(Received for publication, February 27, 1996; and in revised form, March 25, 1996)
From the
Shc proteins (hereafter referred to as ShcA) represent major
substrates of tyrosine phosphorylation by a wide variety of growth
factors and cytokines. We have recently described a novel ShcA-like
protein, ShcC, which like ShcA contains an NH-terminal
phosphotyrosine binding domain (PTB), a central effector region (CH1)
and a COOH-terminal Src homology 2 domain (SH2). Both the SH2 and PTB
domains of ShcC bind a similar profile of proteins as the comparable
regions of ShcA. In an effort to define the functional differences or
similarities between ShcA and ShcC, we have further characterized the
PTB domain of ShcC. Using a degenerate phosphopeptide library screen,
we show that the PTB domain of ShcC preferentially binds the sequence
His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P). This sequence
is similar to the binding site for the ShcA PTB domain, suggesting that
these two proteins may have overlapping specificities. In addition,
random mutagenesis of the ShcC PTB domain has identified several amino
acids important for PTB function (Gly
, Glu
,
Ala
, Gly
, and Asp
). Mutation
of these amino acids dramatically reduces the affinity of the ShcC PTB
domain for the activated epidermal growth factor receptor in
vitro.
Tyrosine phosphorylation represents a critical switch in the
regulation of cell growth, differentiation, and development.
Phosphorylation of cellular proteins on tyrosine residues creates high
affinity binding sites for proteins containing Src homology 2 (SH2) ()domains. SH2 domains recognize tyrosine and the 3-6
amino acids COOH-terminal to the phosphotyrosine. The selectivity of a
particular SH2 domain is dictated by these COOH-terminal amino acids.
Recently another phosphotyrosine binding domain (PTB) has been
described(1, 2, 3) . This domain, also known
as PI (phosphotyrosine interaction domain) and SAIN (Shc and IRS-1
NPXY binding), recognizes phosphotyrosine in the context of
amino acids NH
-terminal to the phosphotyrosine. Thus, PTB
and SH2 domains represent distinct protein modules that recognize
tyrosine-phosphorylated proteins, but under entirely different
contexts.
PTB domains were first described in the adaptor protein
ShcA(1, 2) . ShcA represents a major target of
tyrosine phosphorylation following stimulation by a variety of growth
factors and cytokines(4) . Upon activation of receptor tyrosine
kinases, ShcA becomes physically associated with the receptor and
phosphorylated on tyrosine. This association was initially believed to
occur through the SH2 domain of ShcA(5) . Indeed, the ShcA SH2
binding site on the EGFR was mapped using a combination of in vitro binding and phosphopeptide competition
assays(6, 7) . The peptide selectivity of the ShcA SH2
domain was determined using a degenerate phosphopeptide library screen (8) . These results suggested that a number of receptors had
putative ShcA binding sites. However, some confusion arose as to the
true ShcA binding site due to the finding that ShcA association with
the polyoma virus middle T antigen occurred through an Asn-Pro-Thr-Tyr
sequence and not the consensus ShcA SH2 binding
sequence(9, 10) . In addition, the association of ShcA
with a 145-kDa phosphoprotein in platelet-derived growth
factor-stimulated cells was shown to occur not through the SH2 domain,
but rather through the NH terminus(2) . The
determination that ShcA contains two distinct phosphotyrosine binding
motifs, a COOH-terminal SH2 and a NH
-terminal PTB, provided
an explanation for these observations. PTB recognition sites are also
present in the nerve growth factor receptor (TrkA), the insuln and
insulin-related receptors, interleukin-2 receptor, and the EGFR.
We
have recently described the identification of two shc-like
genes which we called shcB and shcC(11) . shcB is nearly identical in sequence to the partial human shc-like gene sck and most likely represents the
mouse homolog of this gene(11) . shcC, however, has
not yet been found in other organisms. In contrast to the wide
expression of shcA, shcC is restricted in expression
to tissues of neural origin, suggesting a role for this adaptor protein
in brain-specific tyrosine kinase signaling. Like ShcA, ShcC contains
an NH-terminal PTB domain, a central proline-rich region
(CH1) and a COOH-terminal SH2 domain. In addition, ShcC binds to
activated growth factor receptors through both its SH2 and PTB domains.
In this report, we have further characterized the PTB domain of ShcC.
We have determined the phosphopeptide selectivity of the ShcC PTB
domain as well as describe a number of point mutations that
dramatically reduce the affinity of the ShcC PTB domain for activated
growth factor receptors. These mutations occur in conserved regions of
the PTB domain, suggesting an important role for these amino acids in
phosphotyrosine recognition and binding.
