(Received for publication, April 11, 1995; and in revised form, June 8, 1995)
From the
Insulin stimulation of Chinese hamster ovary cells expressing
the human insulin and epidermal growth factor (EGF) receptors
(CHO/IR/ER) resulted in the tyrosine phosphorylation of the 52-kDa Shc
isoform with a relatively low extent of 46-kDa Shc tyrosine
phosphorylation. In contrast, EGF stimulation resulted in the tyrosine
phosphorylation of both the 52- and 46-kDa Shc isoforms. Consistent
with these differences, Grb2 predominantly bound to the 52-kDa Shc
isoform following insulin stimulation, whereas Grb2 associated with
both the 52- and 46-kDa Shc isoforms after EGF stimulation. Further, in vitro kinetic analysis demonstrated that the insulin
receptor has a 4-fold greater V with no
significant difference in the K
for the
purified 52-kDa Shc isoform compared with the 46-kDa Shc isoform.
However, the EGF receptor displayed the identical V
and K
for tyrosine phosphorylation
of both of these species. In direct contrast to the EGF receptor, we
also observed significant differences in binding interactions between
the insulin receptor with the 52- and 46-kDa Shc isoforms in
vitro. These data demonstrate that the predominant
insulin-dependent Shc signaling pathway occurs via the 52-kDa Shc
isoform, whereas the EGF receptor can effectively use both the 52- and
46-kDa Shc species.
Autophosphorylation of tyrosine kinase growth factor receptors
results in the formation of recognition motifs for various Src homology
2 (SH2) ()domain containing effector
proteins(1, 2, 3, 4) . The
association of tyrosine-phosphorylated receptors with these effector
molecules generates distinct receptor-signaling complexes responsible
for downstream biological responsiveness. However, unlike most receptor
tyrosine kinases, the insulin receptor itself does not persistently
associate with these effector molecules but instead
tyrosine-phosphorylates intermediate proteins responsible for the
formation of multisubunit-signaling complexes(5, 6) .
One proximal target of the insulin receptor was originally identified
as a series of proteins (66, 52, and 46 kDa), termed Shc for Src
homology 2/
-collagen-related(7, 8) . The cloning
of cDNAs for this family of proteins has demonstrated that the 46- and
52-kDa species arise from the use of alternative translation initiation
sites within the same transcript. This results in an amino-terminal
59-amino acid truncation of the 46-kDa isoform compared with the 52-kDa
isoform. In contrast, the 66-kDa species most likely arises from an
alternatively spliced message since there is only one Shc gene and
carboxyl-terminal antibodies cross-react with all three molecular
weight
species(7, 8, 9, 10, 11) .
The Shc proteins are tyrosine-phosphorylated on a single tyrosine
residue (Tyr), which serves as a docking site for Grb2 (8, 12) . Grb2 was originally identified as a 25-kDa
growth factor receptor binding protein that contains a single SH2
domain flanked by two Src homology 3 (SH3)
domains(13, 14) . The SH3 domains of Grb2 direct the
association with several proteins containing proline-rich motifs
including the 150-kDa guanylnucleotide exchange factor for Ras, termed
SOS for the Drosophila gene Son-of-Sevenless (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) .
Thus, the tyrosine kinase receptor association of the Shc-Grb2-SOS
complex targets guanylnucleotide exchange activity to the plasma
membrane location of Ras, thereby providing a molecular switch for Ras
activation(26, 27) .
Similar to many SH2 domain-containing proteins, the carboxyl-terminal SH2 domain of Shc was originally assumed to provide the appropriate binding motif for the association of Shc with tyrosine-autophosphorylated receptors(7, 28) . However, recent studies have demonstrated that the amino-terminal 84 amino acids are essential for the association of Shc with the tyrosine-phosphorylated EGF and fibroblast growth factor receptors(29, 30) . Although the tyrosine-phosphorylated insulin receptor does not persistently associate with Shc, the insulin-stimulated tyrosine phosphorylation of Shc appears to function as the major pathway mediating insulin activation of Ras GTP binding and Ras-dependent downstream signaling(31, 32, 33, 34) . In this paper, we demonstrate that the 52-kDa Shc isoform is the predominant substrate for the insulin receptor tyrosine kinase. This is in marked contrast to the EGF receptor, which displays similar substrate specificity for both the 52- and 46-kDa Shc species.
