From the Institut für Pharmakologie, Freie
Universität Berlin, Thielallee 69-73, D-14195 Berlin
(Dahlem), Germany and § Max-Planck-Arbeitsgruppe
"Molekulare Zellbiologie," Friedrich-Schiller-Universität,
Jena, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Class I phosphoinositide 3-kinases (PI3Ks)
regulate important cellular processes such as mitogenesis,
apoptosis, and cytoskeletal functions. They include PI3K, -
,
and -
isoforms coupled to receptor tyrosine kinases and a PI3K
isoform activated by receptor-stimulated G proteins. This study
examines the direct interaction of purified recombinant PI3K
catalytic subunit (p110
) and G
complexes. When
phosphatidylinositol was used as a substrate, G
stimulated p110
lipid kinase activity more than 60-fold
(EC50, ~20 nM). Stimulation was
inhibited by G
o-GDP or wortmannin in a
concentration-dependent fashion. Stoichiometric binding of a monoclonal
antibody to the putative pleckstrin homology domain of p110
did not
affect G
-mediated enzymatic stimulation, whereas incubation of
G
with a synthetic peptide resembling a predicted G
effector domain of type 2 adenylyl cyclase selectively inhibited
activation of p110
. G
complexes bound to N- as well as
C-terminal deletion mutants of p110
. Correspondingly, these
enzymatically inactive N- and C-terminal mutants inhibited G
-mediated activation of wild type p110
. Our data suggest that (i) p110
directly interacts with G
, (ii) the pleckstrin
homology domain is not the only region important for G
-mediated
activation of the lipid kinase, and (iii) G
binds to at least two
contact sites of p110
, one of which is close to or within the
catalytic core of the enzyme.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphatidylinositol 3,4,5-trisphosphate
(PI-(3,4,5)P3)1
is absent in quiescent cells but is rapidly produced upon exposure to
various stimuli (1-4). Hence, PI-(3,4,5)P3 has been
suggested to act as a second messenger eliciting a wide array of
cellular responses (5). PI-(3,4,5)P3-dependent
functions include regulation of cell growth, protein sorting,
exocytosis, and cytoskeletal rearrangements. The enzymes responsible
for the formation of PI-(3,4,5)P3 are phosphoinositide
3-kinases (PI3K; EC 2.7.1.137), which catalyze phosphorylation of
phosphoinositides at the 3 position of the inositol ring (6).
Depending on substrate specificity and enzyme structures, three classes
of PI3Ks have been distinguished. Class I enzymes are involved in
receptor-induced hormonal responses. They utilize PI, PI-4P, and
PI-(4,5)P2 in vitro though within the cell they
exhibit a preference for PI-(4,5)P2. In addition, they show
a moderate serine/threonine protein kinase activity (7-9). Further
discrimination of the class I members is based on their association
with receptor tyrosine kinase (class IA)- or of G
protein-coupled receptor (class IB)-signaling pathways, although very recently one member (p110) has been suggested to respond synergistically to both G
and tyrosine-phosphorylated peptides (10). Additionally a related retrovirus-encoded PI3K causing
hemangiosarcomas was found (11).
Class I enzymes purify as heterodimers with a molecular mass of about
200 kDa containing a catalytic subunit of 110 kDa (p110) and a
regulatory subunit (12-16). Several mechanisms for regulating their
enzymatic activity in response to extracellular stimuli have been
elucidated. Among them the class IA members p110,
, and
are stimulated by tyrosine-phosphorylated proteins through interaction with regulatory PI3K subunits such as p85 or p55 (17, 18).
They in turn bind to the N terminus of the catalytic p110 subunit,
thereby inducing PI3K activity. In contrast, the only known class
IB member p110
does not bind to p85 adaptors, but instead associates with a noncatalytic p101 subunit (19). However, several lines of evidence indicate that G proteins stimulate p110
in
the absence of p101 both in vitro and in vivo
(20-23). G
are thought to be the dominant physiological
stimulus, while G
subunits of the Gi but not
Gq or G12 subfamilies only moderately activate p110
.