A number of groups have defined the sequence requirements of a phosphopeptide for binding the ShcA PTB domain (12, 15, 16, 17, 18) (Table 1). The ShcA and ShcC PTB domains share a high degree of amino acid sequence homology (78% identity; Fig. 1). Given our interest in further defining functional differences or similarities between ShcA and ShcC, we have examined the peptide specificity of bacterially expressed ShcC PTB using a degenerate phosphopeptide library screen (see ``Experimental Procedures''). Using this strategy, the ShcA PTB was shown to select phosphopeptides containing the sequence Asn-Pro-X-Tyr(P)-Phe-X-Arg with the strongest selectivity at positions -3 and -2 relative to the Tyr(P) (12) (Table 1). Indeed, this motif (Asn-Pro-X-Tyr(P)) is present in a number of receptor tyrosine kinases, including TrkA, EGFR, and the insulin receptor as well as polyoma virus middle T-antigen.
Figure 1:
Alignment of PTB domains. Shown is an
alignment of PTB domains from a number of proteins. This alignment does
not include all of the potential PTB containing proteins described thus
far(20) . The alignment was created using the Pileup program of
the Genetics Computer Group software analysis package then imported
into the Maligned multiple sequence alignment program and modified
based on the reported NMR structure(21) . Shown above the
alignment is the predicted secondary structure of the PTB domains based
on the NMR structure of ShcA. We have not included the 1 helix in
this alignment, since our PTB constructs did not contain this region. Asterisks indicate the positions of amino acid changes. The arrow indicates the position of the Arg
mutation
of ShcA(18, 21) . ShcA and ShcC represent the
predicted peptide sequences encoded by the respective mouse genes
(GenBank(TM) accession numbers U15784 and U46854, respectively). The
ShcC sequence begins at amino acid 29. The ShcA sequence begins at
amino acid 46. The accession numbers for the remaining sequences are
listed in (20) .
Using a modification of the above
phosphopeptide library screen, we determined the phosphopeptide
selectivity of the ShcC PTB domain (Table 1). Previous
experiments with ShcA indicated that Asn at the -3 position was
absolutely required for efficient PTB binding(12) . In
addition, most of the selectivity of PTB domains appears to be dictated
by residues NH-terminal to Tyr(P). Therefore, to increase
the sensitivity of our experiments and to examine the importance of
residues at -6 to -4, the -3 position of the
phosphopeptide library was fixed as Asn. The library consisted of
peptides containing the sequence
Met-Ala-X-X-X-Asn-X-X-Tyr(P)-X-Ala-Lys-Lys-Lys,
where X corresponds to any amino acid except for Trp and Cys.
The ShcC PTB domain selects phosphopeptides containing the sequence
His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P) ( Fig. 2and Table 1). There also appears to be some
selectivity at the +1 position for small chain amino acids (Table 1). These data suggest that the ShcC PTB may bind to
similar phosphoproteins as the ShcA PTB. In agreement with these data,
we have shown that the ShcA and ShcC PTB domains bind in vitro to the activated NGFR and EGFR in growth factor-stimulated cells
with relatively equal affinities and this binding can be competed away
with a phosphopeptide modeled on the Tyr
juxtamembrane
autophosphorylation site of TrkA(11) . In addition, both ShcC
and ShcA PTB domains bind a 170-kDa phosphoprotein in EGF-stimulated
A431 cell lysates (Fig. 3A). The identity of the
protein is currently unknown. Thus, the ShcC PTB domain shares common
recognition specificities with the corresponding PTB domain of ShcA.
Figure 2:
Phosphopeptide selectivity of the PTB
domain of ShcC. A degenerate phosphopeptide library was used to examine
the specificity of the ShcC PTB domain (see ``Experimental
Procedures''). The peptides that bound to immobilized GST-ShcC PTB
were purified and sequenced, and the sequence of the purified peptides
was compared with that of the starting peptide library. The data were
normalized such that a value of 1 or less indicates no selectivity for
a given amino acid(33) . A, B, C, D, E and F show, respectively, the
selectivity ot the Tyr(P),
Tyr(P)
, Tyr(P)
,
Tyr(P)
, Tyr(P)
, and
Tyr(P)
positions. Amino acids are given in their
one-letter codes.
Figure 3: Mutations in the PTB domain of ShcC altered the binding to the activated EGFR. A, in vitro binding of the ShcC PTB domain to the activated EGFR. GST fusion proteins for each PTB were purified and used in an in vitro binding experiment, as described previously(11) . Western blots were probed with anti-phosphotyrosine and anti-GST antibodies. Signals were quantitated on a Bio-Rad phosphorimager. B, quantitation of PTB binding data shown in A. Shown are the binding affinities of the various ShcC PTB mutants relative to wild type. These results are the average of three independent binding assays with different preparations of GST-PTB protein. The graph on the right represents the results of binding experiments with the wild-type PTB, mutant 4 PTB, and the mutant 4 PTB derivatives 4a and 4b. Standard errors are indicated with bars.
