(Received for publication, November 2, 1995; and in revised form, December 22, 1995)
From the
Stimulation of the insulin receptor (IR) results in tyrosine phosphorylation of the intermediate molecules insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which then couple the IR to downstream signaling pathways by serving as binding sites for signaling molecules with SH2 domains. It has been proposed that direct binding of IRS-1, IRS-2, and Shc to an NPX-Tyr(P) motif in the juxtamembrane region of the IR is required for tyrosine phosphorylation of these molecules by the IR. In this regard, Shc and IRS-1 contain domains that are distinct from SH2 domains, referred to as the phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domains, which bind phosphotyrosine in the context of an NPX-Tyr(P) motif. To further clarify the role of the Shc PTB/PI domain, we identified a mutation in this domain that abrogated binding of Shc to the IR in vitro. Interestingly, this mutation completely abolished Shc phosphorylation by the IR in vivo whereas mutation of the arginine in the FLVRES motif of the Shc SH2 domain did not affect Shc phosphorylation by insulin. In addition, we identified specific amino acids on the IR that are required for the IR to stimulate Shc but not IRS-1 phosphorylation in vivo. As with the PTB/PI domain Shc mutant, the ability of these mutant receptors to phosphorylate Shc correlates with the binding of the PTB/PI domain of Shc to similar sequences in vitro. These findings support a model in which binding of the PTB/PI domain of Shc directly to the NPX-Tyr(P) motif on the IR mediates Shc phosphorylation by insulin.
A critical early event necessary for the IR ()to
mediate biological functions is receptor autophosphorylation, which
results in increased activity of the receptor's kinase domain.
Autophosphorylation also mediates the interaction of the IR, as well as
other receptor tyrosine kinases, with several signaling molecules,
which must bind autophosphorylated receptors in order to be
activated(1) . The interaction of signaling molecules with
autophosphorylated receptors has now been shown to be mediated by at
least two distinct domains on signaling molecules that bind
phosphotyrosine. The first domains to be described are the SH2
domains(1) . Recently, a second domain that binds
phosphotyrosine was identified in the amino terminus of Shc and has
been termed the PI or PTB domain (also known as the Shc and IRS-1
NPXY-binding (SAIN) domains or IRS homology 2 (IH2)
domains)(2, 3, 4, 5, 6) .
In contrast to the finding that SH2 domains preferentially recognize
amino acids carboxyl-terminal to the phosphotyrosine, the Shc PTB/PI
domain recognizes amino acids amino-terminal to the phosphotyrosine
that consist of a core NPX-Tyr(P)
motif(7, 8, 9, 10, 11, 12) .
IR autophosphorylation results in tyrosine phosphorylation of several intermediate molecules, including IRS-1, IRS-2, and Shc(6, 13, 14, 15) . These molecules, rather than the IR itself, then couple to downstream signaling pathways by serving as binding sites for SH2 domain-containing signaling molecules(16) . For example, binding of GRB2-Sos to tyrosine-phosphorylated Shc is thought to couple the IR to Ras activation(14, 17, 18, 19) . The recognition that Shc interacts with autophosphorylated receptors through its PTB/PI domain, coupled with the finding that IRS-1 and IRS-2 contain a similar domain that binds phosphotyrosine, have helped clarify how the IR likely activates these signaling molecules. These results have led to the suggestion that IRS-1, IRS-2 and Shc share a common NPX-Tyr(P) binding motif on the autophosphorylated insulin and IGF-1 receptors that functions to juxtapose these molecules adjacent to the IR kinase domain, thereby enabling IRS-1 and Shc to become phosphorylated(5, 16, 20) .
While this has become an attractive model to explain the mechanism whereby the IR phosphorylates IRS-1 and Shc, it is still not clear whether phosphorylation of Shc by the IR requires a functional PTB/PI domain or whether binding of Shc directly to the IR is required for Shc phosphorylation in vivo. For example, epidermal growth factor stimulation of a truncated EGFR that does not bind Shc still results in Shc tyrosine phosphorylation(21) . Moreover, although the PTB/PI domain of Shc has been clearly shown by co-immunoprecipitation to associate in vivo with an NPX-Tyr(P) motif on a variety of receptor tyrosine kinases, middle T antigen, and the phosphoprotein p145(4, 8, 9, 22, 23, 24) , the IR has been shown to bind Shc only in vitro(5, 14, 15, 25) . The fact that a mutation in tyrosine 960 in the NPXY motif of the IR impairs tyrosine phosphorylation of Shc by insulin (26) does not prove that Shc is a direct substrate of the IR; this mutation also abrogates tyrosine phosphorylation of IRS-1 by the IR(26, 27) , thereby raising the possibility that activation of a signaling pathway downstream from IRS-1 or binding of an unidentified signaling molecule to the IR NPX-Tyr(P) motif mediates Shc phosphorylation.
