethylsulfonyl fluoride and
benzamidine); protein was eluted with 20-750 mM
K2HPO4 during which the G
-responsive PI
3-kinase was separated from the tyrosine kinase-regulated PI 3-kinase.
Separation of the G
-responsive PI 3-kinase from the tyrosine
kinase-regulated PI 3-kinase can also be achieved by incubating half of
the concentrated enzyme with 2 ml of protein G-Sepharose pre-coupled
with anti-p85 antibodies overnight at 4 °C with gentle agitation.
The G
-responsive PI 3-kinase prepared by either method did not
contain any p85 protein nor any other detectable lipid kinase
activity.
Generally the enzyme activity was
measured by adopting the following assay procedure. 10 µl of platelet
cytosol or column fractions were mixed with 30 µl of lipid vesicles,
which had been premixed with G or their vehicle for 10 min on
ice. 10 µl of MgATP was added to start the reaction. The enzyme
reaction was terminated after incubating at 37 °C for 5 min by
adding 200 µl of 1 M HCl. To prepare lipid vesicles,
equimolar amounts of PS and substrate lipid (PtdIns, PtdIns4P, or
PtdIns(4,5)P2) were dried onto a film under vacuum and
probe-sonicated (3 × 15 s with 1 min on ice between
sonication, at setting 20-30 on a Jencons Ultrasonic Processor) into
kinase assay buffer (40 mM HEPES, pH 7.4, 1 mM
EGTA, 1 mM DTT, 50 mM NaCl, 4 mM
MgCl2). The standard assay contained 100 µM
PtdIns(4,5)P2 and PS, 10-100 µM ATP (10 µM with purified fraction and 100 µM with
crude extract), 1 µM G
or its vehicles and 10 µCi
of [
32P]ATP. Lipid extraction and analysis was
performed as described (24). The products of the PI 3-kinase reactions
were identified by deacylation and separation of their glycerol
derivatives by high performance liquid chromatography and compared with
deacylated 3H-labeled standards. Note that assays were done
under first order conditions with respect to ATP as substrate (to
optimize assay of radioactive product) ensuring that no more than 10%
of ATP was consumed during a reaction.
For assays requiring activated G-proteins, G
-proteins were
incubated for 1 h on ice in the presence of 100 µM
GTP
S and 5 mM MgCl2, mixed with
PtdIns-containing lipid vesicles, and incubated again for 10 min on
ice, before adding to the assay.
Samples of column eluate (for detection of p85 subunit
of PI 3-kinase) or purified G-responsive PI 3-kinase (for
p110
) were mixed with 4 × SDS sample buffer, boiled for 5 min,
and resolved by SDS-PAGE with 7.5% acrylamide in the separating gel.
Proteins were then transferred to polyvinylidene difluoride membranes
using a dry-blotting device (Bio-Rad). Western blots were performed as
described (22) using either anti-p85 antibody (1:1000 dilution) or
p110
antibodies (1:200) followed by horseradish
peroxidase-conjugated secondary antibodies (1:2000 dilution). Blots
were then developed using enhanced chemiluminescence according to the
manufacturer's instructions.
A G-responsive PI 3-kinase was purified
approximately 5000-fold, with an overall yield of 30%, from porcine
platelet cytosol using PEG precipitation, Q-Sepharose, gel filtration,
heparin-Sepharose, and hydroxyapatite. A typical purification is
summarized in Table I. PEG precipitation resulted in a
30-fold enrichment with 100% recovery of G
-responsive activity.
Elution of this sample from Q-Sepharose using a continuous salt
gradient revealed two distinct peaks of G
-responsive PI 3-kinase
(Fig. 1A). The earlier eluting, minor peak of
activity was inconsistently observed in a number of purifications from
different batches of platelets and was not studied further. Analysis of
fractions eluting from the Q-Sepharose column by Western blotting with
an anti-p85 monoclonal antibody revealed that the second peak of
G
-responsive PI 3-kinase co-eluted with p85 immunoreactivity
(Fig. 1B). p85 and G
-responsive activity continued to
co-migrate through gel filtration and heparin-Sepharose (data not
shown). However, separation of p85 and the G
-responsive PI
3-kinase was achieved through hydroxyapatite eluted with a linear
gradient of
K2HPO4/KH2PO4 as shown
in Fig. 2, A and B. Separation
could also be achieved by immunodepletion of p85 using protein
G-Sepharose that had been pre-coupled with anti-p85 antibodies (Fig.
