(Received for publication, October 26, 1995; and in revised form, January 16, 1996)
From the
The phosphotyrosine binding (PTB) or phosphotyrosine interaction
(PI) domain of the proto-oncoprotein p52 binds to an
NPXpY consensus sequence found in several growth factor
receptors (Kavanaugh, W. M., Turck, C. W., and Williams, L. T.(1994) Science 268, 1177-1179). The amino-terminal region of
p52
, which includes the PTB/PI domain, has been
previously shown to associate with protein-tyrosine phosphatase-PEST
(PTP-PEST) in vivo (Habib, T., Herrera, R., and Decker, S.
J.(1994) J. Biol. Chem. 269, 25243-25246). We report
here the detailed mapping of this interaction in a murine context using
glutathione S-transferase fusion protein binding studies and
peptide competition assays. We show that the interaction between murine
SHC and murine PTP-PEST is mediated through the PTB/PI domain of murine
SHC and an NPLH sequence found in the carboxyl terminus of murine
PTP-PEST. Since this interaction is not dependent on the presence of a
tyrosine-phosphorylated residue in the target sequence, this reveals
that the PTB/PI domain of SHC can recognize both
tyrosine-phosphorylated sequences and non-tyrosine-based recognition
motifs.
External stimuli are often transduced into intracellular events
via specific cascades of protein tyrosine phosphorylation and
dephosphorylation, which are modulated by the presence and availability
of adaptor molecules, protein-tyrosine kinases, and PTPases. ()The cytoplasmic adaptor molecule SHC is among many
mediators that act downstream of receptor type and cytoplasmic
protein-tyrosine kinases. SHC can transform fibroblasts and
differentiate PC12 cells in a Ras-dependent manner. SHC regulates these
signaling events through its own tyrosine phosphorylation on residue
Tyr
and by mediating the assembly of
tyrosine-phosphorylated signaling complexes via its SH2 and
phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domains (1, 2, 3) . The p52
NH
-terminal PTB/PI domain is a novel phosphotyrosine
recognition motif that is structurally unrelated to SH2 domains and
that was shown to bind with high affinity to the autophosphorylation
sites of c-Erb B2 Tyr
(4, 5) , TrkA
Tyr
(6, 7, 8) , EGF receptor
Tyr
(6, 7, 9, 10) ,
c-Erb B3 Tyr
(9) , insulin-like growth factor 1
receptor Tyr
(11) , insulin receptor
Tyr
(12) , and the phosphorylated Tyr
residue of polyoma middle T antigen(13) . Peptide
competition assays and screening of phosphotyrosine peptide libraries
have demonstrated that the PTB/PI domain of p52
preferentially binds to the sequence NPXpY (where X represents any amino acid and pY indicates a phosphotyrosine
residue) with high
affinity(5, 9, 10, 13, 14) .
The PTB/PI domain represents a novel mechanism whereby signaling
proteins can interact with tyrosine-phosphorylated protein targets.
MPTP-PEST is a ubiquitously expressed, stable, cytosolic PTPase of
112 kDa that is characterized by the presence of four Pro, Glu, Ser,
and Thr-rich PEST domains within the COOH terminus(15) .
MPTP-PEST is heavily phosphorylated on serine and threonine residues, ()and the enzymatic activity for the human homologue can be
modulated by phosphorylation on specific serine residues(16) .
Another possible mode of regulation by which the activities of
protein-tyrosine phosphatases might be controlled is via association
with other proteins (for review see (17) ). Recently, it has
been demonstrated that the human PTP-PEST protein interacts with the
human p52
protein in vivo(18) . In this
report we demonstrate that the murine p52
protein binds
to MPTP-PEST via its PTB/PI domain and that this binding is not
dependent on the presence of a phosphotyrosine residue in the target
sequence.
Using an affinity-purified antibody that
recognizes SHC proteins, we immunoprecipitated endogenous mSHC from NIH
3T3 cells under conditions where protein complexes have been shown to
co-immunoprecipitate(19) . The anti-mSHC immunoprecipitates
were screened for the presence of endogenous MPTP-PEST by Western
blotting using an anti-MPTP-PEST antibody (Fig. 1). An
immunoreactive band of 112 kDa (corresponding to the electrophoretic
size of MPTP-PEST) is present in the anti-mSHC immunoprecipitates. In
addition, screenings of a mouse embryonic cDNA library in a yeast
two-hybrid system using p52 as a probe resulted in the
isolation of cDNA clones coding for MPTP-PEST (data not shown). These
results indicate that SHC proteins interact with MPTP-PEST in
vivo.