Whereas a precise picture of the molecular mechanisms for
receptor-induced activation of class IA PI3Ks has been
drawn, little is known about how G activates PI3K
and which
structures of the enzyme are involved. Comparison of the deduced amino
acid sequences of the catalytic subunits of class I members revealed several highly conserved regions of homology (HR) but also parts which
are quite diverse (6). All enzymes have a C-terminally located
catalytic domain (HR1). Interestingly, this domain requires N-terminal
regions for enzymatic activity (9). Therefore a horseshoe-like folding
of the p110, enabling interaction between the N- and C-terminal half of
the enzyme, has been assumed (24). HR2 represents a PIK domain found in
all PI3- and PI4-kinases. Other regions of homology are specific for
PI3Ks (HR3) and class I PI3Ks (HR4). All class I PI3Ks also contain a
Ras-binding motif (25). In contrast, only class IA enzymes
have an N-terminal stretch assumed to interact with its p85 subunits,
whereas only p110
exhibits a Ras-GAP homology region, which may fold
to form a pleckstrin homology (PH) domain (6, 20, 26). This region of
p110
has been speculated to be involved in G
-mediated
activation of PI3K
, since PH domains of G
-regulated proteins
such as
-adrenergic receptor kinase or phosducin have previously
been identified to bind G
(27, 28). However, other enzymes with
an inherent PH domain such as phospholipase C
are insensitive to
modulation by G
, while some effectors lacking PH domains,
e.g. adenylyl cyclases and potassium or calcium channels,
are regulated by G
complexes (29-32).
Therefore, the aim of the present study was to examine whether the
putative PH domain of the catalytic subunit of PI3K is critical for
interaction of p110
with G
. Binding of a monoclonal antibody
(mAb) to p110
that blocked the PH domain did not reduce G
-mediated stimulation. Furthermore, results obtained with
deletion mutants of p110
indicated that G
binds to an
N-terminal region as well as to a region near or within the
C-terminally located catalytic core. Correspondingly, inactive N- and
C-terminal mutants inhibited G
-mediated activation of wild type
p110
by sequestration of G
. We conclude that the PH domain of
p110
is not the only region interacting with G
and hypothesize
that N- and C-terminal stretches of p110
contribute to form a common
G
effector region.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction and Purification of PI3K-GST Fusion
Proteins--
Construction of recombinant baculoviruses for expression
of human GST-p110 fusion proteins and mutants thereof and of porcine p101 and GST-p101 were described previously (9, 19, 20, 22).
Recombinant baculoviruses for G
1 and G
2
subunits and for GST-p110
were generous gifts from Drs. M. Lohse
(Würzburg) and M. D. Waterfield (London). For protein
expression cells were infected at a multiplicity of infection of 1 virus per cell. After 48-60 h of infection cells were pelleted by
centrifugation (1,000 × g), washed with
phosphate-buffered saline twice, and resuspended in ice-cold buffer A
containing 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 50 mM Tris/HCl, pH 8.0, 10 mM dithiothreitol, 10 µg/ml each of aprotinin,
benzamidin, leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride. Cells were disrupted by N2 cavitation (30 min at
4 °C, 25 bar) or by forcing the cell suspension through a 22-gauge
needle (20 times) and subsequently through a 26-gauge needle (10 times). Nuclei and debris were discarded. The cytosolic and membranous fraction were recovered by centrifugation at 100,000 × g for 50 min. Membrane extract (0.5% Lubrol PX in buffer A)
and cytosol were incubated overnight with glutathione-Sepharose 4B
beads (Pharmacia Biotech Inc.) prewashed with buffer A. The
Sepharose-bound GST fusion proteins could be stored at
20 °C in
buffer B containing 50% glycerol, 1 mM EDTA, 40 mM Tris/HCl, pH 8.0, 1 mM dithiothreitol, and
1.57 mg/ml benzamidin. For enzymatic assays GST fusion proteins were
freshly eluted with buffer C consisting of buffer A with 10 mM glutathione for 1 h at 4 °C. Purified proteins
were quantified by Coomassie Blue staining following SDS-PAGE with
bovine serum albumin as the standard.
