(Received for publication, February 9, 1996; and in revised form, March 27, 1996)
From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
regulates the growth and function of several myeloid cell types at
different stages of maturation. The effects of GM-CSF are mediated
through a high affinity receptor that is composed of two chains: a
unique, ligand-specific chain and a
common chain
(
) that is also a component of the receptors for
interleukin 3 (IL-3) and IL-5.
plays an essential
role in the transduction of extracellular signals to the nucleus
through its recruitment of secondary messengers. Several downstream
signaling events induced by GM-CSF stimulation have been described,
including activation of tyrosine kinases and tyrosine phosphorylation
of cellular proteins (including
) and activation of
the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A
region within the
cytoplasmic tail (amino acids
517-763) has been reported to be necessary for tyrosine
phosphorylation of the adapter protein, Shc, and for the subsequent
GM-CSF-induced activation of Ras. In this paper, we describe a physical
association between the tyrosine phosphorylated GM-CSF receptor
(GMR)-
chain and Shc in vivo. Using a series
of cytoplasmic truncation mutants of
and various
mutant Shc proteins, we demonstrate that the N-terminal
phosphotyrosine-binding (PTB) domain of Shc binds to a short region of
(amino acids 549-656) that contains
Tyr
. Addition of a specific phosphopeptide encoding amino
acids surrounding this tyrosine inhibited the interaction between
and Shc. Moreover, mutation of a key residue within
the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its
association with
. These observations provide an
explanation for the previously described requirement for Tyr
of
for GM-CSF-induced tyrosine phosphorylation
of Shc and have implications for Ras activation through the GM-CSF,
IL-3, and IL-5 receptors.
The biological effects of granulocyte-macrophage colony
stimulating factor (GM-CSF) ()are mediated by the
interaction of the cytokine with a high affinity receptor expressed on
certain cell types of hematopoietic lineage. The GM-CSF receptor (GMR)
is composed of an
and a
chain. Expression of the
chain alone is sufficient for low affinity binding of ligand. The human
chain does not directly bind ligand; however, its association
with the
chain confers high affinity binding of the cytokine. The
chain of GMR is referred to as
, because it also
interacts with distinct
subunits of the IL-3 and IL-5 receptors
to form heterodimeric high affinity receptors for IL-3 and IL-5,
respectively (reviewed in (1) ). While the cytoplasmic domains
of
and
do not influence ligand binding, they
are essential for transmitting the mitogenic signal induced by
GM-CSF(2) . Although
has no intrinsic kinase
activity, the cytoplasmic domain of
is necessary for
the activation of cytosolic tyrosine kinases, such as JAK2(3) ,
small GTP-binding proteins, such as Ras(4, 5) , and
for induction of c-fos, c-jun, and
c-myc(6) . Mutational analyses have identified regions
of the
cytoplasmic tail that are required for
mediating specific downstream signaling events. Induction of c-myc and activation of JAK2 require the membrane-proximal 60 amino
acids of the cytoplasmic tail of
, while a membrane
distal domain, between amino acids 517 and 763, is necessary for Ras
activation and the induction of c-fos and
c-jun(3, 7, 8) . A site within this
latter domain has been implicated in the tyrosine phosphorylation of
. A specific point mutation at tyrosine 750 abolishes
phosphorylation of
(9, 10) .
Shc
is an adapter protein that has been implicated in linking growth
factor, cytokine and antigen receptors to Ras signaling (reviewed in (11) ). Previous studies have demonstrated that Shc is
phosphorylated on tyrosine following binding of GM-CSF, IL-3, and IL-5
to their receptors(7, 9, 10, 12) .
Phosphorylated Shc subsequently interacts with another adapter protein,
Grb2, which, in turn, interacts with the GTP/GDP nucleotide exchange
factor for Ras, mSOS(13, 14) . Shc contains two
domains capable of interacting with tyrosine-phosphorylated proteins:
an N-terminal phosphotyrosine-binding (PTB) domain and a C-terminal SH2
domain(15, 16, 17) . The interaction of Shc
with tyrosine-phosphorylated receptors and the localization of the
Shc-Grb2-mSOS complex at the membrane have been implicated in Ras
activation. A region of (517-763) that is
necessary for Ras activation is also required for Shc phosphorylation
upon GM-CSF stimulation(7) . A point mutation within
that changes tyrosine 577 to phenylalanine abolished
GM-CSF-induced phosphorylation of Shc and the association of Shc with
Grb2(8, 9) .
One hypothesis for the
Tyr
-dependent Shc phosphorylation during
GM-CSF stimulation is that Shc interacts with phosphorylated
Tyr
through its SH2 or PTB domain and
this interaction leads to Shc phosphorylation. We have used a transient
transfection system to address this question and show here a physical
interaction between Shc and tyrosine-phosphorylated
.
