From the
Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to
play a key role in growth factor signaling pathways, although its
signaling mechanism has not been fully elucidated. Using the yeast
interaction trap system, we have identified Grb2 as a PI 3-kinase
interacting protein. Our experiments demonstrate that p85, the
regulatory subunit of PI 3-kinase, interacts with Grb2 in
vivo, and this interaction is independent of growth factor
stimulation. The direct association between Grb2 and p85 was
reconstituted in vitro with glutathione S-transferase
fusion proteins. Domain analyses and peptide competition indicate that
the association is mediated by the SH3 domains of Grb2 and the
proline-rich motifs of p85 and that only one SH3 domain is required for
minimal binding. The interaction does not displace the catalytic
subunit of PI 3-kinase but is exclusive of Sos. Signaling through PI
3-kinase, therefore, may involve the ubiquitous adapter Grb2, which
serves as a convergence point for multiple pathways.
The discovery of phosphatidylinositol 3-kinase (PI
3-kinase)
Three lines of evidence indicate
that PI 3-kinase plays an important role in growth regulation and
transformation. The first line of evidence comes from genetic analyses
of the binding sites for PI 3-kinase on oncogenes and receptors. For
instance, all mutants of polyoma virus middle T antigen, which either
fail to associate with PI 3-kinase or are unable to elevate the levels
of PI 3-kinase products in vivo, result in a
transformation-defective
phenotype
(10, 18, 19, 20) . Similarly,
point mutations in the PI 3-kinase binding sites of the PDGF receptor
impair the receptor's ability to initiate DNA
synthesis
(21, 22, 23) . Second, recent work by
Roche et al.(24) has shown that microinjection of
antibodies specific for the p110 subunit of the PI 3-kinase into
quiescent fibroblasts inhibited PDGF-induced DNA synthesis. Finally,
studies indicate that inhibition of PI 3-kinase activity by wortmannin,
the specific PI 3-kinase inhibitor, results in blockage of
serum-induced cell proliferation (25).
The precise mechanisms by
which PI 3-kinase regulates cell growth and transformation are not well
defined. Evidence exists to link PI 3-kinase to the regulatory cascade
that controls pp70
Additional information concerning the
proteins that interact with PI 3-kinase is needed to define the
biochemical interactions of PI 3-kinase that are involved in cell
proliferation and cell shape. To this end, we constructed a bait with
full-length human p85 and used it to search for p85 interactors in the
yeast interaction trap system (33). Here we report that an interactor
for the 85-kDa subunit of PI 3-kinase is Grb2. Comprised entirely of
SH2 and SH3 domains, Grb2 is a small adapter protein that binds
phosphotyrosine motifs on activated receptors via the SH2 domain while
the two flanking SH3 domains are used to bind the guanine nucleotide
exchange protein Sos. In this manner, Grb2 links the activation of
receptors to the GTP loading of p21
An
aliquot of GST-beads was washed in PBS once and resuspended in 100
µl of PBS or 20 mM HEPES (pH 7.4). Human thrombin was
added at a concentration of 0.2-0.4 units/µg protein and the
reaction was incubated at room temperature for 15 min. PMSF was added
to a final concentration of 1 mM and the mixture was incubated
for an additional 5 min. The supernatant was separated from the beads
in a microcentrifuge and stored at -80 °C or used immediately.
We have presented several lines of evidence that the p85
subunit of PI 3-kinase interacts directly with the adapter protein Grb2
both in vitro and in vivo. The association was first
revealed in the yeast interaction trap system. The association between
p85 and Grb2 was also observed in quiescent NIH 3T3 cells and Balb/c
3T3 cells on reciprocal immunoprecipitation using anti-p85 and
anti-Grb2 antibodies, independent of PDGF stimulation. Therefore, at
least most of the interaction we observed was not mediated by the
activated receptor. The interaction appears to be direct since it can
be reconstituted in vitro using purified bacterially produced
proteins. Domain analyses demonstrate that the association is mediated
by the SH3 domains of Grb2 and the proline-rich motifs flanking the bcr
homology region of p85.
