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INTRODUCTION |
Phosphoinositide 3-kinases (PI
3-kinases)1 are responsible
for the phosphorylation of inositol phospholipids in the D-3 position of the inositol ring. Their lipid products (PtdIns3P,
PtdIns(3,4)P2, and PtdIns(3,4,5)P3) function as
second messengers in eukaryotic cells. Indeed, PI 3-kinases appear to
be involved in the control of a host of cellular responses ranging from
intracellular transport to cell motility and the suppression of
apoptosis (see Refs. 1-3, for reviews).
Three classes of PI 3-kinases are distinguished (4). Type I PI
3-kinases can be rapidly activated by cell-surface receptors and
in vivo make predominantly PtdIns(3,4,5)P3 (5).
They are heterodimeric enzymes comprising a 110-kDa catalytic subunit
and a regulatory subunit. Type IA PI 3-kinases contain an
,
, or
p110 catalytic subunit (6, 7) and a p50, p55, or p85 (
or
)
regulatory subunit (8-10). The regulatory subunits contain two SH2
domains which allow the enzyme to bind to, and be activated by key
phosphotyrosine residues found in the cytoplasmic tails of growth
factor receptors and various adapter proteins (11).
Type IB PI 3-kinase is made up of a p110
catalytic subunit and a
p101 regulatory subunit (12). This PI 3-kinase seems to be specifically
stimulated by receptors capable of activating heterotrimeric G proteins
(12, 13). It appears, that this effect is mediated by G protein 
subunits which can directly activate p101/p110
PI 3-kinase (12).
Although several reports show that both the biological effects of
p110
and its intrinsic sensitivity to G
are
substantially amplified by the presence of p101, some data suggest that
p110
alone can be substantially catalytically activated by
G
and have clear biological effects (14, 15). We have
begun to address the issue of the role of p101, if any, in these events
by analyzing the regions of p101 involved in interactions with p110
and furthermore, for those constructs that bind, how they affect the
activation of the complex by G
. We analyzed also the
part played by p110
in the process of G
activation
and p101 binding.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis of p101 and p110
--
All point
mutations, novel N or C termini, and internal deletions within porcine
p101 cDNA were done using polymerase chain reaction-based
strategies with mutagenic primers and Taq polymerase (Promega). Polymerase chain reaction-generated fragments were ligated
back into pCMV3 with an N-terminal (EE)- or Myc-tag for expression in
mammalian cells and into pAcOG3 with an N-terminal (EE)-tag for
constructing baculovirus transfer vectors. All polymerase chain
reaction-generated inserts were sequenced.
N- and C-terminal deletions of the human p110
were either done by
digesting with the appropriate restriction enzymes at designated sites.
The isolated DNA fragments were religated into pcDNA3 containing N-terminal Myc-tag linkers. The Ras-binding domain deletions were done
by fusing the internal fragments in-frame to the N-terminal 169 amino
acids, creating a three amino acid linker. Full-length human p101 was
cloned into an N-terminal EE-tag containing pcDNA3 vector.
Cell Culture--
Sf9 cells were grown in TNM-FH medium
with 11% fetal bovine serum in spinner flasks at 27 °C at
0.5-2 × 106 cells/ml. COS-7 cells were grown in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum.
Sf9 Transfections and Production of Recombinant
Protein--
Sf9 cells were lipofected using Insectin
(Invitrogen) liposomes with baculogold (Pharmingen) linearized
baculoviral DNA and a baculovirus transfer vector. Viral particles were
plaque purified prior to amplification. Infection times were 2.3 days
for p101 and 1.8 days for p110
. Harvested cells were pelleted,
washed in 7 mM NaH2PO4 (pH 6.2), 20 mM MgCl2, 0.7% KCl, 2.66% sucrose, snap
frozen in liquid N2 and stored at
80 °C.