Figure 1: SHC interacts with MPTP-PEST in vivo. 500 µg of NIH 3T3 cell lysates were immunoprecipitated using an affinity-purified anti-SHC polyclonal antibody. The immunoprecipitates (IP) and 10 µg of NIH 3T3 cell lysates were subjected to Western blot and analyzed for the presence of bound MPTP-PEST proteins (arrow) using an anti-MPTP-PEST antibody.
Figure 2: MPTP-PEST associates with the PTB domain of mSHC. Schematic representation (A) and Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis (B) of the different GST-SHC fusion proteins used in the MPTP-PEST/SHC binding studies are shown. C, in vitro binding of HA-MPTP-PEST proteins to various GST-SHC fusion proteins. Identical amounts (100 ng) of the different fusion proteins shown in A and B were incubated with COS-1 cell lysate (200 µg) transiently expressing HA-MPTP-PEST proteins (see ``Materials and Methods'') Bound HA-MPTP-PEST proteins were detected using the anti-HA tag antibody 12CA5. The schematic in A is drawn to scale. Some of the GST-Shc fusion proteins shown in B display inherent proteolytic degradation associated with bacterial expression of SHC proteins. Immunoblot analysis in C is representative of three different experiments.
Using a series of GST-MPTP-PEST fusion proteins representing
different portions of the MPTP-PEST COOH terminus (Fig. 3, A and B), the region of MPTP-PEST required for binding to
p52 was determined by an in vitro binding assay.
The segment located between amino acids 576 and 613 of MPTP-PEST is
sufficient for binding to p52
protein (Fig. 3C). Interestingly, this region contains a
sequence (
NPLH
) that closely resembles the
p52
PTB/PI domain binding consensus motif
(NPXpY). In order to verify if the
NPLH
sequence of MPTP-PEST is involved in the interaction with mSHC, a
triple point mutant (N599I,P600A,H602L) was created in the
GST-MPTP-PEST-471-613 fusion protein background (Fig. 3, A-C, lanes labeled NPXH mut) and
analyzed for binding to p52
in vitro. By
mutating all three amino acids (N599I, P600A, and H602L), the binding
of GST-MPTP-PEST-471-613 fusion protein to p52
was
completely abolished (Fig. 3C). These results indicate
that the sequence NPXH, found in the COOH terminus of
MPTP-PEST, is required for binding to SHC in vitro.
Figure 3:
SHC binds to a region of MPTP-PEST
containing an NPXH sequence. Schematic representation (A) and Coomassie Blue-stained SDS-polyacrylamide gel
electrophoresis (B) of the GST-MPTP-PEST fusion proteins used
in the SHC/MPTP-PEST binding studies are shown. C, in
vitro binding of human p52 proteins to various
GST-MPTP-PEST fusion proteins. Identical amounts (100 ng) of the GST
fusion proteins shown in A and B were incubated with
200 µg of extracts from COS-1 cells transiently expressing human
p52
proteins as described under ``Materials and
Methods.'' Bound human p52
proteins (arrow)
were detected using an anti-SHC monoclonal antibody. The schematic in A is drawn to scale. Results shown in C are
representative of three different
experiments.
The same mutations (N599I, P600A, and H602L) were recreated in a full-length HA-MPTP-PEST protein, and the effects of these substitutions were studied in vivo. Immunoprecipitation of SHC proteins in COS-1 cells transfected with either wild type or NPXH mutant HA-MPTP-PEST cDNAs were probed for the presence of associated HA-MPTP-PEST molecules by Western blot analysis (Fig. 4). An immunoreactive band of 120 kDa corresponding to HA-MPTP-PEST co-immunoprecipitated with SHC in cells transfected with the wild type HA-MPTP-PEST cDNA but not in the NPXH mutant cDNA transfectants despite an equal amount of both HA-MPTP-PEST proteins present in the lysates (Fig. 4B). This demonstrates that mutating all three amino acids (N599I, P600A, and H602L) in the context of a full-length HA-MPTP-PEST protein eliminates binding to SHC proteins in vivo.
