From the
Monocyte colony-stimulating factor (M-CSF) is required for the
proliferation of mononuclear phagocytes. The activated M-CSF receptor
associates with phosphatidylinositol 3-kinase (PI 3-kinase). In the
present studies, we demonstrate that M-CSF also induces direct
interaction of PI 3-kinase (p85
The M-CSF
In the present studies, we
demonstrate that M-CSF induces direct association of the p85
Studies in Rat-2 fibroblasts which express the mouse
c- fms gene have demonstrated that M-CSF stimulation is
associated with binding of PI 3-kinase to the activated M-CSF receptor
(4) . In order to confirm these findings in a physiological
system, we stimulated human peripheral blood monocytes with M-CSF and
assayed anti-M-CSF receptor immunoprecipitates for the p85
The SH3 domains of Grb2
bind to Sos
(19, 21, 22, 23, 24, 25) .
This interaction of Sos with Grb2 translocates Sos to the plasma
membrane where it increases the exchange of GDP for GTP on
membrane-bound Ras
(22, 24) . Since p85
subunit) with the SH2/SH3 adaptor
protein Grb2. Tyrosine-phosphorylated PI 3-kinase interacts with the
SH2 domain of Grb2. A pYRNE (pY408) site in PI 3-kinase is potentially
involved in this interaction. The results also demonstrate that the PI
3-kinase
Grb2 complex associates with the guanine nucleotide
exchange protein Sos. Since Sos binds to the SH3 domains of Grb2 and
thereby associates with Ras at the cell membrane, formation of the PI
3-kinase
Grb2
Sos complex provides a potential mechanism for
growth factor-induced interactions of PI 3-kinase and Ras.
(
)
receptor is a transmembrane
protein tyrosine kinase encoded by the c- fms gene
(1, 2) . M-CSF stimulates receptor dimerization and
autophosphorylation at Tyr
, Tyr
, and
Tyr
in the kinase domain
(3, 4) . Studies
have demonstrated that Tyr
serves as a binding site for
PI 3-kinase
(4) , whereas the adaptor protein Grb2 associates
with phosphotyrosine 697
(5) . PI 3-kinase is a heterodimer
consisting of an 85-kDa SH2 domain-containing subunit which links the
catalytic 110-kDa subunit to tyrosine-phosphorylated proteins. PI
3-kinase is responsible for growth factor-induced phosphorylation of
phosphoinositides at the 3` position
(6) . One such
phosphorylated product, phosphatidylinositol 4,5-triphosphate, has been
implicated in the regulation of PKC
(7) . Stimulation of
PI 3-kinase activity has been associated with binding of the 85-kDa
subunit to phosphotyrosine
(8, 9) . While tyrosine
phosphorylation of PI 3-kinase may also be associated with increases in
activity, recent studies have demonstrated that PI 3-kinase is directly
activated by Ras
(10) . The mechanisms, however, responsible for
growth factor-induced PI 3-kinase activity and the basis for the
interaction with Ras are unclear.
subunit of PI 3-kinase with the SH2 domain of Grb2. Our finding that
M-CSF also induces the formation of a PI 3-kinase
Grb2
Sos
complex supports a potential role for PI 3-kinase in Ras signaling
pathways in monocytes.
Monocyte Isolation and Culture
Human monocytes
were isolated from the peripheral blood of healthy volunteers by
Ficoll-Paque separation, followed by adherence for 1 h and removal of
the nonadherent cells
(11, 12) . The monocytes were
treated with 1000 units/ml human recombinant M-CSF (specific activity,
1.90 10
units/ml; Genetics Institute, Cambridge,
MA).
Antibodies and Fusion Proteins
Anti-p85 and
anti-Sos antibodies were purchased from Santa Cruz Biotechnology (San
Diego, CA). Anti-Grb2 and anti-M-CSF receptor antibodies were obtained
from Transduction Laboratory (Lexington, KY) and Upstate Biotechnology,
Inc. (Lake Placid, NY), respectively. GST-Grb2 (full length), GST-Grb2
SH2, GST-Grb2 N-SH3, and GST-Grb2 C-SH3 fusion proteins were purchased
from Santa Cruz Biotechnology.
Immunoprecipitations and Immunoblotting
Cell
lysates were prepared by resuspending cells for 30 min on ice in lysis
buffer (50 mM Tris, pH 7.6, 1% Brij-96, 150 mM NaCl,
1 mM phenylmethylsulfonyl fluoride, 1 mM sodium
vanadate, 1 mM DTT, 10 mM sodium fluoride, and 10
µg/ml each of leupeptin and aprotinin). Equal amounts of proteins
(250-300 µg) were immunoprecipitated by incubation with
anti-M-CSF-receptor, anti-p85, anti-Grb2, or anti-Sos for 2 h at 4
°C and then with protein A-Sepharose (Pharmacia Biotech Inc.) for
an additional 30 min. The resulting precipitates were washed four times
with lysis buffer and resolved by SDS-PAGE under reducing conditions.