The most parsimonious model to explain our
results places both subunits of PI 3-kinase in a position equivalent to
Sos in Grb2 signaling. Although the interaction of Grb2 is with the
85-kDa subunit, PI 3-kinase assays performed on anti-Grb2
immunoprecipitates indicate that p110 remains bound to p85 in the
complex. While only a small fraction of PI 3-kinase binds Grb2 and
vice versa, the interaction presents intriguing possibilities
for linking PI 3-kinase to receptors that lack the ability to bind PI
3-kinase directly. For instance, recent work by Welham et al.(40) has suggested that SHPTP2 may play an important role in
integrating signals from interleukin-3 and GM-CSF receptors to PI
3-kinase. Our data may provide a missing link between SHPTP2 and PI
3-kinase. Previous studies have shown that Grb2 associates with
tyrosine-phosphorylated SHPTP2 via its SH2
domain
(41, 42) . Since Grb2 also interacts with PI
3-kinase p85 subunit through its SH3 domains, it serves as an excellent
candidate connecting the two pathways. Analyses are currently under way
to explore this possibility. In addition to SHPTP2, Shc
(43, 44) and IRS-1 (45) have been shown to interact with the SH2
domain of Grb2, as have non-receptor tyrosine kinases, including
Abl
(46) , Bcr-Abl
(47) , and FAK
(48) .
Alternatively, the interaction between Grb2 and PI 3-kinase might use
p85 to present Grb2 to activated receptors that otherwise lack access
to the adapter protein. Thus, the Grb2-PI 3-kinase interaction has the
potential to play a role in a variety of signaling processes.
The
SH3 domain-mediated interaction provides a novel element for linking PI
3-kinase to molecules that otherwise lack access to the enzyme. The
fact that only one SH3 domain of Grb2 is required for minimal binding
in vitro raises the interesting possibility that Grb2 could
use its free SH3 domain to bind other factors. However, our data
suggest that Sos is not bound in the same complex with PI 3-kinase. The
possibility remains that another molecule could occupy the second SH3
domain and thus would increase the signaling potential of the Grb2/PI
3-kinase system. Further work will need to be devoted to study the
functionality of the interaction and to testing how the interaction
results in specific physiological changes for the cell.
Human p85 (clone p1-74) specifically interacts with Grb2
in yeast. Three colonies from each combination were tested in the X-gal
filter assay as described in the text. B, blue color colony; W, white
color colony.
We thank Tiffany L. Holcombe for technical assistance
and Dr. David R. Kaplan for helpful discussion during preparation of
the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
and its ubiquitous presence in
species from yeast to human has unveiled a new pathway of
phosphatidylinositol (PI) metabolism and intracellular signaling
(1-3). Mammalian PI 3-kinase exists as a heterodimer of an 85-kDa
(p85) regulatory subunit and a 110-kDa (p110) catalytic
subunit
(4, 5, 6) . The structure of the p85
protein is comprised of an SH3 domain, a break point region (bcr)
homology domain flanked by two proline-rich sequences
(7) , and
two SH2 domains flanking a p110-binding region
(5, 6) .
Initially studied in pp60
immunoprecipitates from transformed cells
(8) , PI 3-kinase
phosphorylates PI at the D-3 position of the inositol ring
(1) .
PI 3-kinase was also found to co-immunoprecipitate with middle T
antigen (9, 10) and a variety of protein-tyrosine kinase receptors,
including PDGF receptor
(11, 12) , colony-stimulating
factor 1 receptor
(13) , and insulin receptor
(14) .
Moreover, two of the products of PI 3-kinase, PI-3,4-P
and
PI-3,4,5-P
, are elevated in cells activated by growth
factors
(15) or transformed by
oncogenes
(16, 17) .
, but the actual mechanism is
unknown
(26, 27) . The recent revelation that Ras
(p21
) interacts directly with the catalytic
subunit of PI 3-kinase (p110 subunit) raises the possibility that PI
3-kinase may serve as an effector of Ras
(28) , but again the
details of the process are yet to be elucidated. Finally, other studies
indicate a link between PI 3-kinase and actin polymerization and
depolymerization in vivo(29, 30) and more
recently between PI 3-kinase and the small G proteins Rac and Rho,
suggesting that the enzyme may also play a role in regulating cell
shape
(31, 32) .
(34) .
We have also confirmed that the interaction between Grb2 and PI
3-kinase also occurs in mammalian cells. Genetic and biochemical
analyses indicate that the SH3 domains of Grb2 interact with the
proline-rich motifs of p85. This interaction does not displace the
catalytic subunit (p110) of PI 3-kinase but is exclusive of Sos.