3A). The G
-responsive PI 3-kinase is
related to p110
as it can be recognized by an anti-p110
anti-peptide antibody (Fig. 3B).
|
Characterization of G
The native
molecular mass of the G-responsive PI 3-kinase was determined
using a calibrated Superose 12 size exclusion column. As shown in Fig.
4, the activity eluted at a volume indicating a size of
approximately 210 kDa. The enzyme was not pure at that stage as
revealed by silver-stained SDS-PAGE gel of active fractions (not
shown). This indicated that the enzyme is a very minor protein in
platelet cytosol.
The substrate specificity and kinetic characteristics of the enzyme
were consistent between several different preparations. As the enzyme
after hydroxyapatite cannot survive freezing and thawing, activity
purified through heparin-Sepharose and immunodepleted to remove p85 was
used for most experiments. Such preparations were purified
approximately 2000-fold with respect to platelet cytosol and contained
no detectable PtdIns 4-kinase, PtdIns4P 5-kinase, or PLC (EC 3.1.4.3)
activities. PtdIns, PtdIns4P, and PtdIns(4,5)P2 were all
phosphorylated at the 3-position (see "Experimental Procedures"),
and the phosphorylations of PtdIns and PtdIns4P were inhibited by the
presence of PtdIns(4, 5)P2 as described previously (29)
indicating that a single enzyme activity accounts for the
phosphorylation of all three substrates. Anti-p85 and anti-p110
antibodies were recently shown to co-immunoprecipitate with a
PtdIns(3,4,5)P3-specific 5-phosphatase which was presumed to be present in a complex with this form of PI 3-kinase (30). Analysis
of the partially purified G-sensitive PI 3-kinase using 32P-labeled PtdIns(3,4,5)P3 as substrate or by
monitoring the ratio of
PtdIns(3,4,5)P3/PtdIns(3,4)P2 in PI 3-kinase
assays failed to detect any 5-phosphatase in such preparations (data
not shown).
Km values were determined under basal conditions for
ATP and all three lipid substrates. Similar Km
values for ATP of 25, 44, and 37 µM were determined when
PtdIns, PtdIns4P, or PtdIns(4,5)P2, respectively, were used
as substrates (Fig. 5A and Fig.
6A). Km values for the
lipid substrates were determined using 10 µM ATP and
maintaining a constant mole fraction of 1:1 (substrate/PS). 10 µM ATP (i.e. somewhat less than the Km value) was used to estimate the sensitivity of
the assays. However, since less than 5% ATP was consumed during
incubation, the assays were linear under the conditions determined. The
reactions approximated to Michaelis-Menten kinetics with
Km values of 64, 10, and 15 µM for
PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively (Fig.
5B and Fig. 6B). Similar values were obtained using either Lineweaver-Burk or Wolfe plots.
The effects of increasing concentrations of G subunits were
examined at an ATP concentration of 10 µM with lipid
substrate concentrations of 100 µM. Interestingly the
EC50 values differed according to the lipid substrate as
noted previously for the human platelet cytosol activity (24). When
PtdIns(4,5)P2 was the substrate, an EC50 of
approximately 300 nM was observed, but with PtdIns as the
substrate this increased to about 600 nM
(Fig. 7). The response to G
subunits was
completely blocked in the presence of a 3-fold calculated molar excess
of a preparation of GDP-liganded G
i/o and by 85% at an
equimolar concentration of these proteins (Fig. 8),
suggesting that the activation requires free G
and hence would
require activated heterotrimeric G-proteins in vivo. The
effects of 1 µM G
were next examined over a range
of substrate concentrations. The presence of G
did not
significantly affect the Km for ATP whichever lipid
substrate was employed. However, the effects of increasing lipid
substrate concentrations on activity deviated from Michaelis-Menten
kinetics in the presence of the activator. Thus, G
subunits were
most effective at high concentrations of lipid especially when
PtdIns(4,5)P2 was used as the substrate. The apparent
cooperativity with respect to increasing substrate concentration
prevented the determination of Km values and allowed
only estimation of Vmax values. Nevertheless, G
enhanced Vmax by approximately 2-, 3.5-, and 10-fold with PtdIns, PtdIns4P, and PtdIns(4,5)P2,
respectively, as substrates (Fig. 5, A and
B).