Purification of Proteins from Sf9 Cells--
6H-tagged
porcine p110
and EE-tagged porcine p101 were purified as described
previously (12) with metal ion chelate columns or immunoprecipitation,
respectively. The p110
purification was modified as follows. Buffer
C was 50 mM sodium phosphate (pH 7.1, 4 °C), 1%
betaine, 0.05% Tween 20, 0.1 M NaCl; buffer D was 1% betaine, 30 mM Tris-Cl (pH 7.5, 4 °C), 0.15 M NaCl, 0.02% Tween 20, 10% ethylene glycol; buffer E was
as C supplemented with 7 mM imidazole (pH 7.5, 4 °C);
elution was in buffer F which was as C supplemented with 100 mM imidazole (pH 7.5, 4 °C). Eluted p110
in buffer F
was supplemented with 1 mM dithiothreitol and 1 mM EGTA (F') or, if it was to be bound to p101, passed
through a PD10 column (Pharmacia) equilibrated in buffer H (10 mM Tris-Cl (pH 7.5, 4 °C), 0.15 M NaCl, 1%
betaine, 0.02% Tween 20, 10% ethylene glycol, 1 mM
MgCl2, 1 mM dithiothreitol, 1 mM
EGTA, 1% Triton X-100). In some cases, the p110
(6H)-tag was
cleaved with thrombin (Sigma) at 12 units/ml at 4 °C for 12 h.
The p101 purification was modified as follows, cells were sonicated
into 0.15 M NaCl, 25 mM Hepes (pH 7.2, 4 °C), 2 mM EGTA, 1 mM MgCl2
plus antiproteases; cytoplasmic fractions were not pre-cleared with
anti-Myc beads; washes after the anti-EE beads (Onyx Pharmaceuticals)
were five times in 0.4 M NaCl, 20 mM Hepes (pH
7.4, 4 °C), 1 mM EGTA, 1% Triton X-100, 0.4% cholate,
and three further times in buffer H. To bind the subunits, p101 bound to packed anti-EE beads was mixed end on end for 2.5 h with a 25-fold molar excess of free p110
in a small volume of buffer H. Heterodimer on anti-EE beads was washed in buffer J (1% Triton X-100,
0.15 M NaCl, 20 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA) and in buffer F'. p101-p110
heterodimer was
eluted in buffer F' supplemented with 125 µg/ml EY peptide (EYMPTD).
Typical yields were 2 mg of p110
and 25 µg of p101/500 ml of
Sf9 culture.
GST-p110
(the relevant recombinant baculovirus was a gift from R. Wetzker and encoded the human form of the protein) was purified from
cytosolic fractions or Triton X-100 lysates of Sf9 cells as
described by Leopoldt et al. (Ref. 15 and references therein). This GST construct could not be cleaved by thrombin to yield
full-length untagged p110
.
Transient Transfections in COS-7--
Exponentially growing
COS-7 cells were trypsinized, washed twice in phosphate-buffered
saline, and resuspended in 30 mM NaCl, 0.12 M
KCl, 8.1 mM Na2HPO4, 1.46 mM KH2PO4, 5 mM
MgCl2 at 1 × 107 cells/450 µl. They
were mixed with 30 µg of plasmid DNA (15 µg of pCMV3(EE)101
constructs and 15 µg of pCMV3(myc)110
or 15-30 µg of irrelevant
plasmid DNA) per 107 cells aliquot. Aliquots were placed
into 0.4-cm gap electroporation cuvettes (Bio-Rad) and electroporated
in a single pulse (250 V, 980 microfarads). Cells from each
electroporation were seeded into 175-ml tissue culture flasks in full
growth medium. After 28 h, cells were harvested by trypsinization,
washed once in phosphate-buffered saline, and cell pellets lysed in
1 × phosphate-buffered saline, 1 mM EGTA, 1% Triton
X-100. Cytoplasmic fractions were immunoprecipitated with anti-EE beads
followed by washes in 2 × phosphate-buffered saline, 1 mM EGTA, 1% Triton X-100. Samples for PI 3-kinase assays were washed further in 25 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA. Remaining samples were resolved by SDS-PAGE. Gels
were either Coomassie stained or wet-blotted onto polyvinylidene
fluoride membranes (Millipore) and immunoblotted according to their
tags with anti-Myc antibody (obtained from Onyx Pharmaceuticals) and anti-EE ascites fluid (Babco) or with an anti-p101 antiserum
(Microchemical Facilities, Babraham Institute).