Antibodies
The mouse anti-p85 antibody and the
mouse anti-Grb2 antibody used for immunoblotting were from Transduction
Labs (Lexington, KY). The rabbit anti-p85 antibody used for
immunoblotting and immunoprecipitation was made against glutathione
S-transferase (GST) fusion protein with full-length
p85.(
)
The rabbit anti-Grb2 antibody for
immunoprecipitation was from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). The rabbit anti-mouse Sos1 was from Upstate Biotechnology
Inc. (Lake Placid, NY). The peroxidase-conjugated goat anti-mouse IgG
(Fc
-specific) was from Jackson ImmunoResearch Labs, Inc. (West
Grove, PA), and the alkaline phosphatase-conjugated goat anti-rabbit
IgG and goat anti-mouse IgG were from Promega (New York, NY). The
rabbit anti-LexA antibody was kindly provided by Russ Finley
(Massachusetts General Hospital, Boston, MA).
Library Screening through Yeast Two-hybrid
System
Manipulations of Escherichiacoli and
yeast were performed essentially as described (35). E. coli K-12 strain KC8
pyrF::Tn5,hsdR,leuB600,trpC9830,lacD74,strA,galK,hisB436 was used for
the rescue of yeast plasmids as described
(33) . EGY48 MATa
trp1,ura3,his3,LEU2::pLexAop6-LEU2 was used as a host for all
interaction experiments
(33) . Human PI 3-kinase p85
cDNA
(36) , a gift from Dr. Lewis Cantley (Harvard Medical
School), was linearized with BamHI, ligated to a
BamHI to EcoRI linker, and then excised by
EcoRI digestion. The EcoRI fragment containing the
complete p85 cDNA was inserted into pEG202 at the EcoRI site,
and the generated plasmid pEG-h85 was used as the bait. The
oligo-primed HeLa cDNA yeast expression library was a generous gift
from Dr. Russ Finley (Massachusetts General Hospital) and was screened
essentially as described
(33) .
X-Gal Filter Assay on Yeast Colonies
Yeast
colonies freshly grown on
HisUra
Trp
plates
(glucose or galactose as energy source) were lifted onto nitrocellulose
membranes and lysed by submerging in liquid nitrogen for 30 s to 1 min.
The membranes were then placed gently on Whatman filter paper saturated
with 3 ml of Z buffer (100 mM sodium phosphate, pH 7.0, 10
mM KCl, 1 mM MgSO
, 40 mM
-mercaptoethanol) containing 1 mg/ml X-gal, and the color of
colonies was recorded through the course of 30 min.
Cell Culture and Preparation of Cell Lysates
NIH
3T3, Balb/c 3T3, and bovine kidney cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10%
calf serum. Cells from a confluent 100-mm culture dish were rinsed once
in phosphate-buffered saline (PBS) and then lysed for 15 min in 1 ml of
Nonidet P-40 lysis buffer (135 mM NaCl, 20 mM HEPES,
5 mM EDTA, 1% Nonidet P-40, 10% glycerol, 500 µM
sodium orthovanadate) containing protease inhibitors (aprotinin (1
µg/ml), pepstatin (1 µg/ml), and leupeptin (0.75 µg/ml)).
Lysates were scraped and cleared at 13,000 g.
Clarified supernatants were used fresh or stored at -80 °C.
PDGF Stimulation and Immunoprecipitation
Cells
1-2 days postconfluence were switched to Dulbecco's
modified Eagle's medium containing 0.2% calf serum for an
overnight incubation. PDGF was added to a final concentration of 30
ng/ml for 15 min. The cells were then washed in PBS, and lysates were
prepared as described above. Lysates were incubated with rocking at 4
°C for 2 h with appropriate antibodies and then for 30 min with
protein A-Sepharose (Pharmacia Biotech Inc.). Immune complexes were
washed four times with PBS, 1% Nonidet P-40, 1 mM EDTA and two
times with TNE (10 mM Tris, pH 8.0, 100 mM NaCl, 1
mM EDTA). They were resuspended in 40 µl of 1
electrophoresis sample buffer, boiled for 5 min, and analyzed by
SDS-PAGE.
Immunoblot Analysis
Samples were resolved by
SDS-PAGE, transferred onto nitrocellulose filters (Schleicher &
Schuell), and incubated with primary antibodies at the concentrations
recommended by the manufacturer for 1-2 h at room temperature.