An analysis of the domains of these proteins that are critical for this
interaction revealed that the region spanning amino acids 549-656
of
interacts with the recently described PTB domain
of Shc. Addition of a 16-residue phosphopeptide that encompasses
Tyr
inhibited the interaction between
and Shc. In addition, mutation of a key residue of the
phosphotyrosine-binding pocket in the PTB domain of Shc abolished this
interaction. These observations suggest that the Shc-PTB domain
interacts with the phosphorylated
chain and provide
an explanation for the requirement for
Tyr
in GM-CSF-dependent Shc phosphorylation.
Figure 1:
JAK2-dependent phosphorylation of
and its association with Shc. A, COS-7 cells
were co-transfected with 4 µg of the indicated DNA constructs as
described under ``Materials and Methods.'' After 48 h, cell
lysates were immunoprecipitated with anti-
antibody
and protein A-Sepharose beads.
was co-expressed with
Shc (lane 1) or with JAK2 (lane 2). B, cells
co-transfected with 4 µg of the indicated cDNAs in the presence of
wild type JAK2 (lanes 1-3) and kinase-deficient JAK2 (lanes 4 and 5) were lysed. Proteins were
precipitated with glutathione-Sepharose beads and analyzed by
anti-phosphotyrosine immunoblotting. C, total cell lysates
were analyzed by anti-phosphotyrosine
immunoblotting.
We next determined if
phosphorylation of by JAK2 would lead to an
association between
and Shc. Proteins from cells
co-transfected with
, JAK2, and GST-Shc were
precipitated with glutathione- Sepharose beads, and proteins
co-precipitating with GST-Shc were visualized by anti-phosphotyrosine
immunoblotting. Co-expression of JAK2 and GST-Shc alone, or
and GST-Shc alone, did not lead to an association of
with Shc (Fig. 1B). In the presence of wild type
JAK2,
became phosphorylated and associated with
co-expressed GST-Shc. However, the co-expression of a kinase-deficient
JAK2 with
and GST-Shc failed to lead to an
association of
with Shc, confirming the requirement
for JAK2 kinase activity for this association. Anti-phosphotyrosine
immunoblot analysis of total cell lysates from these transfectants
revealed that
was not phosphorylated in the
kinase-deficient JAK2-expressing cells (Fig. 1C), which
may explain the lack of binding of
to Shc. Thus, we
could obtain JAK2-dependent tyrosine phosphorylation of
and detect an interaction between
and Shc.
To determine the region of necessary for the
interaction with Shc, we expressed deletion mutants of
(schematically shown in Fig. 2A) with JAK2 and
GST-Shc as described above. Deletion mutants of
indicated that amino acids between 549 and 656 are essential for
the association between
and Shc (Fig. 2B). Previous studies based on site-directed
mutagenesis of tyrosines within
have indicated that
Tyr
is necessary for Shc phosphorylation and its
subsequent interaction with Grb2(9) . Tyrosine 577 resides
within the region (549-656) that we find essential for an
interaction between phosphorylated
and Shc.
Immunoblotting with an anti-GST antibody confirmed that comparable
amounts of GST-Shc were precipitated in each sample (Fig. 2C). In addition, immunoblot analysis of total
cell lysates with an anti-HA antibody revealed that all the HA-tagged
deletion mutants were expressed (Fig. 2D).
Figure 2:
A region of the chain
(amino acids 549-656) is essential for its interaction with Shc. A, schematic of
deletion mutants with
C-terminal HA tag. B, cells were transfected as described
above and precipitations were performed using glutathione-Sepharose
beads. Co-precipitation of
with GST-Shc was
visualized by immunoblotting with anti-phosphotyrosine antibody. C, membranes were stripped and immunoblot analysis of GST-Shc
expression with anti-GST antibody was performed. D, total cell
lysates were subjected to immunoblot analysis with anti-HA antibody to
visualize the expression of
mutants. The
549 protein migrates just below a nonspecific band
present in all lanes.
Figure 3:
The PTB domain of Shc is necessary and
sufficient for interaction with .A,
schematic of GST-Shc mutant constructs and summary of their interaction
with
. B, COS-7 cells were transfected and
analyzed as described in Fig. 1and ``Materials and
Methods.'' Co-precipitation of
with GST-Shc
proteins using glutathione-Sepharose beads, was visualized by
anti-phosphotyrosine immunoblotting. C, membranes were
stripped and reprobed with anti-GST antibody to assess the expression
of different GST-Shc proteins. D, cells were transfected with
0.5 µg of the indicated ShcN mutants along with 4 µg of JAK2
and 0.5 µg of
656 as described. Lysates were
subjected to precipitation with glutathione-Sepharose beads and
analyzed by anti-phosphotyrosine immunoblotting. Reprobing the membrane
with anti-GST antibody revealed comparable expression of different ShcN
proteins (data not shown).