Proteins were then transferred to nitrocellulose by semi-dry transfer
(Bio-Rad), blocked by incubation in 5% dry milk in PBST (0.5% Tween 20
in PBS), and then probed with appropriate antibodies. The blots were
developed by ECL (Amersham Corp.).
Fusion Protein Binding Assays
The fusion proteins
GST, GST-Grb2, GST-Grb2 SH2, GST-Grb2 N-SH3, or GST-Grb2 C-SH3 were
purified by affinity chromatography using glutathione-Sepharose beads
and equilibrated in lysis buffer. Cell lysates were incubated with 2
µg of immobilized GST, GST-Grb2, GST-Grb2 SH2, GST-Grb2 N-SH3, or
GST-Grb2 C-SH3 fusion proteins for 2 h at 4 °C. The resulting
protein complexes were washed three times with lysis buffer containing
0.1% detergent and boiled for 5 min in SDS sample buffer. The complexes
were then separated by 7.5% SDS-PAGE and subjected to silver staining
or immunoblot analysis with anti-p85.
Peptide Synthesis and Competition Assays
Peptide
was synthesized using Fmoc
( N-(9-fluorenyl)methoxycarbonyl)-Tyr(POMe
)-OH
for incorporation of phosphotyrosine and subsequently purified by ether
precipitation and preparative reverse-phase high-pressure liquid
chromatography (HPLC). Amino acid analysis was used to confirm the
sequence of the p85
-derived phosphopeptide: LINHpYRNESLAQ.
GST-Grb2 SH2 protein was incubated in the presence or absence of 50
µM tyrosine-phosphorylated synthetic peptide for 1 h at 4
°C. The fusion proteins were incubated with lysates from
M-CSF-treated monocytes and the adsorbates then analyzed by
immunoblotting with anti-p85
.
Second Immunoprecipitation Assays
M-CSF-treated
cell lysates were immunoprecipitated with anti-p85 for 2 h at 4
°C. Immune complexes were released by boiling in 50 mM
Tris-HCl, pH 8.0, 0.5% SDS, and 1 mM DTT and then subjected to
1) a second immunoprecipitation with anti-p85
or 2) precipitations
with GST or GST-Grb2. The resulting protein precipitates were analyzed
by immunoblotting with anti-p85
.
PI 3-Kinase Assays
Assays for PI 3-kinase were
performed as described
(13) . Briefly, PI 3-kinase activity was
measured directly in immune complexes containing 2 µg/ml
phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL), 20
mM HEPES, pH 7.2, 0.5 mM EGTA, 0.5 mM sodium
phosphate, 10 mM MgCl, and
[
-
P]ATP. The reaction was stopped by
addition of 4 N HCl and chloroform/methanol (1:1). The organic
layer was separated and spotted on a Silica Gel-60 plate (Sigma) and
analyzed by thin layer chromatography.
subunit
of PI 3-kinase. While there was no detectable anti-p85
reactivity
in the immunoprecipitates from unstimulated monocytes, M-CSF treatment
rapidly induced binding of p85
to M-CSF receptors
(Fig. 1 A). Reprobing the same filter with anti-Tyr(P)
demonstrated a M-CSF-dependent increase in reactivity with an 85-kDa
protein (Fig. 1 B). These results supported binding of PI
3-kinase to the activated M-CSF receptor and tyrosine phosphorylation
of the p85
subunit.
Figure 1:
Association of PI 3-kinase with M-CSF
receptors. Human peripheral blood monocytes were stimulated with M-CSF
(1000 units/ml) for 5 min at 37 °C. Lysates from control and
M-CSF-treated monocytes were immunoprecipitated with anti-M-CSF
receptor antibody. The proteins were resolved by 7.5% SDS-PAGE and
immunoblotted with anti-p85 ( A) or anti-Tyr(P)
( B). One of the three independent experiments is
shown.
Other studies in Rat-2 fibroblasts have
shown that mutation of Tyr in the M-CSF receptor blocks
binding of PI 3-kinase and inhibits M-CSF-dependent growth
(4, 5) . While these results support the involvement of
PI 3-kinase in growth factor-induced proliferation, recent work has
also demonstrated that PI 3-kinase coimmunoprecipitates with Ras
(14, 15) and that PI 3-kinase is regulated by Ras
(10) . These findings suggest that localization of PI 3-kinase
at the cell membrane, perhaps through SH2 or SH3 interactions, is
necessary for direct activation by Ras. In addressing this issue, we
asked whether the SH2-SH3 adaptor protein Grb2, which binds to the
guanine nucleotide exchange protein Sos (son of sevenless) and thereby
to the Ras activation pathway
(16, 17, 18) ,
interacts with PI 3-kinase. Lysates from control and M-CSF-treated
monocytes were subjected to immunoprecipitation with anti-Grb2.