Immunoblots were subsequently washed and incubated with corresponding
secondary antibodies for 1 h at room temperature, washed three times in
PBS-T (phosphate-buffered saline, 0.2% Tween 20), and washed one time
in attophos buffer (50 mM Tris, pH 9.5, 100 mM NaCl,
0.1 mM MgCl). Filters were then developed for 10
min in attophos buffer containing attophos substrate at 1:20 dilution
and subjected to analysis by Fluorimager (Molecular Dynamics, Inc.,
Sunnyvale, CA). The data were transferred electronically to a Macintosh
computer and printed with a Fujix Pictography 3000 (Fuji Photo Film
USA, Inc., Elmsford, NY).
Preparation of GST Fusion Proteins and Thrombin
Cleavage
A single colony of HB101 transformed with the plasmid
of interest was grown overnight in 50 ml of LB containing 50 µg/ml
ampicillin. Harvesting and purification of the fusion proteins by
affinity to glutathione-Sepharose (Pharmacia) was carried out
essentially as described by Smith and Johnson
(37) .
PI 3-kinase Assay
PI 3-kinase assays were
performed essentially as described
(15) . Briefly,
immunoprecipitates were prepared as described above. Following the
final wash, sonicated lipid substrates were added to the supernatants
at a final concentration of 0.2 mg/ml, and the reaction was initiated
by the addition of 5 mM MgCl and 100
µM [
-
P]ATP at 10
µCi/reaction in 20 mM HEPES (pH 7.2). The reaction was
incubated at room temperature for 5 min and stopped by extraction with
75 µl of 1 M HCl and 180 µl of methanol:chloroform
(1:1). The organic phase was collected and stored at -80 °C
or immediately analyzed by thin layer chromatography (TLC) in an
n-propyl alcohol, 2 M acetic acid solvent system
(65:35).
Protein Kinase Assay
Immunoprecipitates were
prepared as described above. Following the final wash, 45 µl of ATP
mix (20 mM HEPES, 10 mM MnCl, 5
µM ATP, 20 µCi of [
-
P]ATP)
was added to the beads, and the mixtures were incubated at room
temperature for 20 min. The reaction was stopped by adding 40 µl of
sample buffer, boiled for 5 min and analyzed by SDS-PAGE.
Association in Yeast Two-hybrid System
We
screened for p85 interactors from a HeLa cell cDNA library by the
interaction trap technique developed by Zervos et
al.(33) . Fig. 1A shows that the yeast host
cells EGY48, carrying the pEG-h85 bait plasmid, correctly express the
full-length human p85 fused to the LexA-binding domain (lane3). We plated 1.7 10
primary library
transformants onto Leu
galactose selection plates. Of
the colonies that grew, those which gave unambiguous,
galactose-dependent blue color on X-gal filter assay and
galactose-dependent growth on Leu
selection plates
were rescued for sequence analysis. Of twenty clones sequenced, the
majority represent previously uncharacterized proteins. However, one of
these clones, p1-74, was identical to the cDNA sequence of the
adapter protein Grb2. As shown in Fig. 1B, p1-74
interacts specifically with the full-length p85 bait but not with the
control baits of human p110 or the bcr domain of human p85. The same
pattern was observed with the X-gal filter assay (). As
indicated in Fig. 2, p1-74 contains the C-terminal SH3
domain and the majority of the SH2 domain of Grb2.
Figure 1:
Expression and
interaction of human p85 with Grb2 in yeast. A, yeast cells
EGY48 carrying different plasmids were lysed, and 100 µg of total
protein was resolved by SDS-PAGE. Proteins were transferred to a
nitrocellulose membrane and blotted with anti-LexA polyclonal antibody.
Lane1, EGY48 cells alone; lane2,
EGY48 carrying pEG202 and pSH18-34; lane3,
EGY48 carrying pEG-h85 and pSH18-34. B, colonies
containing different combinations of baits and preys were streaked onto
both glucose- and galactose-based
HisUra
Trp
Leu
plates and incubated at 30 °C for 2 days. 1,
3, 5, 7, 9, and 11 represent human p85 bait with a panel of different plasmids as
preys, including pJG4-5, negative control 1 and 2, p1-74,
and positive control 1 and 2, respectively; 2, 4,
6, 8, 10, and 12 represent p110
bait with a panel of preys, including pJG4-5, negative control 1
and 2, p1-74, and positive control 1 and 2, respectively;
13, 14, 15, and 16 represent h85bcr
bait with negative control 1 and 2, p1-74, and positive control
1, respectively.