The fungal metabolite, wortmannin, is a potent inhibitor of several PI
3-kinase isoforms including the G-sensitive PI 3-kinase from U937
cells (22, 26, 27), whereas the quercetin analogue, LY294002, was
developed as a PI 3-kinase inhibitor that lacked the chemical
instability of wortmannin (37). Both of these compounds inhibited the
platelet G
-sensitive enzyme with IC50 values of 10 nM and 2 µM for wortmannin and LY294002,
respectively (data not shown). The value for wortmannin is very similar
to that reported by Stephens et al. (22) for the enzyme from
U937 cells and that for LY294002 is almost identical to its
IC50 when assayed with RBL-2H3 cells (36).
Recombinant p110 was recently shown to respond to both G
and
the GTP
S-liganded
subunits of Gi and transducin. We
repeated these observations using a p110
-glutathione
S-transferase fusion protein expressed in Sf9 cells and
immobilized on glutathione-agarose (Fig. 9B).
1 nM G
i-GTP
S, but not GTP
S alone,
induced a 50% increase in p110
activity, whereas 1 µM
G
enhanced activity 4-fold. By contrast, the platelet enzyme was
insensitive to G
i but was more sensitive to G
being activated approximately 8-fold in the experiment shown in Fig.
9A.
The isolation from platelet cytosol of a G-responsive PI 3-kinase
that is distinct from a p85-associated enzyme appeared to contradict
our previous observation that G
activated a PI 3-kinase which
associated with a biotinylated phosphotyrosyl peptide related to the
p85 binding region of the PDGF receptor. We therefore investigated the
possibility that G
could activate the p85-associated enzyme in a
manner that depended on coincident association with the PDGF receptor
peptides. This was indeed found to be the case. As shown in
Fig. 10A, G
alone did not activate
immunoprecipated p85-associated PI 3-kinase but greatly augmented the
response to the phosphopeptide when assays were carried out under
standard conditions. This effect was even more dramatic when substrate lipid was presented against a background of phosphatidylethanolamine rather than PS as described by Okada et al. (40). With
PIP2/phosphatidylethanolamine vesicles, basal activity was
found to be very low and was slightly activated by either G
or
phosphopeptides. The combination of G
and phosphopeptides,
however, resulted in a greater than 50-fold enhancement of PI 3-kinase
activity (Fig. 10B).
A G-sensitive PI 3-kinase has been purified 5000-fold from
pig platelet cytosol. A second form of G
-responsive enzyme eluted
as an early peak on Q-Sepharose, but the appearance of this peak and
its magnitude relative to the major peak of activity were variable
between platelet preparations. For these reasons the early eluting peak
was not analyzed further in this study; whether it represents a
distinct species of PI 3-kinase, a processing variant, or a proteolytic
fragment are not clear from these studies. The major G
-sensitive
enzyme co-eluted with p85 immunoreactivity through Q-Sepharose, gel
filtration, and heparin-Sepharose but could be separated from the
latter protein on hydroxyapatite. Moreover, separation of the major
G
-responsive enzyme from p85-associated PI 3-kinase can also be
achieved by p85 immunoprecipitation. One further line of evidence that
excludes the association of the major G
-sensitive PI 3-kinase
with p85 was the lack of PtdIns(3,4,5)P3 5-phosphatase
activity which was previously shown to be complexed to p85 in human
platelets (30). Nevertheless, since a polypeptide of approximately 120 kDa was observed on Western blots probed with a p110
-specific
anti-peptide antibody, and its native molecular weight was found to be
in excess of 200 kDa, G
-sensitive PI 3-kinase presumably exists
either as a dimer or in complex with one or more additional
polypeptides. This situation is similar to that reported for the
partially purified enzyme from U937 cells (22).
In agreement with the previous report (24) on the G-responsive PI
3-kinase in human platelet cytosol, the p85-associated PI 3-kinase in
pig platelet cytosol can also be activated by G
under certain
assay conditions. Moreover, this G
-responsive activity can be
further synergistically augmented by phosphotyrosine PDGF receptor
peptide. This may represent a form of coincidence detection through
which the effects on cellular functions of tyrosine kinase and
G-protein-linked receptors might be coordinated. Similar findings have
also been reported in human monocytic THB-1 cells (40).
Although the platelet enzyme that was sensitive to G alone
co-purified with a p110
-immunoreactive component, it differed from
the latter in its native molecular weight as noted above. It also
differed in terms of its regulation, being insensitive to
GTP
S-liganded G
i/o and being stimulated to a greater
degree by G
compared with recombinant p110
. Whether these
distinguishing features reflect the differences between the native and
recombinant proteins, the catalytic subunit itself or the presence of
an additional complexed polypeptide(s) cannot be discerned at
present.
