(Received for publication, May 30, 1995; and in revised form, August 11, 1995)
From the
Binding of human granulocyte/macrophage colony-stimulating
factor (hGM-CSF) to its receptor induces the rapid activation of
phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR)
does not contain a consensus sequence for binding of PI 3-kinase, hGMR
must use a distinct mechanism for its association with and activation
of PI 3-kinase. Here, we describe the identification of a
tyrosine-phosphorylated protein of 76-85 kDa (p80) that
associates with the common subunit of hGMR and with the SH2
domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells.
Src/Yes and Lyn were tightly associated with the p80
PI 3-kinase
complex, suggesting that p80 and other phosphotyrosyl proteins present
in the complex were phosphorylated by Src family kinases. Tyrosine
phosphorylation of p80 was only detected in hGM-CSF or human
interleukin-3-stimulated cells, suggesting that activation of p80 might
be specific for signaling via the common
subunit. We postulate
that p80 functions as an adapter protein that may participate in
linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.
The high affinity receptor for human GM-CSF (hGM-CSF) ()is a heterodimer consisting of two subunits, termed
and
, which are transmembrane proteins of 75-85 and
120-135 kDa, respectively(1, 2, 3) .
The
subunit is specific for the hGM-CSF receptor (hGMR) and binds
hGM-CSF with low affinity(1) . The
subunit, which is
shared by the human receptors for GM-CSF, interleukin (IL)-3 and IL-5,
cannot bind hGM-CSF by itself but is required for high affinity binding
of hGM-CSF(3, 4, 5) . Expression of the
normal hGMR
and
subunits in established murine cells can
generate a potent oncogenic signal in the presence of
hGM-CSF(6) .
The subunit of hGMR is important for
signal transduction(7, 8) . Although the
subunit
itself does not contain consensus sequences characteristic of protein
kinases or protein phosphatases, hGM-CSF induces rapid phosphorylation
of cellular proteins on tyrosine residues (9, 10, 11, 12) , indicating a
functional association with cytoplasmic protein-tyrosine kinases.
Candidates for these non-receptor tyrosine kinases are
p130
(13, 14) ,
p92
(15) ,
p53/p56
(16) , and
p62
(17) .
Activation of phosphatidylinositol-3 kinase (PI 3-kinase) is one of the immediate cellular responses to stimulation by growth factors and cytokines, including hGM-CSF (17-2O). PI 3-kinase is a heterodimer consisting of two subunits: an 85-kDa protein containing SH2 and SH3 domains (p85) and a 110-kDa catalytic subunit (p110)(21) . p85 functions as an adapter molecule that targets p110 to activated growth factor receptors(19, 20, 22, 23, 24) . In most cases, this is mediated by binding of the SH2 domains of p85 to the pYXXM consensus motif in activated tyrosine kinase receptors (25) like the platelet-derived growth factor receptor(26) . However, the hGMR does not have this consensus sequence for PI 3-kinase binding nor the recognition motif pYVXV that has been recently described for binding of PI 3-kinase to the hepatocyte growth factor/scatter factor receptor(27) .
A variation of the direct association of PI 3-kinase with receptor molecules is its indirect binding to activated receptors through a tyrosine-phosphorylated receptor substrate like the insulin receptor substrate 1 (IRS-1). After insulin receptor stimulation and tyrosine phosphorylation of IRS-1, the p85 subunit of PI 3-kinase associates through its SH2 domains with tyrosine phosphorylation sites within pYMXM motifs on IRS-1(28) . Other proteins with SH2 domains like Grb2(29) , Nck(30) , and SH-PTP-2 (31) also associate with IRS-1. Therefore, the role of IRS-1 is to function as a multiside docking protein to link the upstream insulin receptor to several downstream adapter molecules of different signaling pathways, including the PI 3-kinase. Another PI 3-kinase adapter protein is the IL-4 receptor substrate 4PS, which links the activated IL-4 receptor to PI 3-kinase (32) .