To better understand the importance of particular amino acid residues in the PTB domain for recognition of target phosphoproteins, we set out to mutagenize the ShcC PTB domain (Fig. 1). In the absence of structural information regarding the importance of particular regions of the PTB domain, we employed a random mutagenesis approach with the aim of identifying amino acids important for PTB function. Using hydroxylamine mutagenesis and PCR, we isolated a number of PTB mutants, several of which are impaired in their ability to bind the phosphorylated EGFR (Table 2). Mutant 2, which contains a single point mutation (E63G), has dramatically reduced binding (>10-fold) to the EGFR as compared with the wild-type PTB (Fig. 3). This Glu residue at position 63 of ShcC (amino acid 80 of ShcA) is conserved in 14 of 22 PTB domains described thus far ( Fig. 1and (19) ). Four of the remaining eight PTB domains possess a conserved Asp. These findings suggest that a negatively charged side chain amino acid at this position plays a critical role in the recognition of tyrosine-phosphorylated substrates of the ShcC PTB domain.
In addition to the E63G single mutation, PTB mutant 7, which possesses two tandem amino acid substitutions (G139E/D140N), also has dramatically reduced binding (>10-fold) to phosphotyrosine substrates (Fig. 3). These two amino acids occur in a region of the PTB domain which is highly conserved in Shc family members as well as other PTB containing proteins (Fig. 1). In particular, Asp is present in 15 of the 22 PTB domains(19, 20) , suggesting an important role in PTB function. PTB mutant 4 also has dramatically reduced affinity (>9-fold) for the activated EGFR. This mutant contains three amino acid substitutions (G32R, A136T, C166R; Table 2). The Gly is conserved in 11 of 19 PTB domains, and the Ala is present in 6 of 22 PTB domains, suggesting a conserved role for these amino acids in PTB function. The Cys, however, is only present in 3 of 21 PTB domains, which suggests that this amino acid may not be critical for PTB function(19, 20) . We have separated this triple mutant into a single mutant containing the G32R mutation and a double mutant containing the A132T and the C166R mutations. The relative binding affinities of these two mutants compared with the triple mutant and wild-type PTB domains were determined. Although the triple mutant had approximately a 90% decrease in binding the activated EGFR, neither the single nor double mutant were severely impaired in binding. The G32R mutation resulted in approximately a 20% decrease in binding, and the A136T/C166R double mutation resulted in approximately a 50% reduction in binding. We have not assessed the individual contributions of the A136T or C166R mutations. We believe the severity of the triple mutant is due to a synergistic effect of the three mutations on PTB binding function. We do not believe that the triple mutant is defective in binding due to a decrease in the stability of the protein, since equivalent amounts of fusion protein are obtained for mutants 4, 4a, and 4b (data not shown).
During the course of this study, the NMR
solution structure of the PTB domain of ShcA complexed to a TrkA
phosphopeptide was described(21) . The tertiary structure of
the PTB domain is composed of two antiparallel -sheets formed by a
series of seven
strands and three
helices. The overall
topology of the PTB domain bears a striking resemblance to that of
another modular domain, the pleckstrin homology (PH) domain, although
these two domains lack any sequence homology. The ShcA and ShcC PTB
domains share a high degree of sequence identity. Overall these two
domains are 78% identical particularly in the regions that form
specific contacts with the phosphopeptide as determined by
NMR(21) . For example, the
5 strand is 100% identical in
ShcA and ShcC and forms four contacts with the phosphopeptide ligand (21) . These findings suggest that the solution structure of
the ShcC PTB domain may be very similar to that of ShcA. Therefore, we
have analyzed our mutations using the ShcA PTB structure as a framework
for comparison. The E63G mutation occurs in the middle of
2 helix,
which connects the
1 and
2 strands. These
strands
comprise part of a
-sheet that forms a hydrophobic pocket into
which the phosphopeptide binds. Thus, the E63G mutation likely disrupts
important ionic interactions with the
2 helix, thereby abrogating
phosphopeptide recognition. The G139E/D140N mutations occur in a loop
between the
5 and
6 strands. The
5 strand forms several
contacts with the phosphopeptide backbone(21) . Gly
appears important for forming a proper turn between these two
strands, which allows for their antiparallel arrangement. Thus,
the G139E/D140N double mutant likely disrupts these contacts by
restricting the ability of the loop to form a turn, thereby disrupting
the alignment of
5 and
6 and diminishing the affinity of the
PTB for phosphopeptide.