Figure 1: The insulin receptor primarily tyrosine-phosphorylates the 52-kDa Shc isoform in CHO/IR cells. CHO/IR cells were incubated in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100 nM insulin for 5 min at 37 °C. Whole cell detergent lysates were prepared and either immunoprecipitated with a Shc antibody (lanes1 and 2) or a Grb2 antibody (lanes3 and 4) as described under ``Experimental Procedures.'' The immunoprecipitates were then Western blotted with the Shc antibody (A) or the PY20-HRP phosphotyrosine antibody (B). IP, immunoprecipitate; IB, immunoblot.
Figure 2: Differential tyrosine phosphorylation of the 52- and 46-kDa Shc isoforms by insulin and EGF in CHO/IR/ER cells. CHO/IR/ER cells were incubated in the absence (lanes1 and 4) and in the presence of either 100 nM insulin (lanes2 and 5) or 100 ng/ml EGF (lanes3 and 6) for 5 min at 37 °C. Whole cell detergent lysates were prepared and either immunoprecipitated with a Shc antibody (lanes1-3) or a Grb2 antibody (lanes4-6) as described under ``Experimental Procedures.'' The immunoprecipitates were then Western blotted with the Shc antibody (A) or the PY20-HRP phosphotyrosine antibody (B). IP, immunoprecipitate; IB, immunoblot.
Similarly, Shc immunoblotting of the Grb2 immunoprecipitates demonstrated the predominant association of the 52-kDa Shc isoform with Grb2 following insulin stimulation (Fig. 2A, lane5). In contrast, EGF stimulation resulted in a greater extent of 46-kDa Shc association with Grb2 compared with insulin-stimulated CHO/IR/ER cells (Fig. 2A, lane6). In parallel, phosphotyrosine immunoblotting of the Grb2 immunoprecipitates also demonstrated the predominant insulin-stimulated co-immunoprecipitation of the tyrosine-phosphorylated 52-kDa Shc isoform (Fig. 2B, lane5), whereas EGF stimulated the co-immunoprecipitation of the tyrosine-phosphorylated 52- and 46-kDa Shc species (Fig. 2B, lane6). In addition, we also observed a relatively small extent of EGF-stimulated tyrosine phosphorylation and Grb2 co-immunoprecipitation of the 66-kDa Shc isoform (Fig. 2B, lanes3 and 6). Furthermore, both Shc and Grb2 immunoprecipitations resulted in the co-immunoprecipitation of the tyrosine-phosphorylated EGF receptor (Fig. 2B, lanes3 and 6), whereas insulin stimulation only resulted in the Grb2 co-immunoprecipitation of tyrosine-phosphorylated IRS1 (Fig. 2B, lane5). As observed in the CHO/IR cells, we were unable to detect the presence of the tyrosine-phosphorylated insulin receptor in either the Shc or Grb2 immunoprecipitates (Fig. 2B, lanes2 and 5).
Figure 3:
The insulin and EGF receptor display Shc
isoform-specific substrate kinase activity in vitro. The
insulin and EGF receptors were isolated from CHO/IR and 3T3/ER cells,
respectively, by detergent solubilization and wheat germ agglutinin
affinity chromatography as described under ``Experimental
Procedures.'' The insulin (A) and EGF (B)
receptors were incubated with a mixture of 10 µg of the GST-52Shc
and 10 µg of the GST-46Shc fusion proteins in the presence of 100
nM insulin or 100 ng/ml EGF, respectively. The reaction was
initiated by the addition of [-
P]ATP (100
µM, 3 µCi/nmol) for 0 (lane1), 2 (lane2), 5 (lane3), 15 (lane4), 30 (lane5), and 60 (lane6) min. The reaction was terminated by the addition of
Laemmli sample buffer, and the samples were then subjected to
SDS-polyacrylamide gel electrophoresis and
autoradiography.
Figure 5: Differential in vitro binding of the insulin and EGF receptors to the 52- and 46-kDa Shc isoforms. CHO/IR (A) and 3T3/ER (B) cells were incubated with 100 nM insulin or 100 ng/ml EGF for 5 min at 37 °C. The cells were detergent-solubilized, and the lysates (2 mg) were incubated with 10 µg of GST (lanes1 and 2), 10 µg of GST-52Shc (lanes3 and 4), or 10 µg GST-46Shc bound to glutathione-Sepharose beads for 60 min as described under ``Experimental Procedures.'' The samples were then precipitated by microcentrifugation, and the resulting precipitates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was then Western blotted for the presence of the insulin (A) or the EGF (B) receptors.