Figure 2:
Complete nucleotide and peptide sequence
of human Grb2 (34). The SH2 and SH3 domains are underlinedinboldface. The sequence underlinedinlightface represents clone
p1-74.
Direct Association of p85 to Grb2 in Vitro Mediated by
SH3 and Proline-rich Domain
To determine whether p85 and Grb2
can interact directly, we examined complex formation in vitro with bacterially produced p85 and Grb2. We found that soluble p85
released from GST-p85 fusion protein by thrombin cleavage was able to
bind to GST-Grb2 immobilized on glutathione-Sepharose beads but not to
the GST-2T control protein (Fig. 3A). Similarly,
thrombin-cleaved Grb2 forms a complex with GST-p85 immobilized on beads
but not with GST-2T (Fig. 3B).
Figure 3:
Direct association of GST-85 and GST-Grb2
in vitro. A, soluble p85 was prepared by thrombin
cleavage as described under ``Experimental Procedures.''
Approximately 5 µg of soluble p85 was incubated with GST-2T
(lane1) or GST-Grb2 (lane2) beads
for 30 min at 4 °C. The complexes were washed and separated by
SDS-PAGE and immunoblotted with anti-p85 monoclonal antibody.
B, soluble Grb2 was prepared by thrombin cleavage, and
approximately 5 µg of soluble Grb2 was incubated with GST-2T
(lane1) or GST-Grb2 beads for 30 min at 4 °C.
The complexes were washed and analyzed by SDS-PAGE and immunoblotted
with anti-Grb2 antibody.
To examine the
molecular basis for the association, we prepared GST-fusion proteins
that contained the N-terminal SH3, C-terminal SH3, and SH2 domain of
Grb2 and the bcr, bcr with the flanking proline-rich sequences (bcrP),
and SH3 domain of p85. When different fragments of p85 were examined
for their ability to bind soluble Grb2 thrombin-cleaved from GST-Grb2
beads, only the bcrP domain was able to bind Grb2
(Fig. 4A), suggesting that the proline-rich sequences
are involved in the association. We synthesized the two proline-rich
peptides corresponding to the two proline-rich motifs in p85 (amino
acids 83-100 and 313-329, respectively)
(7) . The
effects of these peptides on the binding of the bcrP fragment to Grb2
were investigated (Fig. 4B). At 60 molar excess,
either peptide partially inhibited the binding of bcrP to Grb2. At 400
molar excess, they blocked the binding to near completion. An
additive effect was observed when both peptides were used in
combination.
Figure 4:
Domain analysis for p85 and Grb2
association. A, soluble Grb2 generated by thrombin cleavage
was incubated with full-length (GST-p85, lane1), bcr region (GST-bcr, lane2), bcrP region (GST-bcrP, lane3), and the SH3 domain (GST-SH3, lane4) of p85. The complexes were separated by SDS-PAGE and
immunoblotted with anti-Grb2 antibody. B, soluble Grb2
generated by thrombin cleavage was incubated with GST-bcrP beads alone
(lane1), or bcrP beads plus 400 molar excess
(lanes 2-4) of N-terminal (lane2),
C-terminal (lane3), N- and C-terminal proline-rich
peptides (lane4), or bcrP beads plus 60
molar excess (lanes 5-7) of N-terminal (lane5), C-terminal (lane6), N- and
C-terminal proline-rich peptides (lane7). The
complexes were resolved by SDS-PAGE and immunoblotted with anti-Grb2
antibody. C, soluble p85 was incubated with GST-2T (lane1), GST-Grb2 (lane2), the N-terminal
SH3 domain (lane3), the SH2 domain (lane4), the C-terminal SH3 domain (lane5),
or both N- and C-terminal SH3 domains of Grb2 (lane6). The complexes were resolved by SDS-PAGE and
immunoblotted with anti-p85 monoclonal antibody. D, soluble
p85 generated by thrombin cleavage was incubated with GST-2T (lane1), GST-SH2 of Grb2 (lane2), GST-Grb2
(lane3), or GST-W36K/W193K (K36/193)
(lane4). The complexes were analyzed by SDS-PAGE and
immunoblotted with anti-p85 monoclonal
antibody.