PI 3-Kinase Assays--
Free, purified protein from Sf9
cells or COS-7 derived protein on anti-EE beads was diluted in sample
dilution buffer (2 mg/ml fatty acid free bovine serum albumin, 0.1 M KCl, 20 mM Hepes (pH 7.4, 4 °C), 1 mM dithiothreitol). Lipid mixtures containing
phosphatidylethanolamine and PtdIns(4,5)P2 were dried down
and sonicated into 0.1 M NaCl, 25 mM Hepes (pH
7.4, 4 °C), 1 mM EGTA, 0.1% cholate for final concentrations of 50 µM phosphatidylethanolamine (Sigma)
and 5 µM PtdIns(4,5)P2 (13). They were
supplemented with G protein 
subunits (prepared as in Ref. 16) or
its vehicle (1% cholate, 0.15 M NaCl, 1 mM
dithiothreitol, 5 mM EGTA, 0.2 mM EDTA) to a final concentration of 0.3 µM G
.
[
-32P]ATP was diluted to a concentration of 5 µCi/10
µl into 0.1 M NaCl, 25 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA. For the assay, 5 µl of diluted
protein were kept on ice until 20 µl of lipid mixture with or without
G
was added and the mixture transferred to a 30 °C
waterbath. 4 min later, 50 µl more sample dilution buffer
supplemented with MgCl2 to give a final concentration of 3.5 mM was added, another 4 min later, 10 µl of diluted
ATP was added. Assays were stopped after 12 min incorporation time by addition of 160 µl of 1.25 M HCl. Assays were extracted
with 800 µl of CHCl3:MeOH (2:1) and then with
CHCl3, MeOH, 1 M HCl (3:48:47). Lipids were
deacylated and resolved on PEI TLC plates as described before (13).
Alternatively, assays were conducted precisely as described by Leopoldt
et al. (15) with PtdIns acting as the substrate.
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RESULTS |
First, we simply reproduced the results of previous work showing
purified p110
could bind p101 both in vitro and in
vivo and that this association substantially increased the scale
of activation of the PI 3-kinase with G
from 1-2-fold to 50-150-fold, but now using modified procedures and porcine and human
versions of the proteins. Human p101 (GenBank accession number
AF128881) was 88% identical to porcine p101 (c.f. human p110
is 94% idential to porcine p110
). We found the species orthologs behaved interchangeably in these assays and gave results identical to those of the earlier work (Ref. 12, data not shown).
Analysis of p101 Structure/Function in Vitro--
In order to
understand the interactions between p101 and p110
and the ability of
the heterodimer to respond to G
subunits, we made
panels of p101 and p110
constructs. Analysis of p101 structures
involved in binding to p110
was first approached with the following
assay. NH2-terminal (EE)-tagged p101 constructs were
purified from Sf9 cells using anti-(EE)-beads. After washing, an
excess of purified, NH2-terminal (6H)-tagged full-length
p110
from Sf9 cells was mixed with the immobilized p101
constructs. The beads were washed again and eluted with an epitope (EY)
peptide. The released proteins were analyzed by SDS-PAGE and PI
3-kinase assays with and without G
subunits. This
analysis revealed that all of the p101 constructs tested (Fig.
1A) had reduced, but still
significant capacity to specifically bind p110
relative to
full-length. Thus both the large N-and C-terminal truncations of p101
(
1-163 and 1-733) resulted in a 50% reduction in the recovery of
p110
, while
283-581 reduced recovery by about 25% (Fig.
1B). Some p110
specifically bound to N-terminal (p101
1-163) and C-terminal fragments of p101 (p101
1-574) (about 10 and
30% of wild-type, respectively) (Fig. 1C).

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Fig. 1.
In vitro study of p101 association
with p110 and activation of complexes by
G . A,
panel of p101 constructs used for expression and purification from
Sf9 cells. All p101 proteins were N-terminal (EE)-tagged ( ).
B and C, in vitro association of the
(EE)-p101 proteins to independently purified, N-terminal (6H)-tagged
p110 as detailed under "Experimental Procedures." Fractions of
eluted heterodimer were visualized by SDS-PAGE and Coomassie staining.
p101b was prepared from an equivalent extract of Sf9 cells
infected with wild-type baculovirus and thus represents a control for
nonspecific binding of p120 to the anti-(EE) beads. All data shown is
representative of at least four independent experiments. D,
PI 3-kinase assays of fractions of the heterodimers shown in
B and C using PtdIns(4,5)P2 as
substrate, with and without addition of G as
indicated. The data are mean ± range (n = 2-5)
drawn from independent experiments. The activity in the absence of
G was normalized to the amount of p110 protein in
each assay (estimated from the equivalent SDS-PAGE gels).