Here, we describe the identification of a
new PI 3-kinase adapter protein of 80 kDa (p80), which is the major
tyrosine-phosphorylated substrate in hGM-CSF-stimulated cells. p80
coprecipitates with the common subunit and binds directly to the
p85 subunit of PI 3-kinase. Our data suggest that p80 is a new adapter
protein, which may participate in linking the activated hGM-CSF
receptor to the PI 3-kinase pathway.
Cell lysates were immunoprecipitated with
the indicated antibodies, and the immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel
electrophoresis and Western blot analysis were carried out as described
previously(39) . Filters were developed using the ECL system
according to the manufacturer (Amersham). For reprobing, bound
antibodies were removed from the nitrocellulose membrane by incubation
in stripping buffer (62.5 mM Tris, pH 6.7, 2% SDS, and 100
mM -mercaptoethanol) for 30 min at 50 °C. Stripped
filters were washed five times for 10 min in TTBS before blocking and
incubation with other antibodies. The same filter was used up to four
times.
Figure 1: A protein of 76-85 kDa (p80) is the major tyrosine-phosphorylated protein in hGM-CSF-stimulated cells. A, starved TF-1 cells were stimulated at 37 °C for the indicated times with 25 ng/ml hGM-CSF. After incubation, cells were extracted in CHAPS lysis buffer, and total cell lysates containing 50 µg of protein were analyzed by immunoblotting using anti-phosphotyrosine antibodies. B, TF-1 cells were stimulated for 5 min with the indicated concentrations (Conc.) of hGM-CSF, and total cell lysates were analyzed by anti-phosphotyrosine immunoblotting. Small arrows indicate the positions of phosphotyrosyl proteins induced by hGM-CSF. The sizes of the molecular mass markers are given in kDa. C, total cell lysates of unstimulated (lane 1), hGM-CSF-stimulated (lane 2), or hIL-3-stimulated (lane 3) TF-1 cells were analyzed for tyrosine-phosphorylated proteins.
Figure 2:
Association of p80 with the common
subunit of hGMR. After starvation for 18 h, TF-1 cells were stimulated
with hGM-CSF for 2 min and lysed in CHAPS buffer. Anti-
subunit
immunoprecipitates (lanes 1 and 2) or total cell
lysates (lanes 3 and 4) from unstimulated (lanes
1 and 3) or hGM-CSF-stimulated (lanes 2 and 4) TF-1 cells were analyzed by anti-phosphotyrosine
immunoblotting. The location of the
subunit of hGMR (
-GMR),
p80, and immunoglobulin heavy chain (Ig) is
indicated.
Figure 3: p80 interacts with the p85 subunit of PI 3-kinase through the N- and C-terminal SH2 domains of p85. A, starved TF-1 cells were stimulated for 2 min with hGM-CSF, and cells were lysed in Nonidet P-40 buffer. Total cell lysates from unstimulated (lanes 1, 3, 5, and 7) or hGM-CSF-stimulated (lanes 2, 4, 6, 8, and 9) cells were analyzed by anti-phosphotyrosine immunoblotting either directly (lanes 1 and 2), after immuno-precipitation with anti-p85 antibodies (lanes 3 and 4), after adsorption with glutathione S-transferase fusion proteins containing either the C-terminal SH2 domain (lanes 5 and 6) or the N-terminal SH2 domain of p85 (lanes 7 and 8), or after adsorption with vector glutathione S-transferase protein (lane 9). The sizes of the identified proteins are indicated on the right. Molecular weight markers are indicated on the left. B, anti-p85 immunoprecipitates from unstimulated or hGM-CSF-stimulated TF-1 cells were first analyzed by anti-phosphotyrosine immunoblotting (lanes 1 and 2) (also shown in Fig. 3A, lanes 3 and 4), and the filters were then stripped and reprobed with anti-p85 antibodies (lanes 3 and 4). The position of p80 and p85 is indicated on the right. C, starved TF-1 cells were lysed in CHAPS buffer after stimulation for the indicated times, and anti-p85 immunoprecipitates were analyzed by anti-phosphotyrosine immunoblotting (lanes 1-8). D, total cellular lysates (TCL) or anti-p85 immunoprecipitates (IP:p85) of unstimulated (lane 1) or hGM-CSF-stimulated (lane 2) TF-1 cells, unstimulated (lanes 3 and 5) or CSF-1-stimulated (lanes 4 and 6) BAC1.2F5 cells, and unstimulated (lanes 7 and 9) or EGF-stimulated (lanes 8 and 10) pCO12-EGFR cells were analyzed by anti-phosphotyrosine immunoblotting (lanes 1-10).