Preparation of G Proteins--
For isolation of bovine retinal
transducin as well as G
o subunits and G
complexes from bovine brain, we employed standard techniques with
modifications (33, 34). Bovine brain G protein subunits were purified
to apparent homogeneity in the presence of aluminum fluoride. Isolation
and final purification of G
o and G
was achieved
using a Mono Q (Pharmacia) fast protein liquid chromatography column
(35). G protein subunits were identified by their immunoreactivity.
Contamination by other pertussis toxin-sensitive G
subunits was
excluded by analysis of autoradiographic signals after pertussis
toxin-mediated [32P]ADP-ribosylation with a BAS 1500 Fuji-Imager (Raytest, Straubenhardt, Germany) (36). Concentrations of G
protein heterotrimers and their
subunits were determined by binding
of [35S]GTP
S (35), amounts of G protein
subunits
were determined by the method of Lowry et al. (37) and by
Coomassie Blue staining following SDS-PAGE with bovine serum albumin as
the standard (38). Purified proteins were stored at
70 °C
until use.
Gelelectrophoresis, Immunoblotting, and
Antibodies--
Generation of the monoclonal antibody against p110
and antiserum AS 398 against G
subunits were detailed elsewhere (22, 39). For detection of p110
or the G protein subunit, preparations were fractionated by SDS-PAGE transferred to nitrocellulose or polyvinylidene difluoride membranes (Millipore, Eschborn,
Germany). Visualization of specific antisera was performed using the
ECL chemiluminescence system (Amersham, Braunschweig, Germany) or the CDP-Star chemiluminescence reagent (Tropix, Bedford, MA) according to the manufacturers' instructions.
Lipid Kinase Assay--
The assays were conducted in a final
volume of 50 µl containing 0.1% bovine serum albumin, 2 mM EGTA, 0.2 mM EDTA, 10 mM
MgCl2, 120 mM NaCl, 40 mM HEPES, pH
7.4, 1 mM dithiothreitol, 1 mM
-glycerophosphate as described previously (20) with some
modifications. Briefly, 30 µl of lipid vesicles (320 µM
phosphatidylethanolamine, 300 µM
phosphatidylethanolserine, 140 µM
phosphatidylethanolcholine, 30 µM sphingomyelin, and 320 µM PI) were mixed with either G
complexes or their
vehicle and incubated on ice for 8 min. Thereafter the enzyme fraction
(1-10 ng) was added and the mixture was incubated for further 10 min
at 4 °C in a final volume of 40 µl. The assay was then started by
adding 40 µM ATP (1 µCi of [
-32P]ATP)
in 10 µl of the above assay buffer (30 °C). Water-dissolved peptides (Eurogentec, Brussels, Belgium) used in this study were incubated with G
complexes before adding lipid vesicles.
Wortmannin was stored in dimethyl sulfoxide (20 mM) in the
dark at
20 °C and added to the kinase immediately before the
experiment. After 15 min the reaction was stopped with ice-cold 150 µl of 1 N HCl and placing the tubes on ice. The lipids
were extracted by vortexing samples with 450 µl of
chloroform/methanol (1:1). After centrifugation and removing of the
aqueous phase, the organic phase was washed twice with 200 µl of 1 N HCl. Subsequently, 40 µl of the organic phase were
resolved on potassium oxalate-pretreated TLC plates (Whatman, Cliffton,
NJ) with 35 ml of 2 N acetic acid and 65 ml of 1-propanol
as the mobile phase. Dried TLC plates were exposed to Fuji-Imaging
plates, and autoradiographic signals were quantitated with a BAS 1500 Fuji-Imager.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our initial experiments with the purified recombinant p110
yielded a 5-6-fold stimulation with 1 µM G
and an
EC50 of 200 nM for transducin
(20). As
this effect of G
is moderate compared with effects on other G
protein-regulated cellular targets, we optimized the conditions for
p110
activation. Human recombinant p110
was expressed as GST
fusion protein in Sf9 cells. Purified cytosolic enzyme exhibited
a basal specific activity of 1-3 nmol of PI-3P/min/mg of p110
(Fig.