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All p101-p110
heterodimers were also assayed for PI 3-kinase
activity in the presence or absence of G
(Fig.
1D). All of the heterodimers created containing p101
truncations and deletions had reduced activation by G protein 
subunits. It is striking, however, that the N-terminal truncation
(
1-163) completely abrogated activation by G
,
while the remaining truncations resulted merely in decreased ability of
the respective heterodimers to be activated by G
.
Neither N- nor C-terminal p101 peptides bound to p110
yielded
considerable activation when assayed in the presence of
G
subunits. Overall this suggests a number of regions
in p101 are involved in its ability to interact with p110
,
particularly the N and C termini. Similarly, multiple areas in p101 are
required for heterodimers to give maximal activation by
G
subunits, however, the N terminus is absolutely
required for this process.
Analysis of p101 Structure/Function in Vivo--
To address issues
such as (a) the possibility that purification and our
handling of the p101 constructs had resulted in varying levels of
denaturation and that this effect generated the differential binding we
observed, and (b) that some binding only occurs in vitro in the absence of competing proteins found in the cell, we
examined the ability of p101 derivatives to bind to p110
when the
proteins are transiently co-expressed in COS-7 cells. In addition, to
allow for more detailed mapping of the impact of different areas of
p101 on both binding to p110
and activation of the heterodimer with
G
, a number of further constructs were introduced (Fig. 2A).

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Fig. 2.
In vivo association of p101
constructs with p110 and activation of
resulting heterodimers by
G . A,
further p101 constructs for use in COS-7 transient transfection assays
included further N- and C-terminal peptides and larger deletions.
Furthermore, an unusual acidic region was replaced by a stretch of
serines, threonines, and prolines (DE328-341STP); a region bearing a
vague similarity to PH domains of known signaling proteins as well as
potential key residues therein were mutated ( 161-263, Y193C,
W252A); a possible spliced variant of p101 is also included (p84, this
sequence diverges from p101 at residue 733 resulting in a truncated
version of the protein; P. Hawkins and A. Eguinoa, unpublished data).
Again, all p101 constructs included N-terminal (EE)-tags ( ).
B, COS-7 cells were transiently transfected with mammalian
expression vectors encoding a (EE)-p101 construct and a (Myc)-p110
(for controls, the total amount of DNA was made up to with irrelevant
DNA). Cytoplasmic fractions of transfected cells were subjected to
immunoprecipitations with (EE)-beads. The amount of
co-immunoprecipitated (Myc)-p110 was visualized on Coomassie-stained
protein gels. Binding ratios were estimated by eye with a minimum of
three independent transfections being taken into account for each p101
construct. C, graphical illustration of all binding data
obtained from the COS-7 transient transfection assays. D,
aliquots of the COS-7-derived p101-p110 heterodimers (on the beads)
were assayed for PI 3-kinase activity in the presence or absence of
G . Fold activation obtained with G
for the different constructs is detailed comparatively in this graph.
The legend on the y axis indicates with which construct
p110 had been expressed.
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All p101 constructs were (EE)-tagged and co-expressed with (Myc)-tagged
p110
in COS-7 cells in transient transfection assays. Cytosolic
fractions of harvested cells were subjected to anti-(EE) immunoprecipitations, half of which were used to estimate binding stoichiometries on gels or blots (see Fig. 2B, for an
example) while the other half was assayed for PI 3-kinase activity in
the presence or absence of G protein 
subunits.
The data describing binding of p101 derivatives to p110
in this
assay is summarized in Fig. 2C. Binding to p110
was
affected in all p101 constructs except for the point mutations and
DE328-341STP constructs, supporting the conclusion from the in
vitro studies indicating that more than one area of p101
contributes to binding p110
. It appears, however, that in the COS-7
cell assays compared with the in vitro assays, the
N-terminal deletions lead to a larger decrease in p110
binding
ability than C-terminal deletions (25 versus 70% binding)
and N-terminal peptides can rescue more binding than C-terminal
peptides (50 versus 12% binding).