To characterize the interaction between p80 and p85, we examined whether this association was mediated through phosphotyrosine-SH2 interactions. To examine this question, the C-terminal and N-terminal SH2 domains of p85 were expressed as glutathione S-transferase fusion proteins in bacteria. Immobilized C-terminal (SH2-C) and N-terminal (SH2-N) SH2 domains of the p85 subunit bound phosphorylated p80, and this association was dependent on stimulation with hGM-CSF (Fig. 3A, lanes 5-8). The C-terminal SH2 domain had higher affinity for p80 than the N-terminal SH2 domain (Fig. 3A, lanes 6 and 8). In addition to p80, two other phosphotyrosyl proteins with apparent molecular masses of 110 and 130 kDa were bound to the C- and N-terminal SH2 domains, respectively (Fig. 3A, lanes 6 and 8, respectively). None of these proteins were recognized by a glutathione S-transferase vector protein (Fig. 3A, lane 9). The association of p85 with p80 followed the same time course as the tyrosine phosphorylation of p80 (Fig. 3C and 1A), suggesting that p80 associated with p85/PI 3-kinase primarily through phosphotyrosine-SH2 interactions. However, at the present time we cannot rule out the possibility that p80 was already associated with p85 before hGM-CSF stimulation through other interactions not involving phosphotyrosine. This will be clarified when antibodies to p80 become available.
Stimulation of cells with other growth factors that also stimulate PI 3-kinase activity, namely CSF-1 and EGF did not induce tyrosine phosphorylation of p80 or its association with the p85 subunit of PI 3-kinase (Fig. 3D). Instead, p85 coprecipitated with an unknown tyrosine-phosphorylated protein of 110 kDa in CSF-1-stimulated BAC1.2F5 cells (Fig. 3D, lane 6), and, as previously shown(20) , p85 coprecipitated with activated EGF receptor in EGF-stimulated pCO12-EGFR cells (Fig. 3D, lane 10).
We conclude that after hGM-CSF stimulation, p80 becomes rapidly phosphorylated on tyrosine and associates with the p85 subunit of PI 3-kinase by binding preferentially to the C-terminal SH2 domain of p85. Thus, p80 represents a novel signaling molecule that is capable of interacting with the p85 adapter protein in cells stimulated with hGM-CSF.
Figure 4: Association of PI 3-kinase activity with tyrosine-phosphorylated proteins in hGM-CSF stimulated cells. Starved TF-1 cells were incubated in the absence(-) (lanes 1 and 3) or presence (+) (lanes 2 and 4) of 50 ng/ml hGM-CSF for 2 min at 37 °C. Cells were lysed in Nonidet P-40 buffer, and cell lysates containing equal amounts of protein were immunoprecipitated with anti-phosphotyrosine (lanes 1 and 2) or anti-p85 antibodies (lanes 3 and 4). Immunoprecipitates were then subjected to an in vitro PI 3-kinase assay, and the final products were resolved by thin layer chromatography. The position of phosphatidylinositol 3-phosphate (PI-3P) is indicated. Exposure times were 4 h at -70 °C (lanes 1 and 2) or 30 min at room temperature (lanes 3 and 4).