Figure 4:
The NPXH sequence of MPTP-PEST is
required for binding to SHC proteins in vivo. A, SHC
proteins were immunoprecipitated from identical amounts of extracts
derived from COS-1 cells transiently expressing either wild type
HA-MPTP-PEST (WT HA-MPTP-PEST) or HA-MPTP-PEST with NPLH
mutated to
IALL
(NPXH mut HA-MPTP-PEST) and untransfected cells as a
control. SHC immunoprecipitates were analyzed for the presence of bound
HA-MPTP-PEST proteins (upper panel) and for p46
,
p52
, and p66
proteins (lower
panel) using the anti-HA tag antibody 12CA5 and an anti SHC
monoclonal antibody, respectively. B, 40 µg of total cell
lysate (TCL) from the different transfectants were analyzed
for the presence of HA-MPTP-PEST proteins (wild type and NPXH
mutant) by Western blot analysis using the anti-HA tag 12CA5 antibody
as described under ``Materials and Methods.'' The results
shown in A and B are representative of two
independent experiments.
Figure 5:
Competition by peptides for HA-MPTP-PEST
binding to the PTB/PI domain of SHC. 200 µg of total cell lysate
from COS-1 cells transiently expressing HA-MPTP-PEST proteins were
incubated with 100 ng of GST-SHC-1-209 fusion protein in the
presence of the indicated concentrations of peptides (see
``Materials and Methods''). The noncompeted, bound
HA-MPTP-PEST was visualized by Western blot analysis using the anti-HA
tag antibody 12CA5. The blots were then stripped and reprobed with an
anti-GST antibody to assess loading (data not shown). The amino acid
sequences of the synthetic peptides are as follows: EGFR-1148, QISLDNPDYQQDF
; EGFR-P-1148,
QISLDNPDpYQQDF
; PEST-WT,
PLSFTNPLHSDDWH
; and Scrambled,
DPSTHSHPLWLNFD. The results shown are representative of at least three
independent experiments.
Similar peptide competition assays (10, 13) and
phosphopeptide library screenings (14) have previously
demonstrated that the -3 Asn, -2 Pro, and the
phosphorylated tyrosine residue in the NPXpY motif are
essential for SHC PTB/PI domain interaction with the autophosphorylated
sites of growth factor receptors. To determine the contribution of
these residues in the NPXH-mediated binding of SHC to
MPTP-PEST, mutant peptides were used in the same in vitro competition binding assays. An MPTP-PEST mutant peptide bearing a
N599D substitution is incapable of competition (Fig. 5). A
peptide with a P600G mutation is also inefficient at competing for
binding even at the highest concentration (Fig. 5). This
suggests that both the Asn and Pro
residues
are involved in the binding of HA-MPTP-PEST to the PTB/PI domain of SHC in vitro. However, substituting the His
for an
Ala residue did not alter the competing potential of the peptide when
compared with the wild type peptide. Finally, a peptide with a
scrambled amino acid sequence is incapable of competing for binding.
These results indirectly demonstrate that the binding of the PTB/PI
domain of SHC to the
NPLH
sequence of
MPTP-PEST in vitro is dependent on both the Asn
and Pro
amino acid residues.
In order to determine the importance of the
His residue for binding to SHC proteins in vivo,
we mutated the histidine to an alanine residue in the context of a
full-length HA-MPTP-PEST. This H602A mutant was assayed for binding to
SHC proteins in vivo under the same conditions described in
the legend to Fig. 4. Anti-HA-MPTP-PEST immunoblot analysis of
SHC immunoprecipitates reveals that the wild type but not the H602A
mutant co-immunoprecipitated with SHC proteins (Fig. 6). Upon
longer exposure of Fig. 6(upper panel), less than 5%
of the H602A HA-MPTP-PEST co-immunoprecipitated with SHC proteins (data
not shown). This demonstrates that changing the histidine 602 to an
alanine residue drastically reduces (>95%) the binding of SHC
proteins to HA-MPTP-PEST in vivo, suggesting that the
His
residue is essential for binding to SHC proteins in vivo.