Next, we examined the three domains of Grb2 for their
ability to bind p85. Each domain of Grb2 was expressed as a GST fusion
protein. As shown in Fig. 4, C and D, both N-
and C-terminal SH3 domain but not the SH2 domain of Grb2 independently
bound to p85. A double mutant of Grb2 (W36K/W193K), which contains
point mutations in each SH3 domain, lost its ability to bind p85. Taken
together, this evidence indicates that the association of p85 and Grb2
is mediated by the proline-rich sequences of p85 and the SH3 domains of
Grb2.
Association of p85 and Grb2 in Vivo
To test
whether complex formation between p85 and Grb2 also occurs in mammalian
cells, we examined whether these two proteins co-immunoprecipitate.
Cell lysates were prepared from one to two day postconfluent NIH 3T3
cells and subjected to immunoprecipitation with anti-p85, anti-Grb2,
and control antibody. As shown in Fig. 5A, p85 can be
precipitated with anti-Grb2 but not the control antibody. Similarly,
Grb2 can be precipitated with anti-p85 antibody but not the preimmune
serum (Fig. 5B). Identical results were obtained from
Balb/c 3T3 and Bovine kidney cells (results not shown). These results
provide the first evidence for an in vivo association between
the PI 3-kinase p85 subunit and the adapter protein Grb2 in
unstimulated mammalian cells.
Figure 5:
Association of p85 with Grb2 in quiescent
NIH 3T3 cells. A, immunoprecipitates were prepared from
confluent NIH 3T3 cells using control serum (lane1)
or anti-Grb2 antibody (lane2). The samples were
washed four times with 1% Nonidet P-40, PBS, 1 mM EDTA and two
times with 10 mM Tris, pH 8.0, 100 mM NaCl, 1
mM EDTA and were subjected to SDS-PAGE. Immunoblotting was
performed with anti-p85 antibody. B, immunoprecipitates were
prepared from confluent NIH 3T3 cells using preimmune serum (lane1) or anti-p85 antibody. The samples were separated by
SDS-PAGE and immunoblotted with anti-Grb2 antibody. C,
immunoprecipitates were prepared from fresh lysates of confluent NIH
3T3 cells using anti-p85 antibody (lane1), p85
preimmune serum (lane2), or anti-Grb2 antibody
(lane3). Protein kinase assay was performed as
described under ``Experimental Procedures,'' and the samples
were boiled in the presence of 0.5% SDS and 10 mM
-mercaptoethanol for 5 min. The supernatants were diluted with
lysis buffer and immunoprecipitated with anti-p85
antibody.
Recent findings demonstrated that PI
3-kinase is a dual specificity kinase, phosphorylating both lipids and
protein serine residues
(38, 39) . This enabled us to
confirm the above interaction with a more sensitive method.
Immunoprecipitates using anti-p85 antibody, p85 preimmune serum, and
anti-Grb2 antibody were prepared from fresh NIH 3T3 cell lysates and
were subjected to Mn-dependent protein kinase assay.
The reactions were boiled, diluted, and subjected to an additional
immunoprecipitation with anti-p85 antibody. As shown in
Fig. 5C, both anti-p85 and anti-Grb2 antibodies but not
the control serum precipitated p85 from the lysates.
The Interaction Is Independent of PDGF
Stimulation
Since both Grb2 and p85 can be found in PDGF
receptor immunoprecipitates, it is possible that PDGF receptor might be
involved in bringing the two proteins together. The fact that the
association occurs in quiescent cells suggest a receptor-independent
interaction. Our in vitro data also support a direct binding
mechanism. In an additional experiment to test this hypothesis, the
effect of PDGF stimulation on the association was investigated. As
shown in Fig. 6A, while PDGF stimulation of NIH 3T3
cells significantly increased the level of anti-phosphotyrosine
immunoprecipitated p85, it did not appear to increase the amount of p85
brought down by anti-Grb2 antibody. The amount of Grb2 in
anti-phosphotyrosine immunoprecipitates also increased significantly
after PDGF treatment, whereas it remained essentially constant in
anti-p85 immunoprecipitates (Fig. 6B). Taken together,
these results indicate that PDGF receptor is not involved in mediating
the interaction.