In preliminary experiments, we found
that p60, p62
,
and p53/p56
were expressed in TF-1 cells (data not
shown). We then used an anti-Src antibody (SRC2) raised against
residues 509-533 in the C-terminal region of human
p60
, which cross-reacts with
p62
and p59
, for
immunoprecipitation and Western blot analysis. Immunoprecipitation of
TF-1 lysates with anti-SRC2 antibodies resulted in precipitation of two
proteins of 60 and 62 kDa (Fig. 5A), which were
identified in separate gels as p60
and
p62
, respectively (data not shown). To
analyze whether p80 and other tyrosine-phosphorylated proteins
coprecipitated with p60
and/or
p62
, we reprobed the same filter with
anti-phosphotyrosine antibodies. This analysis showed that after
hGM-CSF stimulation, phosphotyrosyl proteins of 51, 56, 68,
76-85, 140, and 160 kDa were present in the anti-SRC2
immunoprecipitates (Fig. 5B). The broad band of
76-85 kDa in anti-SRC2 precipitates from hGM-CSF-stimulated cells (Fig. 5B) had the same electrophoretic mobility as p80,
suggesting that p80 may be a substrate of p60
and/or p62
. The 51-, 56-, and 68-kDa
proteins (Fig. 5B) had the same electrophoretic
mobility as the three isoforms of the adapter protein Shc,
p46
, p52
, and
p66
(41) . Stripping and reprobing of the filter
with anti-Shc antibodies confirmed that p52
(Fig. 5C) and p66
(observed after
long exposure) were present in anti-SRC2 immunoprecipitates from
hGM-CSF-stimulated cells. Whether p46
is also in the
anti-SRC2 precipitate could not be ascertained because the IgG band
interfered with detection of p46
.
Figure 5: Src family members form a complex with p80 and p85/PI 3-kinase in hGM-CSF-stimulated cells. A, starved TF-1 cells were either not stimulated(-) or stimulated with hGM-CSF (+) for 2 min and lysed in Nonidet P-40 buffer; cell lysates were then immunoprecipitated with anti-Src antibody SRC2. Immunocomplexes were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with SRC2 antibodies. Proteins detected are indicated on the right. Molecular weight markers are indicated on the left. B, the filter shown in A was stripped and reprobed with anti-phosphotyrosine antibodies (P.Tyr). C, the filter shown in B was stripped and reprobed with anti-Shc antibodies (Shc). D, the filter shown in C was stripped and reprobed with antibodies directed against the p85 subunit of PI 3-kinase (p85/PI3K).
Since p80 associates
with the p85 subunit of PI 3-kinase (Fig. 3), the filter shown
in Fig. 5, A-C, was reprobed with anti-p85
antibodies to determine if p85 was also present in this complex. As
shown in Fig. 5D, similar amounts of p85 were present
in anti-SRC2 precipitates from unstimulated and stimulated TF-1
lysates. Thus, one or more Src kinases were associated with p85 before
hGM-CSF stimulation. Recent reports that Src family kinases can
associate with p85 in a phosphotyrosine-independent manner (42, 43) are consistent with the idea that
p60 and/or p62
may be already complexed with p85 before hGM-CSF stimulation.
p80 also associated with p53/p56 in a
hGM-CSF-dependent manner. As shown in Fig. 6, the time course of
p80 association with Lyn was similar to the time course of tyrosine
phosphorylation of p80 (Fig. 1A).
Figure 6:
Association of Lyn with
tyrosine-phosphorylated p80. Starved TF-1 cells were either not
stimulated (lane 1) or stimulated with 25 ng/ml hGM-CSF (lanes 2-8) for various times (lane 2, 0.5 min; lane 3, 2 min; lane 4, 5 min; lane 5, 10
min; lane 6, 30 min; lane 7, 60 min; lane 8,
120 min). Nonidet P-40 lysates were immunoprecipitated (IP)
with anti-Lyn antibodies and analyzed by anti-phosphotyrosine
immunoblotting (P.Tyr). The positions of p80,
p56, p53
, and
immunoglobulin heavy chain are indicated on the left.
Molecular weight markers are indicated on the right.
Since
p53/p56 are not recognized by the anti-SRC2 antibody,
these data indicate that at least two members of the Src family, i.e. p53/p56
and p60
and/or p62
, associate with p80.
Therefore, these kinases are very strong candidates for phosphorylation
of p80 and other phosphotyrosyl substrates present in the p80
PI
3-kinase complex (e.g. Shc, p140, p160).