Figure 6:
The His residue of MPTP-PEST
is necessary for binding to SHC proteins in vivo.A,
SHC proteins were immunoprecipitated from equal amounts of lysates
derived from COS-1 cells transiently expressing either wild type (WT) HA-MPTP-PEST or HA-MPTP-PEST with an H602A mutation and
untransfected cells as a control. SHC immunoprecipitates were probed
for the presence of bound HA-MPTP-PEST proteins (upper panel, Western:
HA) and for SHC proteins (lower panel, Western:
SHC) as described under ``Materials and
Methods.'' B, 20 µg of total cell lysate (TCL) from the different transfectants were probed for the
presence of HA-MPTP-PEST proteins (wild type and H602A mutant) by
immunoblot analysis as described above.
Our data show that the PTB/PI domain of SHC binds to the
murine protein-tyrosine phosphatase-PEST via an NPXH sequence.
Computer aided searches of sequence data bases have revealed that
several proteins contain regions that have sequence similarity to the
PTB/PI domain of p52(22) . However, their ability
to bind to tyrosine-phosphorylated residues in a sequence-specific
manner and/or to non-phosphotyrosine-containing recognition sequences
remains to be determined. The amino acids immediately surrounding the
NPLH sequence in human and murine PTP-PEST proteins are identical (data
not shown) and could underscore the importance of this region given the
relatively low overall identity between the two COOH termini.
Previous peptide competition assays reveal that distinct amino acids
located at different positions in the SHC PTB/PI domain binding sites
of the EGF receptor, polyoma middle T antigen, and c-Erb B2 proteins
are essential for binding(5, 10, 13) . In all
three examples, the Asn at position -3 and the phosphorylated
tyrosine residue are absolutely essential for high affinity binding,
but the location of other required amino acids seems to be
protein-specific (data not shown). Although these experiments were
performed by different groups and under slightly different conditions,
the results nevertheless demonstrate that the modality of binding for
the PTB/PI domain of SHC does not appear to be universal. This
diversity and the recent NMR studies of both the PTB/PI domain of SHC (23) and of a PTB/PI domain recognition binding sequence (13) suggest that structural components of the SHC PTB/PI
domain and its recognition binding site are critical. Therefore, a
detailed mutagenic analysis of the surrounding amino acids of the NPLH
sequence of MPTP-PEST could define the
requirements for binding of the SHC PTB/PI domain to the
nonphosphotyrosine target sequence of MPTP-PEST. Such analysis could
unmask unique residues required for binding in a
non-phosphotyrosine-dependent manner.
The necessity for the
His residue in MPTP-PEST for binding to SHC proteins in vivo, in addition to the lower binding affinities displayed
by the nonmodified wild type and H602A mutated MPTP-PEST peptides in vitro, suggest that the His
could be
post-translationally modified. Phosphohistidine is an important
intermediate in prokaryotic signal transduction pathways involved in
processes such as chemotaxis (24) and porin
expression(25) . Phosphohistidine has been found in many
eukaryotic proteins and has recently been implicated in eukaryotic
signal transduction in platelets(26) . The cytoplasmic tail of
P-selectin (a leukocyte adhesion molecule) undergoes rapid and
transient histidine phosphorylation on the peptide sequence
NPHSH
, where both histidine residues are
potential sites of phosphorylation following thrombin or collagen
stimulation of platelets(26) . It is therefore possible that
the sequence
NPLH
of MPTP-PEST could be
phosphorylated on the His
residue and that this
post-translational modification is required to achieve high affinity
binding to SHC proteins much in the same way phosphorylation of
tyrosine residues leads to an increase in binding affinity.
Phosphorylation, be it on histidine or other phosphorylatable amino
acids, may be an essential recognition parameter by which PTB/PI domain
binding occurs in a tyrosine-independent manner. Our data demonstrating
a phosphotyrosine-independent binding for this domain serves to
underscore the need to re-evaluate the specificity of this interaction.
In conclusion, we demonstrate that the PTB/PI domain of SHC interacts with the non-phosphotyrosine-based NPLH sequence of MPTP-PEST. In vivo and in vitro studies established that the Asn and Pro residues at positions -3 and -2, respectively, are essential for binding and that the histidine residue at position 0 may require post-translational modification for high affinity binding to SHC proteins.