The three mutations present in PTB mutant 4
occur in different regions of the PTB domain. Of particular interest is
the fact that the A136T mutation occurs in the 5 strand, which
forms part of the cleft into which the phosphotyrosine
binds(21) . Several amino acids in this
strand, including
Ala
, are in close proximity with the phosphopeptide.
Mutation of Ala
likely disrupts these contacts, thereby
abrogating binding to the activated EGFR. Thus, the A136T mutation
likely accounts for the majority of the reduction in EGFR binding by
mutant 4. The A136T/C166R double mutant does not appear to be as
impaired in binding as the triple mutant (Fig. 3C).
Although we have not assessed the individual effects of these two
substitutions, we believe that the C166R mutation may have some
compensatory effect in the context of the double mutant. This
compensation in binding may not occur in the context of the triple
mutant.
In addition to mutations which affect phosphotyrosine binding, a number of PTB mutants are unaffected in their interaction with the activated EGFR (Fig. 3). Many of these mutations represent conservative substitutions that likely do not have a profound affect on the structure or interactions with other amino acids within the PTB domain itself or the phosphopeptide. Many of the nonconservative substitutions occur in loop regions that tend to be more resistant to mutational effects due to the ability of these regions to move freely in space.
We have identified several mutations in the PTB domain of a novel adaptor protein, ShcC, which dramatically reduce the affinity of its PTB domain for the activated EGFR. Based on the predicted structure of the ShcA PTB domain, these mutations likely disrupt regions important in phosphopeptide recognition and binding. Several groups have identified additional mutants in the ShcA PTB domain that affect PTB binding to phosphotyrosine containing proteins(18, 20, 21) . Interestingly, Yajnik et. al. (20) independently isolated an Ala to Thr mutation at amino acid 153 in ShcA, which is identical to the A136T mutation present in mutant 4 of ShcC. This mutation resulted in a 74% reduction in binding of the ShcA PTB to the activated EGFR, further supporting the notion that the A136T mutation of mutant 4 is indeed the critical amino acid mutation affecting binding.
Mutation of
Arg of ShcA to either Glu, Met, or Lys completely
abolishes phosphotyrosine binding, suggesting a critical role for this
Arg in substrate binding(18, 21) . Based on the
recently described structure of the ShcA PTB domain, Arg
directly participates in binding the phosphotyrosine residue of
the phosphopeptide ligand. Interestingly, this Arg is not absolutely
conserved in all PTB domains(19) . This finding is in contrast
to SH2 domains, which contain an absolutely conserved Arg, mutation of
which blocks SH2 binding to tyrosine-phosphorylated
proteins(22, 23) . In addition to Arg
,
mutation of Phe
drastically reduces binding (<1% of
wild type)(20) . Phe
, in contrast to
Arg
, is conserved in the majority of PTB domains
described thus far(19, 20) . Interestingly, the IRS-1
PTB domain binds to a similar sequence as the ShcA and ShcC PTB
domains, yet shares no apparent sequence homology. These observations
suggest that although different PTB domains may lack primary sequence
homology, they may adopt similar three-dimensional structures. Indeed,
the PTB domain of ShcA and the PH domain of pleckstrin share a similar
three-dimensional structure in the absence of sequence
homology(21) . Alternatively, PTB domains lacking a comparable
Arg
as found in the PTB domains of Shc family members may
adopt a different structure and, thus, employ a different mechanism for
phosphopeptide recognition and binding. Determining the structures of
other PTB domains will address these possibilities.
We have examined the peptide selectivity of the ShcC PTB domain. This PTB domain has a similar selectivity as compared with the PTB domain of ShcA. Similar results were obtained with the SH2 domains of Shc family members(11) . The similarity in the sequence and the peptide selectivities of both ShcA and ShcC PTB and SH2 domains suggests that these two adaptor proteins may share overlapping functions, but in different cell types. Indeed, both proteins interact with similar receptors and tyrosine-phosphorylated proteins in vitro(11) . The identification of mutant PTB domains presents the possibility of designing ShcC dominant interfering mutants that may block signaling from tyrosine kinases as well as other proteins that signal through Shc family members.
In addition to binding tyrosine-phosphorylated proteins, the PTB domain of ShcA has also been shown to bind phospholipids(21) . Furthermore, the SH2 domains of phosphatidylinositol 3`-kinase, as well as Src and Abl, have been shown to bind phospholipids(24) . This interaction with lipids provides a possible explanation for how ShcA may translocate to the membrane to activate Ras in the absence of direct binding to activated growth factor receptors(25, 26, 27, 28) . In addition, the interaction of ShcA with phospholipids may play a role in the regulation of phospholipid metabolism.