1A) and was much more
sensitive to G
than the enzyme isolated from Sf9 membrane
extracts (not shown). To investigate the stimulatory effect of G
complexes we used a mixture of highly concentrated purified bovine
brain G
(see Fig. 1A) instead of using the less potent
transducin
. Under these experimental conditions with
phosphatidylinositol (PI) as substrate, G
complexes stimulated
purified cytosolic p110
up to 60-fold with an EC50 of
~20 nM in a bimodal manner (see Fig. 1, B and
C). As expected, p110
was not stimulated by G
(not
shown). G
increased the Vmax of the
recombinant p110
for ATP, corresponding to the data obtained with
the native purified PI3K
(23). Some variation in the efficiency of
G
on p110
activity was probably due to either a well known
variability in the quality of PI-liposome preparations used as
substrates (40) or to the degree of autophosphorylation of p110
. The
specific covalent inhibitor wortmannin decreased G
-stimulated
enzymatic activity half-maximally at 15 nM and completely
at 100 nM (Fig. 2A). An excess of GDP-bound
G
o also inhibited G
-mediated stimulation of
p110
activity, most likely by sequestration of free G
(Fig. 2B).
|
|
To examine the involvement of the putative PH domain of p110 we took
advantage of a mAb raised against the purified enzyme (22). The mAb
binds specifically to an amino acid stretch of the enzyme corresponding
to the PH domain postulated between amino acids 87 and 302 (6, 26).
Fig. 3 shows that the mAb did not recognize constructs lacking amino acids 75-398 (see Fig.
3B, lane 8) or amino acids 1-739 (lane
4), whereas it bound to constructs lacking amino acids 1-97
(lane 7), 335-1068 (lane 6), or 741-1068 (lane 5). Preparations containing active p110
were
incubated with different volumes of antibody solution to ensure binding of saturating amounts of the mAb to the enzyme. The p110
-antibody complex was purified on glutathione-Sepharose beads (Fig.
4, upper panels) and
subsequently stimulated with G
. As seen in Fig. 4 (lower
panel), blocking the p110
PH domain by the mAb did not prevent
G
-mediated stimulation. Conversely, a peptide derived from the
adenylyl cyclase 2 (AC2) (amino acids 956-982; Fig.
5, upper part) not containing
a PH domain did compete with p110
for the G
complex (41, 42).
Increasing concentrations of this peptide completely reversed
G
-mediated stimulation of p110
with an IC50 of 50 µM (see Fig. 5, lower part). The adenylyl
cyclase 2 peptide did not change basal activity, and a control peptide derived from the corresponding region of the G
-insensitive
adenylyl cyclase 3 (AC3) did not compete for the
G
-stimulated activity (see Fig. 5).
|
|
|
To encircle regions of p110 required for G
-elicited activation
of PI3K, we studied the copurification of
G
1
2 with various GST-p110 constructs
following coexpression of recombinant proteins in Sf9 cells.
First, to test the specificity of this experimental approach we
coexpressed G
1
2 with GST-p110
and
GST-p110
(Fig. 6, center
and upper panels). Since G
does not activate p110
it should not copurify with p110
, whereas it should copurify with
the G
-activated p110
. p110 isoforms were isolated from cell
homogenates on a glutathione affinity matrix. Subsequently proteins
were subjected to SDS gels, and copurified G
complexes were
detected by immunoblotting with a G
-specific antibody (see Fig. 6,
lower panel). This assay revealed that
G
1
2 indeed copurified with p110
,
whereas no G
immunoreactivity was detected after purification of
p110
or GST. Further experiments showed that G
copurified only
with membrane-bound p110
but not with cytosolic p110
, as could be
explained by the membrane association of G
complexes (not
shown).
|
Next, we coexpressed wild type p110 or different p110
mutants as
GST fusion proteins together with G
1
2 and
studied copurification. As shown in Fig.