We also assayed all constructs immunoprecipitated from COS-7 cells for
PI 3-kinase activity. The resulting data (Fig. 2D) underlines the data already obtained from the in vitro
assays from Sf9-produced protein. Again, we found that both C
and N termini of p101 contribute to full activation of the complex by G
protein 
subunits. Any deletion within p101 interfered with the
ability of the heterodimer to be completely activated. Interestingly, deletion of the C-terminal 150 amino acids (1-733 and p84) and deletion of much larger C-terminal portions (1-314, 1-265, and 1-163) lead to a similar decrease in activation by G
to less than 25% of that of full-length p101, stressing the role for
the very C-terminal part. The most dramatic result, however, is induced
by deletion of the very N-terminal portion of p101 (constructs
1-153 and
1-263) which lead to complete loss of activation,
confirming the conclusions from the in vitro assays that
this region of p101 is essential for activation of the complex by G
protein 
subunits.
Analysis of p110
Structure/Function--
To define the sites in
p110
involved in both interaction with p101 and activation by
G
, we prepared a set of constructs shown in Fig.
3 for expression in COS-7 and Sf9
cells. These constructs were used in assays aimed at defining the
regions that interacted with p101 that were relevant to the
G
activation of p101-p110
heterodimers and were
required for basal PI 3-kinase activity.

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Fig. 3.
p110 domain
structure; all truncation and deletion constructs. p110
contains two regions of high homology with p110 / . These include
the N-terminal Ras-binding domain (RBD) and the C-terminal
kinase domain. All constructs used in our study are drawn to scale;
they were N-terminal tagged (Myc or 6H) except where stated otherwise
in the main text.
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PI 3-Kinase Activity of p110
Constructs--
Increasing
N-terminal truncation of p110
systematically reduced its basal
catalytic activity (Fig. 4A).
Deletions beyond residue 369 had no detectable PI 3-kinase activity.
N-terminal tags seemed to increase the basal catalytic activity of
p110
. The activity of the N-terminal (6H)-tagged p110
(6H-p110
) was reduced 2-fold by removal of the tag with thrombin and
the activity of defined p110
constructs was reduced by half by
switching from an N-terminal to a C-terminal (6H)-tag. Furthermore,
N-terminal (GST)-tagged p110
(GST-p110
) possessed an
approximately 10-15-fold higher specific activity than wild-type
p110
. In contrast, and as previously reported (12), binding of p101
reduced the basal catalytic activity of p110
by 5-fold.

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Fig. 4.
The intrinsic activity of
p110 and its activation by
G are affected by
deletions of the very N-terminal portion of the molecule.
A, comparison of p110 constructs basal catalytic
activities. All proteins were derived from Sf9 cells, and
purified according to their tags. The basal catalytic activity was
compared with that of N-terminal (6H)-tagged p110 for each
individual construct. Note that thrombin cleavage of the (6H)-tag
results in loss of 26 N-terminal amino acids due to an internal
thrombin cleavage site (R. Williams, personal communication).
GST-p110 could not be successfully cleaved by thrombin.
B, porcine p101, porcine (6H)-p110 , porcine
p101/(6H)-p110 , and human GST-p110 were purified from Sf9
cells as described under "Experimental Procedures" and aliquots
were assayed for PI 3-kinase activity in the presence or absence of
G according to the protocol of Leopoldt et
al. (15). Samples were normalized for their basal activity (this
is not equivalent to amount of p110 in the assay, see A
for this comparison).
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Reports that GST-p110
can be substantially activated by
G
in vitro (15) lead us to test the effect
of manipulating p110
structure on its responsiveness to
G
, despite the fact that we have never detected
significant effects of G
on porcine p110
activity
in the absence of p101. Testing a range of catalytically active porcine
and human p110
constructs with or without N-terminal or C-terminal
(6H) tags in the presence and absence of G
showed up
to 2-fold activations of human (but not porcine) proteins with either
our standard assay procedure or that of Leopoldt et al. (15;
see below). However, with human GST-p110
we found that although our
assay procedure (using PtdIns(4,5)P2 and 3.5 mM
MgCl2) failed to reveal any activation by
G
, using PtdIns and 10 mM
MgCl2 in the assay (15), we could reproducibly detect
6-7-fold activations by G
(Fig. 4B); this
was unaffected if the constructs were purified from membrane or
cytosolic fractions (data not shown).