Figure 6:
Association of p85 and Grb2 is independent
of PDGF stimulation. A, confluent NIH 3T3 cells 1-2 days
postconfluence were switched to 10 ml of Dulbecco's modified
Eagle's medium containing 0.2% calf serum overnight. The cells
were then incubated for 15 min in the presence or absence of PDGF at a
final concentration of 30 ng/ml. Immunoprecipitates were prepared from
the lysates using anti-Grb2 antibody (lanes1 and
2) or 4G10 anti-phosphotyrosine antibody (lanes3 and 4). Immunoblotting was performed with anti-p85
antibody. B, confluent NIH 3T3 cells were stimulated with PDGF
as described in panelA. Immunoprecipitates were
prepared from the lysates using anti-p85 antibody (lanes1 and 2) or 4G10 antibody (lanes3 and
4). Immunoblotting was performed with anti-Grb2
antibody.
The Catalytic Subunit of PI 3-kinase Remains Bound to p85
after Its Association with Grb2
The nature of the p85-Grb2
complex was examined further by testing whether p110 was present.
Fig. 7A shows the result of PI 3-kinase assay performed
on anti-Grb2 immunoprecipitates. A significant amount of PI 3-kinase
activity was precipitated by both anti-p85 and anti-Grb2 antibodies
(lanes1 and 3), compared with the preimmune
serum, which had background levels of phosphatidylinositol 3-phosphate
and phosphatidylinositol 3,4,5-trisphosphate (lane2). A variation of the experiment is shown in
Fig. 7B where different GST-fusion proteins were
examined for their ability to bring down PI-3 kinase activity from cell
lysates. Compared with the vector control and the SH2 domain of Grb2,
which had background level of PI 3-kinase activity, the full-length
Grb2 fusion was able to precipitate a significant amount of activity.
The double mutant W36K/W193K was comparable with the vector control.
These results indicate that the catalytic subunit p110 is present in
the same complex with p85 and Grb2.
Figure 7:
The
catalytic subunit p110 of PI 3-kinase co-exists in the complex of p85
and Grb2. A, immunoprecipitates were prepared from confluent
NIH 3T3 cells using anti-p85, preimmune serum, or anti-Grb2 antibody
(lanes1, 2, 3) and washed four
times with 1% Nonidet P-40, PBS, 1 mM EDTA and two times with
10 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA.
The beads were subjected to PI 3-kinase assay as described under
``Experimental Procedures'' and analyzed on a TLC plate.
B, GST-fusion constructs, each containing 10 µg of
protein, were incubated with fresh lysates prepared from confluent NIH
3T3 cells for 1 h at 4 °C. The beads were washed as in panelA and subjected to PI 3-kinase analysis. Lane1, GST-2T; lane2, GST-Grb-SH2;
lane3, GST-W36K/W193K (K36/193); lane4, GST-Grb2. PIP and PIP,
phosphatidylinositol 3-phosphate and phosphatidylinositol
3,4,5-trisphosphate, respectively.
p85 and Sos Do Not Co-exist in a Single Complex with
Grb2
We also attempted to test whether Sos and p85 could
co-exist in the complex with Grb2. We were unable to detect Sos in
anti-p85 immunoprecipitates. We next performed experiments using GST
fusion proteins. Fig. 8shows that only GST-Grb2 (lanes1, 2, and 3), but not GST-bcrP
(lanes4, 5, and 6) or the vector
control (lanes7, 8, and 9), binds
Sos from NIH 3T3 cells. Furthermore, thrombin-cleaved soluble bcrP was
not able to displace Sos from binding Grb2 (lane1).
Similarly, while soluble Grb2 was brought down by GST-bcrP (lane4), there was no detectable Sos in the precipitates,
indicating that Sos could not displace bcrP from binding Grb2.
Figure 8:
p85 and Sos do not co-exist in a single
complex with Grb2. Lysates prepared from confluent NIH 3T3 cells were
incubated for 1 h at 4 °C with different combinations of GST fusion
proteins on beads or in thrombin-released soluble form. GST-Grb2 beads
were mixed with soluble bcrP (lane1), 2T control
(lane2), or by themselves (lane3); GST-bcrP beads were mixed with soluble Grb2 (lane4), 2T control (lane5), or by
themselves (lane6); GST-2T beads were mixed with
soluble bcrP (lane7), Grb2 (lane8), or by themselves (lane9). The
complexes were resolved on SDS-PAGE and immunoblotted with anti-Sos
antibody (upperpanel) and stripped and reblotted
with anti-Grb2 antibody (lowerpanel).
Table:
Human p85 specifically interacts with Grb2 in
yeast
-D-galactoside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.