In order to clarify the role of Shc's PTB/PI domain in mediating Shc phosphorylation by insulin, we determined whether a single amino acid substitution in the PTB/PI domain of Shc that impairs binding of Shc to the IR in vitro also impairs the tyrosine phosphorylation of Shc by the IR in vivo. In addition, we determined whether changes in the amino acids surrounding the NPXY motif in the juxtamembrane region of the IR modulate the ability of insulin to stimulate Shc phosphorylation in vivo. The findings reported in this paper support a model in which binding of the PTB/PI domain of Shc directly to the NPX-Tyr(P) motif on the IR is required for Shc phosphorylation by the IR in vivo.
To
overexpress the Trk/IR receptor in 3T3-L1 cells, the wild type and
mutant Trk/IRs were subcloned into the retroviral vector SR using EcoRI-HindIII(19) . Helper-free infectious
retrovirus was produced by transiently transfecting the various
constructs into the retroviral packaging cell line BOSC 23 using
Ca
PO
precipitation(31) . Trk/IR
containing retroviruses were used to infect 3T3-L1 fibroblasts, and
G418 (1 mg/ml)-resistant pools were selected. Expression of Trk/IR was
confirmed by immunoblotting using an antibody to the IR
-chain. To
overexpress the IR in NIH 3T3 fibroblasts (NIH/IR), the complete cDNA
of the human IR was subcloned into the retroviral expression vector
pBABE-puro. Retrovirus was obtained as above and used to infect NIH 3T3
cells, and puromycin (1 µg/ml)-resistant pools were selected. Wild
type (wt) and mutant murine p52 Shc proteins containing a point
mutation in either the PTB/PI domain (replacement of phenylalanine at
position 198 by valine, F198V) or the SH2 domain (replacement of
arginine at position 397 for lysine in the highly conserved FLVRES
motif, R397K) were tagged with the myc epitope and subcloned
into the retroviral expression vector pLEN(32) . Helper-free
infectious retroviruses were obtained and used to infect NIH/IR cells.
Shc requires an intact PTB/PI domain to be tyrosine-phosphorylated in insulin-stimulated cells. To identify residues in the PTB/PI domain of Shc that are important in binding phosphorylated peptides, random mutagenesis of Shc's PTB/PI domain was performed, and the ability of these mutants to bind the tyrosine-phosphorylated EGFR was determined (35) . One of the PTB/PI mutants identified that decreased binding to the autophosphorylated EGFR contained valine in place of phenylalanine at amino acid position 198 (F198V) of p52 Shc. This residue, which is highly conserved in most PTB/PI domains described so far, has recently been shown by NMR to interact with the NPXY asparagine(36) . We found that F198V Shc also impairs the binding of Shc to the IR using the yeast two-hybrid system (Fig. 1). In agreement with previous studies(7) , wt Shc bound the IR as demonstrated by the selection for growth of yeast on medium lacking histidine in the presence of 5 mM 3-aminotriazole. However, while the efficiency of yeast transformation of the F198V Shc is similar to that of wt Shc, F198V Shc did not support growth on medium lacking histidine. These results indicate that the F198V mutation impairs the binding of Shc to the IR in vitro.
Figure 1: Wild type Shc but not F198V Shc interacts specifically with IR in the yeast two-hybrid system. Saccharomyces cerevisiae strain L40 containing plasmid pBTM116 that expresses a fusion protein containing the LexA DNA-binding domain and cytoplasmic domain of the IR was transfected with either the plasmid pVP16 alone or pVP16 containing wt p52 Shc or F198V Shc as fusion proteins with the VP16 activation domain. The efficiency of transformation was determined by selecting yeast transformants on medium lacking tryptophan, leucine, and uracil. The interaction between the IR and Shc was determined by plating yeast on similar plates lacking histidine and containing 5 mM 3-aminotriazole.