Interaction of p110
Constructs with p101 and Activation of
Heterodimers by G
--
Experiments performed with
proteins expressed in either COS-7 or Sf9 cells indicated that
the N terminus of p110
was critical for binding to p101. p110
deletions through the series
1-122,
1-133, and
1-144 (Fig.
5A, for examples) showed
decreasing ability to bind p101, a
1-169 construct showed no
detectable binding (Fig. 5B). However, other regions were
clearly involved in binding p101 (see Fig. 5C for an
overview). Hence, N-terminal peptides (e.g. 1-169) had low
p101 binding potential and further deletion of either the Ras-binding
domain (178-330) or the central regions (330-775) of p110
also
significantly reduced p101 binding. It is significant that those
p110
constructs capable of binding p101, once bound, were all very
similarly activated by G
(Fig. 5D).
Catalytically active p110
constructs incapable of binding p101
remained insensitive to G
when assayed in the
presence of p101 (Fig. 5C).

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Fig. 5.
Interaction of p110
constructs with p101. A, in vitro
interaction assays to assay binding ability of free (6H)-p110
constructs to immobilized (EE)-p101 as described under "Experimental
Procedures" are shown here with full-length p110 and two
N-terminal truncations in comparison. Left-hand lanes show
p110 derivatives after two different purification steps and
right-hand lanes show the p110 derivatives obtained via
binding to the (EE)-p101 beads. Binding of 1-144 could not be
detected on the gel, since the proteins co-migrated, however, activity
assays showed that a small degree of binding did take place (see
below). B, Myc-tagged human p110 and EE-tagged human p101
were transiently co-transfected into COS-7 cells as indicated and the
protein complexes were co-immunoprecipitated and immunoblotted.
I, p101 expression: anti-p101 Western blot of an anti-EE
immunoprecipitation; II, p101 binding to p110 : anti-p101
Western blot of an anti-Myc immunoprecipitation; III,
p110 expression: anti-Myc Western blot of the cell lysates.
C, overview of the ability of all p110 constructs to bind
p101 as estimated by eye from Coomassie-stained gels or Western
blottings derived from in vitro interaction assays and COS-7
transient transfection assays. It is indicated also, which constructs
are catalytically active and where complexes are activated by
G . D, extent of fold-activation of
catalytic activity by G for aliquots of the three
p101-p110 heterodimers prepared in vitro from
Sf9-derived proteins and shown in Fig. 2A.
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 |
DISCUSSION |
It is clear from the above results that multiple regions of p101
are involved in either direct (i.e. physical contact) or indirect interactions with p110
. Interestingly, the
G
sensitivity of the p110
heterodimers formed by
using various p101 constructs clearly correlated with how tightly they
formed heterodimers, but absolutely depended on the presence of the N terminus of p101. We have previously published some binding data showing G
can apparently bind about five times more effectively to p101 than p110
(12), but we cannot discount important
binding of G
to p110
, as others have identified clear effects of G
on GST-p110
(13, 15; see also
Fig. 4B). There are, therefore, two types of explanation for
this data. First, that a lot of the contacts between p101 and p110
are required for G
to have their full effects on
p110
catalytic activity (whether the G
bind to
p101, p110
, or both). Second, that the primary sites of
G
binding that influence the activity of the
heterodimer are located on p101 and are "co-incidentally" disrupted
by changes which impinge on p101-p110
interaction. This apparently
highly interwoven structure-function relationship between p101,
G
, and p110
is not surprising if it is accepted
that binding of p101 and G
can have such a profound
effect on the catalytic site in p110
. The binding site for p101 in
p110
, although primarily involving the N terminus of the protein, is
a far larger region extending deeper into the protein. Considering the
fact that several parts of p101 are involved in binding to p110
, it
would seem reasonable that a substantial piece of p110
is involved
in binding p101 (note that in p110
a relatively short region of the
N terminus is thought to bind to a short inter-SH2 domain segment of
p85 (17, 18)). In contrast to the situation with p101, where deletions
affecting the total contact area and strength of binding to p110
also affected the sensitivity of the heterodimer to
G
, deletions through the N terminus of p110
, that
reduced the efficiency of binding to p101, had no effect on the
activation of heterodimeric enzyme that could be recovered. Clearly,
this analysis cannot be complete because deletions removing more than
200 N-terminal residues begin to strongly reduce the intrinsic
catalytic activity of p110
, such that deletions beyond residue 369 activity is completely lost (see below), yet these deeper regions are
clearly involved in binding to p101. However, the implication of this
data is that some of the contacts between p101 and p110
, involved in
their binding, can be eliminated by disruptions on the p110
side
without effect on G
activation of the heterodimer,
but that contacts broken by p101 disruptions reduce the ability of
G
to activate the enzyme. The simplest explanation
for these observations is that p101 disruptions are also interfering
with G
-binding sites that are most important for
activation of the heterodimer; i.e. that the latter of our
proposed models (see above) is most likely to be correct.