7, G
copurified with different
mutants expressing the N-terminal PH domain (lower panel
1-97, lane 5;
335-1068, lane 7;
741-1068, lane 9), but
also with a mutant lacking the PH domain (
75-398,
lane 6) and with a C-terminal mutant (
1-739, lane 8) bearing only the catalytic core of the enzyme. These
results indicate that G
specifically interacts with at least two
binding sites of p110
. One binding site is localized N-terminally,
whereas the other one is found at the C terminus.
|
As has been shown before, all p110 deletion mutants lacking more
than the first 97 amino acids were enzymatically inactive as well as a
p110
construct containing a Lys
Arg mutation at position 799 (K799R), which abolishes wortmannin binding (9). To support the
assumption that G
interacts with two regions of p110
, we
tested whether the inactive N- and C-terminal mutants would compete
with wild type p110
for G
. This was done by coinfecting Sf9 cells with a constant amount of recombinant baculovirus
encoding enzymatically active wild type p110
-GST fusion protein and
increasing amounts of viruses encoding the N terminus (
741-1068) or
the C terminus (
1-739) of p110
as GST fusion proteins. The
coexpressed proteins were affinity-purified from Sf9 cytosol on
glutathione-Sepharose beads and stimulated by addition of bovine brain
G
complexes, and the fold activation of p110
was calculated.
The C-terminal fragment bearing only the catalytic domain of p110
(
1-739) as well as the N terminus of p110
(
741-1068)
inhibited G
-mediated stimulation of wild type p110
in a
concentration-dependent manner (Fig.
8). GST alone did not affect
G
-mediated stimulation of p110
. The enzymatically inactive
K799R p110
point mutant capable of binding to G
(see Fig. 7,
lower panel, lane 10) inhibited enzymatic
activity to the same extent as the deletion mutants (see Fig. 8). This
observation is in accordance with results from the copurification
experiments (see above) and underlines the hypothesis that p110
exhibits two domains interacting with G
. Therefore, we suppose
that both the N and C terminus bearing the catalytic domain of p110
are important for direct interaction with G
.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study examines the direct interaction between p110 and
G
. We show that the catalytic subunit of PI3K
is greatly
stimulated by G
in the absence of the recently reported p101
subunit. It extents our previous findings (20-22) supported by Tang
and Downes (23) and points against an indispensable function of p101
for the stimulation of p110
by G
as recently hypothesized
(19). Furthermore, preliminary results employing a purified recombinant heterodimer consisting of p110
and p101 showed no further increase in PI-3P formation in response to G
compared with p110
alone. At present there is no conclusive evidence for an essential role of
p101 as an adaptor protein linking G protein-coupled receptors and
p110
in a similar way as p85 mediates interaction of p110
with
receptor tyrosine kinases (43). In addition, the recent observation
that G
can bind directly to the related p110
supports an
interaction with p110
as well (10). Accordingly, in reconstituted neutrophils we recently observed stimulation of p110
lipid kinase activity by the agonist of the G protein-coupled fMet-Leu-Phe receptor
in the absence of p101 (21). A possible explanation for this apparent
discrepancy is the observation that purified recombinant human p110
degrades rapidly in the absence of p101, this subunit could stabilize
the enzymatic activity of p110
. In addition, p101 could also affect
the extent of autophosphorylation of p110
which may influence
G
-mediated activation of PI3K
. Furthermore, the possibility
that a small fraction of the recombinant human p110
associated with
a putative insect cell-derived p101-like protein mediated all the
G
-sensitive activity is unlikely, since the basal specific
activities of recombinant p110
recorded in this study was roughly 1 and 2 orders of magnitude larger than for recombinant p110
and
p110
/p101, respectively, as estimated from published results
(19).
In our study we noticed that geranyl-geranylated G
complexes from bovine brain were 10 times more potent in stimulating p110
than previously used farnesylated bovine transducin
(20). This difference in potency was also seen in studies with other G
-regulated effectors (31). We found that half-maximal
stimulation of the PI3K
catalytic subunit required only low
nanomolar concentrations (~20 nM) of bovine brain
G
. These concentrations are similar to those required for
regulation of other effectors such as phospholipase C-
, potassium,
or calcium channels, but are much lower than those reported previously
by other groups studying native or recombinant heterodimeric PI3K (14,
16, 19, 23, 31, 40, 44, 45).
Only cytosolic but not membrane-extracted purified p110 was
significantly stimulated by addition of exogenous G
. The marginal responsiveness of the membrane-derived enzyme corresponds to the observation that recombinant G
1
2
copurified with membrane-extracted but not cytosolic p110
. G
complexes may therefore function as a membrane anchor for p110
. This
in turn could facilitate the access of the enzyme to its lipid
substrates thereby enhancing p110
activity. Interestingly, for the
purified native PI3K
Tang and Downes (23) proposed cooperative
kinetics for lipid substrates in the presence of G
.