Consistent with the EGF-dependent Shc phosphorylation in vivo, the EGF receptor demonstrated essentially an identical rate and extent of 52- and 46-kDa Shc phosphorylation in vitro (Fig. 3B). As observed for the insulin receptor, the EGF receptor was unable to phosphorylate the GST fusion protein itself (data not shown). In addition, the autophosphorylated EGF receptor was also found to co-precipitate with the GST-Shc fusion proteins (Fig. 3B) but not with GST alone (data not shown).
To determine if the enhanced insulin receptor
phosphorylation of the 52-kDa Shc isoform was due to a differences in V or K
, we next
determined the substrate concentration dependence of Shc tyrosine
phosphorylation (Fig. 4A). In the presence of insulin,
the insulin receptor had essentially an identical K
(0.45 versus 0.40 µM) for both the 52-
and 46-kDa Shc isoforms. In contrast, the insulin receptor had an
approximate 4-fold increase in V
for the 52-kDa
Shc isoform (4.0 pmol/min) compared with the 46-kDa Shc species (1.0
pmol/min). In comparison, we also determined the kinetic properties of
the EGF receptor-mediated tyrosine phosphorylation of the Shc proteins (Fig. 4B). As expected, there was no significant
difference in either the K
(0.70 versus 0.85 µM) or V
(4.4 versus 5.0 pmol/min) values between the 52- and 46-kDa Shc
isoforms as substrates for the EGF receptor.
Figure 4:
Kinetic analysis of insulin and EGF
receptor tyrosine phosphorylation of the 52- and 46-kDa Shc isoforms.
The initial rates of hormone-stimulated insulin (A) and EGF (B) receptor tyrosine phosphorylation of the GST-52Shc ()
and GST-46Shc (
) fusion proteins were determined as described in
the legend to Fig. 3. The initial rate of substrate
phosphorylation was determined at various fusion protein concentrations
and graphically analyzed in the form of a Lineweaver-Burke
plot.
In comparison, the EGF receptor isolated from either control or EGF-stimulated 3T3/ER cells also did not associate with the GST protein alone (Fig. 5B, lanes1 and 2). In addition, control cell extracts did not significantly associate with either the GST-52Shc or GST-46Shc fusion proteins (Fig. 5B, lanes3 and 5). However, extracts from EGF-stimulated cells demonstrated the co-precipitation of the EGF receptor to a similar extent with both the GST-52Shc and GST-46Shc fusion proteins (Fig. 5B, lanes4 and 6). Thus, although tyrosine phosphorylation of both the insulin and EGF receptor was required for in vitro association, the EGF receptor does not strongly discriminate between the 52- and 46-kDa Shc isoforms compared with the insulin receptor.
It has been well established that hormone-stimulated tyrosine kinase activity of growth factor receptors is a necessary event in the propagation of growth factor-dependent downstream signaling (for reviews see (38, 39, 40) ). Although several specific combinations of proximal effectors have been identified for numerous growth factor tyrosine kinase receptors, they all appear to have in common the tyrosine phosphorylation and/or association with the Shc proteins. Since tyrosine-phosphorylated Shc directs its association with the Grb2-SOS complex, this provides a pathway directly linking tyrosine kinase receptors to Ras activation(14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . Consistent with this model, several studies have demonstrated that Shc is the predominant route for the Ras-dependent activation of the mitogen-activated protein kinase pathway(31, 32, 33, 34) .