In the process of engineering and assaying a range of p110
constructs it became clear that the N terminus of the protein had
significant potential to influence catalytic activity. Hence the
addition of N-terminal GST and (6H)-tags apparently increased the
activity of the enzyme. In contrast, binding of p101, which appears to
interact most strongly with the N terminus of p110
lead to a 5-fold
inhibition of basal catalytic activity. This effect of p101 appears to
have some parallels with observations that p85 binding to p110
suppresses its PI 3-kinase catalytic activity (19). Clearly the scale
of activation of the p101/p110
heterodimer by
G
in vitro indicates this "simple
model" cannot apply here, however, this phenomenon lead us to test
whether N-terminal tagging also influenced the sensitivity of p110
alone to G
subunits.
We found that of a large range of constructs GST-p110
alone was
clearly activated by G
, but only under the conditions previously reported to show this effect (15). The very fact that
G
can have some significant effect on GST-p110
in
the absence of p101 may be taken to suggest one primary point of
interactions of G
with p101/p110
is through direct binding to the p110
subunit. This gains some support from work indicating G
can bind to p110
alone. We have
previously found that although five times more G
could be rescued from in vitro binding assays associated
with p101 than with p110
we could detect above background binding to
p110
(12). Furthermore, Leopoldt et al. (15) have shown
G
can be recovered with p110
immunoprecipitated
from Sf9 cells, and they went on to map this binding to two
distinct regions within p110
. However, there are major problems with
these approaches (15). First, neither directly demonstrated that the
G
-binding sites were relevant to the activation of
the PI 3-kinases and second as both analyses depended on an
"immunoprecipitation wash" protocol they are subject to selective
recovery of interactions with appropriate affinities and on/off rates;
meaning much more physiologically important binding sites could be
missed. As G
are notoriously prone to nonspecific
hydrophobic interaction and for binding to effectors with low affinity
but high on/off rates this means these assays are of very limited value
in defining the regions that bind G
that lead to
activation of the enzyme.
As a consequence of these considerations and in the light of (i)
lack of effect of G
on other, non-GST-tagged p110
constructs, (ii) the unphysiological nature of the substrate and ionic
conditions required to see G
activation of
GST-p110
, (iii) the inevitable problem that in using p110
alone
rather than in a complex with p101 further hydrophobic regions of
protein interaction may be exposed, and (iv) that the presence of the GST fusion will drive homodimerization of p110
, we consider the evidence that the effects of G
on p101/p110
PI
3-kinase are only via direct interaction with p110
to be very weak.
Overall, our data suggests that p101 and p110
interact primarily
through large areas covering the N and C termini of p101 and the
N-terminal half of p110
and that the areas which bind G
giving the major effect on PI 3-kinase activity are probably located on p101. Given the substantial difficulties
encountered in studying specific, low affinity interactions of
G
subunits with various effectors in vitro
(e.g. studying G
activation of PLC
s can
be seen as an analogous problem), it is likely that a combination of
further technologies, including detailed structural information, will
be required to yield further insight into the mechanism of action of
G
on p101/p110
PI 3-kinase.