Immunoblotting of the precipitates with anti-p85
revealed
increased reactivity following M-CSF stimulation
(Fig. 2 A). In the reciprocal experiment, analysis of
anti-p85
immunoprecipitates with anti-Grb2 confirmed an
M-CSF-dependent association between these two proteins
(Fig. 2 B).
Figure 2:
Association of PI 3-kinase with Grb2.
A, monocytes were stimulated with M-CSF for 5 min. Lysates
were subjected to immunoprecipitation with anti-Grb2 antibody. The
immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and analyzed
by immunoblotting with anti-p85 antibody. B,
anti-p85
immunoprecipitates from control and M-CSF-treated cell
lysates were analyzed by 10% SDS-PAGE and immunoblotting with anti-Grb2
antibody. One representative experiment out of four is
shown.
In order to determine whether the
interaction between Grb2 and p85 is direct, we prepared
anti-p85
immunoprecipitates from M-CSF-treated monocytes. The
precipitates were subjected to SDS-PAGE, transferred to nitrocellulose,
and then incubated with GST-Grb2 (full length). After washing the
filters thoroughly, we assayed Grb2 binding by immunoblotting with
anti-Grb2. The finding that anti-Grb2 reactivity is detectable at a
molecular mass of 85 kDa supports a direct interaction of Grb2 with the
p85
subunit of PI 3-kinase (Fig. 3 A). In order to
confirm these findings, we prepared anti-p85
immunoprecipitates
from M-CSF-treated monocytes and then released the proteins by boiling
the immune complexes in 0.5% SDS and 1 mM DTT. After diluting
SDS to 0.1% by lysis buffer, secondary protein precipitations were
performed using GST or GST-Grb2. Analysis of the second precipitates by
immunoblotting with anti-p85
confirmed direct interaction of Grb2
with p85
(Fig. 3 B). Secondary anti-p85
immunoprecipitates were used as a positive control in this experiment
(Fig. 3 B).
Figure 3:
Direct
binding of PI 3-kinase and Grb2. A, lysates from
M-CSF-stimulated monocytes were subjected to immunoprecipitation with
anti-p85. The proteins were separated by 7.5% SDS-PAGE and
transferred to nitrocellulose. The filters were then incubated with 50
µg/ml GST-Grb2 (full length) or GST at 4 °C for 2 h. After
washing thoroughly with PBST, immunoblotting was performed by anti-Grb2
antibody. B, lysates from M-CSF-treated monocytes were
immunoprecipitated by anti-p85
. Proteins were released from the
precipitates by boiling the immune complexes in 0.5% SDS, 1 mM
DTT buffer. Secondary protein precipitations were performed by
incubating either with GST, GST Grb2, or anti-p85
and the
resulting protein complexes were analyzed by immunoblotting with
p85
. Two independent experiments showed similar results.
C, lysates from control ( lanes 2, 4, 6, and
8) and M-CSF-treated ( lanes 1, 3, 5, 7, and
9) monocytes were incubated with GST, GST-Grb2 (full length),
GST-Grb2 N-SH3, GST-Grb2 SH2, and GST-Grb2 C-SH3 proteins immobilized
on glutathione-Sepharose. The bound proteins were resolved by 7.5%
SDS-PAGE and immunoblotted with anti-p85
antibody. D,
GST-Grb2 SH2 fusion protein was preincubated (1 h, 4 °C) in the
absence (-) or presence (+) of 50 µM
tyrosine-phosphorylated synthetic peptide (LINHpYRNESLAQ). The fusion
protein was then incubated with lysate from M-CSF-treated monocytes and
the adsorbate analyzed by immunoblotting with
anti-p85
.
This interaction was further analyzed
using GST fusion proteins prepared from full-length Grb2, the SH3
(carboxyl and amino-terminal) and SH2 domains of Grb2. Adsorbates
obtained with GST-Grb2 (full length) revealed increased binding of
p85 when using lysates from M-CSF-stimulated, as compared with
control, monocytes (Fig. 3 C). A low level of p85
binding to the Grb2 SH3 domains was obtained when using lysates from
both control and M-CSF-treated cells (Fig. 3 C). In
contrast, adsorbates obtained with GST-Grb2 SH2 demonstrated a
M-CSF-dependent increase in binding of p85
(Fig. 3 C). The SH2 domain of Grb2 interacts with
proteins that contain the pYXNX motif
(18, 19, 20) . To define the site in PI 3-kinase
responsible for the association with the Grb2 SH2 domain, we identified
a potential candidate sequence at Tyr
which is followed
by RNE. A chemically phosphorylated synthetic peptide corresponding to
this site (amino acids 404-415) was used in competition assays.