Direct interaction of p110 with G
raises the question which
region of p110
is critical for interaction with G
. Since p110
significantly differs from the receptor tyrosine
kinase-regulated enzymes by an inherent PH domain it was speculated
that this stretch may function as a G
-binding site (6, 20, 26).
Blocking the PH domain of enzymatically active p110
by an specific
antibody reacting with this p110
domain did not reduce the
stimulatory activity of G
on p110
. Although the N-terminal
half of p110
(
741-1068) did bind G
and inhibited
G
-mediated activation of the fully processed p110
, we present
evidence that interaction of G
with the putative p110
PH
domain is not exclusively responsible for stimulation of PI3K
activity. In particular, p110
deletion mutants lacking the PH domain
and adjacent stretches still bound G
. Furthermore, coexpression
of p110
with an enzymatically inactive C-terminal deletion mutant
(
1-739) significantly inhibited activation of fully processed
p110
by G
. Similar results were obtained after mixing of
separately purified p110
with increasing concentrations of mutants
(not shown). Our results suggest that the catalytic core is important
for G
-mediated activation. This is not unlikely since the amino
acid identity with p110
and -
in this region is only 45 and
46.5%, respectively. Recently, the motif QXXER has been
proposed as a consensus binding site for G
(41). p110
but not
p110
, -
, or -
contains these amino acids within the catalytic
core (amino acids 888-893), although they lack the correct spacing of
this consensus motif. Unfortunately, peptides derived from
corresponding regions of p110 isozymes strongly inhibited basal
enzymatic activity preventing further analysis. In this context it
should be noticed that a recent study on G protein-regulated calcium
channels identified two G
-interacting domains that do not contain
a QXXER motif (32). Nevertheless, a peptide derived from the
adenylyl cyclase 2, which is thought to interact with a G
domain,
that for its part does not interact with PH-like structures, blocked
G
-induced activation of p110
.
In summary, this study shows that the N as well as the C terminus of
p110 interacts with G
. This could be explained by a folding of
the enzyme which results in proximity of N and C terminus. Indeed,
several lines of evidence point to a horseshoe-like structure of p110
(24) since N-terminal regions outside the C-terminal catalytic core are
mandatory for enzymatic activity and wortmannin binding (9). Based on
the proposed horseshoe-like structure of p110
one may suggest that
N- and C-terminal portions of p110
form a common G
-effector
region for regulation of enzymatic activity. The putative PH domain may
be a part of this effector region but additional structures are
required for G
-mediated p110
activation. This finding supports
the emerging concept in molecular and cell biology of PH structures
being not sufficient do define molecules as G
-regulated
effectors.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Antje Tomschegg for excellent
technical assistance. We are grateful to Drs. Michael Waterfield for
providing a p110-encoding virus, Martin Lohse for G
- and
G
-encoding viruses, as well as Len Stephens and Phil Hawkins for
providing us with the pcDNA3-p101 construct. Valuable discussions
with Drs. Doris Koesling and Alan V. Smrcka are appreciated. We are
indebted to Günter Schultz for critical reading of the manuscript
and for his support.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.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.
¶ Present address: Institut für Neurobiologie, Otto von Guericke Universität Magdeburg, Magdeburg, Germany.
Recipient of the Stiftung Stipendien-Fonds des Verbandes
der Chemischen Industrie.
** To whom correspondence should be addressed. Tel.: 49-30 838 4210; Fax: 49-30 831 5954; E-mail: bnue{at}zedat.fu-berlin.de.
1
The abbreviation used are: PI,
phosphatidylinositol (locants of other phosphates on the inositol ring
shown in parentheses); PI3K, phosphoinositide 3-kinase; GST,
glutathione S-transferase; p110, catalytic subunit of PI3K;
p101, subunit associated with p110; p85, regulatory subunit of
receptor tyrosine kinase activated PI3K; PH, pleckstrin homology; HR,
homology region; G
o,
-subunit of the major G protein
in mammalian brain; G
,
-subunit from bovine brain; PIK,
lipid kinase domain; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; GTP
S, guanosine
5'-O-(thiotriphosphate).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|