After determining that F198V Shc does not interact with the IR in the yeast two-hybrid system, we determined whether this mutation affected tyrosine phosphorylation of Shc by the IR in vivo. We reasoned that if binding of Shc to the IR is required for Shc phosphorylation in cells, then F198V Shc would not undergo tyrosine phosphorylation in response to insulin stimulation. NIH/IR cells were infected with retroviruses containing either Myc epitope-tagged wt or F198V Shc, and G418-resistant pools of cells expressing the various Shc proteins were isolated. Cells were then stimulated with insulin, and Myc epitope-tagged Shc was immunoprecipitated using the anti-Myc antibody 9E10. Insulin stimulated tyrosine phosphorylation of wt p52 Shc (Fig. 2, lanes c and d) but did not stimulate tyrosine phosphorylation of F198V Shc (Fig. 2, lanes e and f) although a similar amount of F198V Shc as compared with wt Shc was immunoprecipitated. We also determined whether mutation of the arginine in the highly conserved FLVRES motif of the Shc SH2 domain (R397K) impaired tyrosine phosphorylation of Shc. This mutation has previously been shown not to inhibit the binding of Shc to the IR in vitro(7) . In contrast to the results obtained with F198V Shc, insulin stimulated increased tyrosine phosphorylation of R397K Shc (Fig. 2, lanes g and h). GRB2 also co-immunoprecipitated with tyrosine-phosphorylated Shc (Fig. 2, lanes d and h). These findings demonstrate that Shc requires an intact PTB/PI domain to be tyrosine-phosphorylated in insulin-stimulated cells.
Figure 2: F198V Shc is not tyrosine-phosphorylated by the IR. NIH/IR cells were infected with either vector alone, Myc-tagged wt, F198V, or R397K Shc, and G418-resistant pools of cells were obtained. Cells were either left unstimulated or stimulated with insulin and lysed. Cell lysates were either used directly or immunoprecipitated with the anti-Myc antibody 9E10 as indicated. The washed immunoprecipitates (IP) were then separated by SDS-PAGE (10%), transferred to nitrocellulose membranes, and immunoblotted with antibodies as indicated. The positions of the IR, p52 Shc, p46 Shc, and GRB2 are indicated on the right. Ptyr, phosphotyrosine.
Figure 3: Construction and expression of Trk/IR chimeras in 3T3-L1 fibroblasts. A, schematic diagram of mutant Trk/IR constructs. The restriction site introduced with each mutation is indicated. EC, extracellular domain of the human TrkA receptor; TM, transmembrane domain of the human IR; IC, intracellular domain of the human IR. B, alignment of the amino acids surrounding several NPXY motifs on different receptors and middle T antigen that phosphorylate IRS-1 and/or Shc. The position of the tyrosine in the NPXY motif is indicated. Consensus amino acid sequences for Shc and IRS-1 binding domains analyzed in this study are indicated in boldface. The core NPXY motif is underlined. h, indicates hydrophobic amino acid; *, amino acid position corresponds to the precursor form of the protein. C, expression of wt and mutant Trk/IR constructs in 3T3-L1 fibroblasts. 25 µg of lysates from G418-resistant pools of 3T3-L1 fibroblasts expressing the various Trk/IR constructs were separated by SDS-PAGE (7.5%) and immunoblotted with antibodies to the IR or phosphotyrosine (Ptyr) as indicated.
Figure 4: Tyrosine phosphorylation of Shc and IRS-1 by wt and mutant Trk/IRs. 3T3-L1 fibroblasts overexpressing various Trk/IR constructs were either left unstimulated or stimulated with 1 µM insulin (I) or 100 ng/ml NGF (N), lysed, and immunoprecipitated with antibodies to Shc or IRS-1. The washed immunoprecipitates were then separated by SDS-PAGE (Shc immunoprecipitate, 10%; IRS-1 immunoprecipitate, 7.5%), transferred to nitrocellulose membranes, and immunoblotted with antibodies as indicated. Ptyr, phosphotyrosine. C, control.