Preincubation of GST-Grb2 SH2 with the peptide inhibited binding of PI
3-kinase from lysates of M-CSF-treated monocytes
(Fig. 3 D). These findings indicate that PI 3-kinase
interacts directly with the SH2 domain of Grb2 and that the pYRNE
(Y408) in p85
may be the binding site.
binds to
the SH2 domain of Grb2, and PI 3-kinase is khown to be a direct target
of Ras
(10) , we asked whether PI 3-kinase associates with Sos.
Immunoblot analysis of p85
immunoprecipitates with anti-Sos
demonstrated an increased association of p85
and Sos in
M-CSF-treated, as compared with control, monocytes
(Fig. 4 A). Moreover, analysis of anti-Sos
immunoprecipitates with anti-p85
demonstrated a M-CSF-dependent
increase in the association of these proteins (Fig. 4 B).
These findings and the demonstration that PI 3-kinase and Sos bind to
the SH2 and SH3 domains, respectively, of Grb2 support the formation of
a PI 3-kinase
Grb2
Sos complex. Since Ras regulates PI
3-kinase
(10) , we asked whether PI 3-kinase exhibits an
increase in activity when associated with the Grb2
Sos complex.
The results demonstrate that anti-Grb2 immunoprecipitates exhibit
M-CSF-dependent increases (approximately 20-fold) in activity of PI
3-kinase (Fig. 4 C). Similar findings were obtained with
the anti-Sos immunoprecipitates (Fig. 4 D). These results
demonstrate that activation of PI 3-kinase in the anti-Grb2 or anti-Sos
immunoprecipitates occurs in response to M-CSF (Fig. 4, C and D).
Figure 4:
M-CSF-dependent activation of PI 3-kinase
in anti-Grb2 and anti-Sos immunoprecipitates. A, lysates from
control and M-CSF-treated monocytes were subjected to
immunoprecipitation with anti-p85. Lysates from M-CSF-treated
monocytes were also subjected to immunoprecipitation with anti-Grb2.
The immunoprecipitates were analyzed by immunoblotting with anti-Sos.
B, anti-Sos immunoprecipitates from control and M-CSF-treated
cell lysates were subjected to immunoblotting with anti-p85
.
C, lysates from control and M-CSF-treated monocytes were
immunoprecipitated with anti-Grb2. M-CSF-treated cell lysates were also
subjected to immunoprecipitation with anti-p85
. The immune
complexes were extensively washed and assayed for precipitable PI
3-kinase activity by the addition of PI and
[
-
P]ATP. The lipids were extracted with
chloroform, separated by TLC, and analyzed by autoradiography. The
origin and position of phosphatidylinositol phosphate ( PIP)
are indicated. D, lysates from control and M-CSF-treated
monocytes were immunoprecipitated with anti-Sos antibody. M-CSF-treated
cell lysates were also subjected to immunoprecipitation with
anti-p85
and normal rabbit serum ( NRS). The immune
complexes were assayed for precipitable PI 3-kinase activity as
described above. One of the three independent experiments is
shown.
PI 3-kinase directly associates with and is
stimulated by activated receptor PTKs
(26) . The interaction of
p85 with receptor phosphotyrosines may induce structural
alterations that result in activation of the p110 catalytic subunit
(8, 27) . Tyrosine phosphorylation of p85
may also
be responsible for increases in activity of PI 3-kinase
(28, 29) , whereas other work has implicated Ras as a
regulator of the intrinsic phosphoinositide kinase activity
(10) . The present findings demonstrate that M-CSF induces
tyrosine phosphorylation of p85
and that a pYXNX-like motif
potentially contributes to binding of p85
to the SH2 domain of
Grb2. While the tyrosine kinases involved in phosphorylation of PI
3-kinase could be of either the receptor or nonreceptor types, this
event would be necessary for the formation of a complex with Grb2. The
demonstration that PI 3-kinase binds to a pool of Grb2 associated with
Sos also provides a potential mechanism for PI 3-kinase to complex with
Ras through Sos/Ras interactions. Other studies have demonstrated that
the insulin receptor substrate 1 interacts with both PI 3-kinase
p85
and the SH2 domain of Grb2
(25, 30) . Insulin
receptor substrate 1 or related proteins, such as 4PS
(31) , may
therefore also associate with a PI 3-kinase
Grb2 complex by
binding to p85
. In any event, the present results provide the
first evidence for binding of PI 3-kinase to an adaptor protein and
support a mechanism for growth factor-induced regulation of PI 3-kinase
by Ras. Since other studies have suggested that PI 3-kinase can also
function upstream to Ras
(32) , the formation of a PI
3-kinase
Grb2
Sos complex could similarly contribute to the
regulation of Ras.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.