The Jak2 tyrosine
kinase associates with the subunit and has been implicated in the
tyrosyl phosphorylation of the
subunit(14) . Since p80
also associates with the
subunit, we analyzed the possibility
that p80 is a substrate of Jak2. We confirmed that Jak2 becomes
tyrosine phosphorylated after stimulation of TF-1 cells with hGM-CSF;
however, we could not detect p80 in Jak2 immunoprecipitates, suggesting
that p80 may not be a direct substrate of Jak2 (data not shown).
Similarly, the tyrosine kinase Fps/Fes, which has been implicated in
hGM-CSF signaling(15) , was not present in the p80
PI
3-kinase complex (data not shown), suggesting that p80 was not a
substrate of Fps/Fes either.
To analyze molecular events involved in hGM-CSF signaling
that may also occur in vivo, we have used a human
GM-CSF-dependent cell line that responds to physiological
concentrations of the ligand. Our experiments resulted in the
identification of a new phosphotyrosine protein (p80) that associates
with the SH2 domains of the p85 subunit of PI 3-kinase and with the
common subunit of the hGM-CSF/IL-3/IL-5 receptor.
We postulate that p80 is a
tyrosine-phosphorylated adapter protein that may link PI 3-kinase to
the hGMR directly or indirectly. This is based on the observations that
p80 was found in immunoprecipitates of the subunit of hGMR and
that phosphotyrosyl p80 was tightly bound to the p85 subunit of PI
3-kinase. Our experiments showed that the C-terminal and N-terminal SH2
domains of p85 recognized phosphorylated p80, strongly suggesting that
this protein binds directly to p85 through SH2-phosphotyrosine
interactions. The C-terminal SH2 domain of p85 had higher affinity for
phosphorylated p80 than the N-terminal SH2 domain, as has been shown
for the association between p85 and the activated platelet-derived
growth factor receptor(45) . Clarification of the nature of the
association between p80 and the
subunit will require further
analysis.
In
addition to p80, other tyrosine-phosphorylated proteins were detected
in anti-Src/Yes precipitates, i.e. Shc, p140, and p160,
suggesting that these proteins might also be substrates of Src or Yes.
p140 is a protein that associates with Shc after stimulation with
several cytokines (46) including hGM-CSF ()through a
new phosphotyrosine binding domain in the N terminus of
Shc(47) . p160 is an unknown protein. Interestingly, while the
association of Src and/or Yes with tyrosine-phosphorylated p80, Shc,
p140, and p160 was dependent on hGM-CSF, Src/Yes was already associated
with the p85 subunit of PI 3-kinase in the absence of hGM-CSF
stimulation. Since we did not detect tyrosine phosphorylation of the
p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells, the interaction
between Src and/or Yes with p85 may not be dependent on
SH2-phosphotyrosine interactions. This finding is consistent with
recent reports that the SH3 domain of Src kinases binds directly to a
proline-rich region of the p85 subunit of PI
3-kinase(42, 43) . The presence of Src kinases in the
PI 3-kinase complex before stimulation makes these kinases good
candidates for phosphorylation of p80 or other proteins in this
complex.
We also analyzed other tyrosine kinases for their
association with p80. Jak2 was found to be tyrosine phosphorylated
after hGM-CSF treatment of TF-1 cells(13, 14) . However, no tyrosine-phosphorylated p80 was detected in anti-Jak2
immunoprecipitates (data not shown). Similarly, no phosphorylated p80
was found in immunoprecipitates of Hck or Fps/Fes from
hGM-CSF-stimulated cells (data not shown). Therefore, we conclude that
these kinases are unlikely to be involved in tyrosine phosphorylation
of p80.
In summary, we have identified a new adapter protein, p80,
that associates with the subunit of the hGMR and with the p85
subunit of PI 3-kinase. p80 may link the hGMR to the PI 3-kinase
signaling pathway. So far, tyrosine phosphorylation of p80 seems to be
specific for signaling via the common
subunit of the
hGM-CSF/IL-3/IL-5 receptors. We are now attempting to clone p80 to
clarify its function and mechanism of action in hGM-CSF signaling.