The partially purified platelet enzyme phosphorylated PtdIns, PtdIns4P,
and PtdIns(4,5)P2 to give the corresponding
3-phosphorylated lipids as determined by co-chromatography of their
deacylation products with authentic standards. The phosphorylation of
PtdIns and PtdIns4P could be inhibited by an excess of
PtdIns(4,5)P2 suggesting that a single enzyme species was
responsible for the observed phosphorylation of all three substrates
(29). However, the efficiency with which these substrates were utilized
varied substantially, with polyphosphoinositides exhibiting lower
Km values than PtdIns and with
Vmax values being greatest with
PtdIns(4,5)P2 as substrate. These features were exaggerated
in the presence of G subunits that enhanced
PtdIns(4,5)P2 phosphorylation more markedly than either
PtdIns or PtdIns4P. Assuming these features are relevant to the
situation in cell membranes, then this enzyme would be expected to
synthesize mainly PtdIns(3,4,5)P3 in vivo, consistent with the observed effects of thrombin on 3-phosphorylated inositol phospholipids in intact platelets (5).
In the absence of G the PI 3-kinase activity obeyed
Michaelis-Menten kinetics with respect to both ATP and lipid
substrates. This was not the case in the presence of G
which
induced sigmoidal kinetics for lipid substrates, especially in the
presence of PtdIns(4,5)P2. This suggests that this form of
PI 3-kinase might possess more than one PtdIns(4,5)P2
binding site; by analogy with
(31) and
(32) isoforms of
phospholipase C (PLC) the additional site(s) might be non-catalytic and
function to associate PI 3-kinase at a substrate-bearing membrane and
thus allow processive catalysis to occur. Because we only observed
sigmoidal kinetics in the activated state, it is proposed that G
regulates the binding of substrate lipid at such a non-catalytic site.
The results with different lipid substrates predict that the putative
regulatory lipid site prefers PtdIns(4,5)P2 over PtdIns4P
which in turn is preferred over PtdIns. Interestingly this matches the
expected rank order of binding of these lipids to some PH domains (33),
a structural feature that has been proposed to occur in p110
but not
other published forms of PI 3-kinase. A further interesting regulatory feature was the observation that the EC50 for activation by
G
also was affected by the nature of the lipid substrate
suggesting that the binding of lipid (especially
PtdIns(4,5)P2) and G
are mutually cooperative. Such
an observation is again reminiscent of the ligand binding properties of
some PH domains and closely associated sequences C-terminal to the PH
domain proper that appear to possess distinct binding sites for anionic
lipids and G
subunits, respectively. However, it should be
pointed out that without structural studies, the proposal that p110
possesses a PH domain remains speculative.
An alternative explanation for the observed cooperative kinetics for
lipid substrates in the presence of G might be that more G
is complexed with vesicles at the higher lipid concentrations. There
are two lines of evidence suggesting that the membrane localization of
G
is important for its function. Katz et al. (34)
reported that transfection of COS-7 cells with cDNA for PLC
2 and
G-protein
1
1 subunits caused an increase in PLC activity as
evidenced by the accumulation of inositol phosphates. The use of a
mutant cDNA encoding a
subunit lacking the essential cysteine
residue required for isoprenylation resulted in a shift of the
complex to the cytosol and prevented the increase in cellular inositol phosphates. Furthermore, the purification of
dimers from
baculovirus transfected insect cells has shown that only C-terminally
modified
subunits confer PLC
2-activating function on
complexes (35), suggesting that the isoprenylation and carboxyl
methylation of
subunits may be important for both membrane location
and functionality of the complex. However, the extent of membrane
insertion of G
is unlikely to explain the marked differences
between lipid substrates in terms of the cooperativity observed and the
extent of activation of PI 3-kinase.
Much can be learned about the molecular diversity of protein families,
such as the PI 3-kinases, using cloning strategies that exploit
sequence relationships among the family members. Such approaches
identified p110 and have also revealed a wider family that
encompasses both inositol phospholipid and protein serine/threonine
kinases. The initial observations that revealed the presence of
G-protein-regulated forms of PI 3-kinase, however, were made using
partially purified protein preparations from cell extracts. Since the
major G
-sensitive forms of PI 3-kinase present in both platelets
and myeloid cells appear to be complexed to at least one other
polypeptide, which is distinct from p85 and which confers unknown
properties on the enzymes, further studies are required to define the
molecular components of these native proteins. Such studies are also
required to understand the structural basis for the regulatory
mechanisms that we have described.
We are very grateful to Dr. Reinhard Wetzker
for providing us with anti-p110 antibodies and recombinant p110
protein.