In agreement with our results, several
studies have shown that, in addition to NPX-Tyr(P), a leucine
or hydrophobic amino acid at a position -5 from the NPXY
phosphotyrosine (Tyr(P)-5) (equivalent to Ser-955 of the IR) is
critical for binding the PTB/PI domain of Shc. Deletion of this leucine
or substitution of alanine or glycine markedly reduced binding of the
PTB/PI domain of Shc to NPXY containing
phosphopeptides(7, 11, 12, 22) . In
addition, Kavanaugh et al. (9) found that a
hydrophobic or aromatic residue at the (Tyr(P)+1) position
(equivalent to Leu-961 of the IR) was important for the binding of the
PTB/PI domain of Shc to c-ErbB-2(9) . The three-dimensional
structure of Shc complexed to a phosphopeptide as recently determined
by nuclear magnetic resonance (NMR) may help explain the role of the
Tyr(P)-5 and Tyr(P)+1 positions in binding of Shc's
PTB/PI domain to NPXY phosphopeptides (36) . A
hydrophobic amino acid at the Tyr(P)-5 position contacts a
hydrophobic pocket formed by the PTB/PI domain of Shc, and the amino
acid at the Tyr(P)+1 position forms a hydrophobic interaction with
the aliphatic side chains of arginine 67 of Shc. These structural
findings, together with the finding that phosphorylation of Shc by the
mutant Trk/IRs correlates with binding of Shc's PTB/PI domain to
other NPX-Tyr(P)-containing phosphopeptides, support a model
in which binding of Shc via its PTB/PI domain directly to the IR is
critical for insulin-stimulated Shc phosphorylation in cells. In
addition, the inability of L961R Trk/IR to tyrosine phosphorylate Shc
may explain why the IL-4R binds and mediates tyrosine phosphorylation
of IRS-1 but not Shc. The IL-4R subunit, which phosphorylates
IRS-1 but not Shc(19, 37) , is similar to L961R Trk/IR
in that it contains an arginine at the Tyr(P)+1 position rather
than the hydrophobic amino acid, which is present in receptor tyrosine
kinases that bind the PTB/PI domain of Shc (Fig. 3B).
While this paper was in preparation, He et al. (38) reported an analysis of the interaction of Shc and IRS-1 with IRs containing a series of amino acid substitutions surrounding the NPXY motif using the yeast two-hybrid system. We have been able to extend some of their observations to Shc phosphorylation in vivo. However, in contrast to the findings reported here, He et al. (38) did not find that serine at position 955 was important for binding the PTB/PI domain of Shc. Interestingly, He et al. (38) also demonstrated that substitution of leucine at position 952 and tyrosine at 953 with alanine led to reduced IRS-1 but not Shc binding. These findings led the authors to conclude that hydrophobic residues at these two positions are critical for binding and tyrosine phosphorylation of IRS-1 by the insulin and IL-4Rs. Our demonstration that substitution of tyrosine 953 with phenylalanine (Y953F) does not impair tyrosine phosphorylation of IRS-1 (Fig. 4, lanes m-o) is consistent with their conclusion that a bulky hydrophobic residue at this position is critical for in vivo binding and phosphorylation of IRS-1. However, in contrast to their results, substitution of alanine for leucine in Trk/IR (L952A) did not reduce IRS-1 phosphorylation in vivo by NGF (data not shown).
Shc and IRS-1 appear to bind the IR with a relatively low affinity compared with the binding of SH2 domains to their target proteins or the binding of the PTB/PI domain of Shc to other targets such as the EGFR or middle T antigen. For example, as stated above we and others have been unable to co-immunoprecipitate Shc with the IR from insulin-stimulated cell lysates(17, 18, 25) . In addition, we have been unable to demonstrate binding of a GST-Shc fusion protein containing amino acids 1-209 of Shc to either the activated wt IR or the chimeric Trk/IR by far Western analysis, although binding to the activated EGFR could be easily detected (data not shown). The presence of a serine rather than a hydrophobic amino acid at the Tyr(P)-5 position in the IR may account for the low affinity binding of Shc to the IR. It is possible that this low affinity binding facilitates signaling. The interaction of Shc, IRS-1, and IRS-2 with the IR, while being of a sufficient affinity to localize these molecules adjacent to the IR kinase to promote phosphorylation, may be of a low enough affinity to allow these molecules to rapidly dissociate from the activated receptor following phosphorylation. The binding affinity of Shc and IRS-1 to the IR may be reduced as a result of a rapid dissociation rate from the activated IR. Such rapid dissociation of Shc, IRS-1, and IRS-2 from the IR may ensure that all these molecules that compete for the same binding site on the IR have a chance to bind and become phosphorylated. In addition, the rapid rate of dissociation from the receptor may enable IRS-1 to target active associated signaling molecules such as phosphatidylinositol 3-kinase to specific locations in the cell.
In summary, the findings reported here support the idea that Shc must bind directly to the activated IR via its PTB/PI domain to become tyrosine-phosphorylated in vivo. Analysis of the mutants reported in this study together with those reported by other investigators (38) support a model in which both Shc and IRS-1 recognize a core NPX-Tyr(P) motif on the IR, with amino acids surrounding this motif being critical for binding either IRS-1 or Shc in cells. The mutant IRs described in this report should provide a powerful tool to uncover biological functions that are predominantly mediated by Shc or IRS-1 in insulin-stimulated cells.