In the case of EGF signaling, Shc directly associates with the tyrosine-phosphorylated EGF receptor, targeting the Shc-Grb2-SOS ternary complex to the plasma membrane location of Ras(12, 21, 25, 41, 42, 43, 44) . This model is consistent with studies demonstrating that EGF has no effect on SOS or RasGAP activity and that expression of SOS proteins that are directly targeted to the plasma membrane via myristoylation or prenylation constitutively activate Ras GTP loading(26, 27) . Although this is an appealing model, tyrosine kinase receptor docking of Shc cannot account for the ability of insulin to target the Shc-Grb2-SOS complex to the plasma membrane since the insulin receptor does not persistently associate with Shc in vivo(8, 41, 42, 45) . Consistent with this conclusion, we have not been able to observe any significant association of Shc with the insulin receptor under identical conditions that readily detect Shc association with the EGF receptor and Grb2 with IRS1. Furthermore, point mutations and carboxyl-terminal deletions of the EGF receptor that completely abolish Shc binding have no effect on EGF receptor-mediated activation of the Ras-dependent mitogen-activated protein kinase pathway(46, 47, 48, 49) . Thus, it appears that the tyrosine phosphorylation of Shc is the essential feature necessary to mediate growth factor tyrosine kinase receptor-dependent Ras activation.
Several studies have observed
that activation of various growth factor receptor tyrosine kinases
results in the tyrosine phosphorylation of both the 52- and 46-kDa Shc
isoforms and, in some studies, the 66-kDa
isoform(8, 11, 43, 50) . In these
studies, we have not detected significant insulin or EGF-stimulated
tyrosine phosphorylation of the 66-kDa Shc isoform by phosphotyrosine
immunoblotting. However, there was a marked decrease in electrophoretic
mobility of the 66-kDa Shc protein, most likely reflecting
serine/threonine phosphorylation. In any case, we have observed that
insulin stimulation predominantly resulted in the tyrosine
phosphorylation of the 52-kDa Shc isoform compared with the 46-kDa Shc
species. This difference in substrate specificity was also
recapitulated in vitro, demonstrating that the 52-kDa Shc
isoform was a significantly better substrate for the insulin receptor
compared with the 46-kDa Shc isoform. Although we have been unable to
detect the co-immunoprecipitation of Shc with the insulin receptor, in vitro binding demonstrated that the 52-kDa Shc isoform was
more efficiently associated with the insulin receptor than the 46-kDa
Shc isoform. Similarly, binding interactions between the insulin
receptor subunit and Shc have been observed by high level
expression in the yeast two-hybrid system(51) . We therefore
hypothesize that the observed in vitro binding between the
insulin receptor and Shc was indicative of association at the level of
substrate-enzyme interactions but does not reflect a stable interaction in vivo.
In contrast, the tyrosine-phosphorylated EGF receptor was stably associated with Shc, which was directly detected by co-immunoprecipitation. Since Shc contains a carboxyl-terminal SH2 domain, it was previously assumed that this domain was responsible for the docking of Shc to the EGF receptor. However, recent studies have demonstrated that the amino-terminal 84 amino acids of Shc provide this function independent of the carboxyl-terminal SH2 domain(29, 30) . Consistent with these findings, we also have not observed any significant difference in the binding of the tyrosine-phosphorylated EGF receptor to the 52- and 46-kDa Shc isoforms, the latter differing by the deletion of 59 amino-terminal residues. Together, these data suggest that amino acid residues 59-84 define the Shc protein tyrosine binding domain for the EGF receptor. This is in contrast to the insulin receptor, which preferentially utilized the 52-kDa Shc isoform as a tyrosine kinase substrate both in vivo and in vitro. Thus, although the amino-terminal 84 residues of Shc were necessary for receptor association and tyrosine phosphorylation, specific determinants within this domain are apparently necessary to impart substrate specificity between the EGF and insulin receptors.
To date, there does not appear to be any specific difference in the signaling role played by the 52- and 46-kDa Shc isoforms. Both species, when tyrosine-phosphorylated, associate with Grb2 and thereby presumably link SOS to the activation of Ras. However, since all cells express both the 52- and 46-kDa Shc isoforms, albeit at different levels, the contribution of these complexes to downstream signaling events has not been quantitatively determined. Based upon the apparent specificity of the insulin receptor, we speculate that the 52- and 46-kDa Shc isoforms have defined signaling and/or regulatory roles.
In summary, we have observed that the EGF receptor associates with and tyrosine phosphorylates both the 52- and 46-kDa Shc isoforms with equal efficacy both in vivo and in vitro. In contrast, the insulin receptor preferentially tyrosine phosphorylates the 52-kDa Shc isoform, resulting in the predominant targeting of Grb2 to this species. Whether or not the tyrosine phosphorylation and/or receptor association of the 52- and 46-kDa Shc isoforms underlies physiological specificity in the signaling pathways between the EGF and insulin receptors remains to be determined.