We have recently obtained the solution
structure for the Shc-PTB domain in complex with a phosphopeptide
derived from TrkA and identified several residues important for the
interaction with phosphoproteins (18) . Site-directed
mutagenesis of the Shc-PTB domain has revealed that Arg,
which lies within the phosphotyrosine-binding pocket, is essential for
interactions with phosphoproteins. To further characterize the
interaction between
and ShcN, we co-transfected wild
type GST-Shc-N or mutant forms of GST-Shc-N (R175K or R112Q) with
656 and JAK2 (Fig. 3D). While the wild
type GST-Shc-N domain bound to
656, the R175K mutant
failed to interact with
. In contrast, a mutation at
another arginine residue (R112Q), which lies outside the phosphoprotein
binding pocket, did not affect binding of GST-ShcN to
656. These results confirm that the physical
association of Shc with
is dependent upon an intact
phosphotyrosine-binding pocket within the PTB domain of Shc.
Recent
studies have indicated that the PTB domain of Shc recognizes amino acid
residues N-terminal to the phosphotyrosine and preferentially interacts
with -turn forming sequences containing a critical asparagine
residue at the -3 position relative to the
phosphotyrosine(16, 18, 28, 29, 30, 31) .
The only tyrosine within the
cytoplasmic domain that
fits such a tyrosine-based recognition motif is Tyr
(SFDFNGPYLGP), which has been implicated in GM-CSF-dependent Shc
phosphorylation. Our observations reported here demonstrate that the
Shc-PTB domain interacts with tyrosine phosphorylated
through a region that encompasses Tyr
. To provide
direct evidence that Shc interacts with
through
Tyr
, we synthesized a phosphopeptide encoding Tyr
and tested its inhibition of the Shc-
interaction. Increasing concentrations of peptide were incubated
with GST-ShcN followed by the addition of the lysates from
656- and JAK2-expressing cells. In the absence of
peptide, or the presence of 100 µM EGFR control peptide
(previously shown not to inhibit the Shc-PTB interaction with
phosphoproteins(28) ), association of
with
Shc was unaffected (Fig. 4). In contrast, a peptide encoding
Tyr
(QASSFDFNGPpYLGPPH) specifically inhibited the
interaction between
and Shc. Titration of the peptide
indicated that 10 µM of the specific peptide significantly
affected the association, while at 50 µM the association
was completely disrupted. These observations are consistent with
previously reported affinities of Shc-PTB for
phosphopeptides(28, 29) .
Figure 4:
Addition of a specific phosphopeptide
encoding amino acids surrounding Tyr inhibits the
association of
with Shc. GST-ShcN proteins were
precipitated from cells transfected with ShcN alone, washed, and
incubated without peptide, with 100 µM EGFR control
peptide or the indicated concentration (1-100 µM) of
a 16-residue
phosphopeptide encompassing Tyr
for 30 min. Lysates from cells co-expressing
656
and JAK2 were added and mixtures incubated for an additional 2 h.
Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and
immunoblotting with anti-phosphotyrosine
antibody.
Although Tyr of
was originally implicated in Shc
phosphorylation, it has subsequently been reported that this may be an
indirect effect due to the failure to phosphorylate
(9, 10) . This is consistent with our
data that truncation mutants that lack Tyr
, but are
phosphorylated at Tyr
, can still interact with Shc. Thus,
we favor the hypothesis that recruitment of Shc to
occurs through Tyr
, which is further supported by
our peptide inhibition studies.
Taken together, these data suggest a
model whereby GM-CSF stimulation leads to recruitment of Shc to GMR
first, followed by tyrosine phosphorylation of Shc. This situation is
analogous to the IL-2R chain, which shares a high degree of
structural homology in its cytoplasmic tail with
of
GMR. Interestingly, Shc also interacts with phosphorylated Tyr
of IL-2R
through its PTB domain. Mutation of
Tyr
, the Shc binding site on the IL-2R
chain, failed
to lead to Shc phosphorylation(27, 32) . An intriguing
possibility is that the interaction of the Shc-PTB domain with
or IL-2R
allows for the SH2 domain of Shc to
recruit other phosphotyrosine-containing proteins (to the proximity of
the receptor) that may also contribute to intracellular signaling. It
remains to be established if this reflects a pattern of Shc-mediated
signaling that is common to type I cytokine receptor superfamily
members.