(Received for publication, December 15, 1994)
From the
Src homology 2 (SH2) domains are phosphotyrosine binding modules found within many cytoplasmic proteins. A major function of SH2 domains is to bring about the physical assembly of signaling complexes. We now show that, in addition, simultaneous occupancy of both SH2 domains of the phosphotyrosine phosphatase SH-PTP2 (Syp, PTP 1D, PTP-2C) by a tethered peptide with two IRS-1-derived phosphorylation sites potently stimulates phosphatase activity. The concentration required for activation by the tethered peptide is 80-160-fold lower than either corresponding monophosphorylated peptide. Moreover, the diphosphorylated peptide stimulates catalytic activity 37-fold, compared with 9-16-fold for the monophosphorylated peptides. Mutational analyses of the SH2 domains of SH-PTP2 confirm that both SH2 domains participate in this effect. Binding studies with a tandem construct comprising the N- plus C-terminal SH2 domains show that the diphosphorylated peptide binds with 60-90-fold higher affinity than either monophosphorylated sequence. These results demonstrate that SH-PTP2 activity can be potently regulated by interacting via both of its SH2 domains with phosphoproteins having two cognate phosphorylation sites.
Many cell surface receptors signal their effects by initiating a
cascade of tyrosine phosphorylation reactions. Often the receptors are
themselves tyrosine kinases (e.g. insulin, PDGF, ()and epidermal growth factor receptors), whereas in other
cases the receptors are non-covalently associated with cytoplasmic
tyrosine kinases (e.g. B- and T-cell and cytokine receptors).
In each case, engagement of the extracellular ligand binding site leads
to the phosphorylation of intracellular receptor and/or substrate
tyrosines, which frequently act as docking sites for proteins with Src
homology 2 (SH2) domains. SH2 domains are phosphotyrosine binding
modules associated with a wide variety of cytoplasmic enzymes,
including phospholipid kinases and lipases, protein tyrosine kinases
and phosphatases, and enzymes that regulate Ras proteins. The cell
surface receptors are thus physically coupled via SH2 domains to
various enzymes that effect changes in such diverse cellular functions
as differentiation, growth, and metabolism.
Tyrosine kinases initiate signaling events by phosphorylating proteins, SH2 domain proteins bind to the phosphorylation sites to propagate the signals, and protein tyrosine phosphatases (PTPases) catalyze the dephosphorylations that can terminate the signals. SH-PTP2 (also called Syp, PTP1D, and PTP2C) is a cytoplasmic PTPase with two SH2 domains(1, 2, 3, 4) , which embodies all three levels of regulation. SH-PTP2 is phosphorylated by and thus is a substrate of tyrosine kinase receptors(2, 3, 5) . Once phosphorylated SH-PTP2 provides a docking site for Grb2, a protein with SH2 and SH3 domains that is coupled to Ras activation(6, 7) . In addition, the SH2 domains of SH-PTP2 bind directly to phosphorylation sites on tyrosine kinases like the PDGF receptor and kinase substrates like IRS-1(2, 3, 5, 8) . The PTPase domain of SH-PTP2 may act to dephosphorylate SH2 domain-bound proteins like IRS-1(9) , although additional studies are needed to more fully characterize the catalytic targets of SH-PTP2. Several recent reports indicate that SH-PTP2 is a positive mediator of the effects of insulin and growth factors (6, 7, 10, 11) and suggest that its cellular substrates might include proteins that are inhibited rather than activated by phosphorylation (such as c-Src), although alternative mechanisms for positive signaling by SH-PTP2 certainly exist.
In addition to having multiple mechanisms for exerting biologic effects, studies conducted in vitro suggest several biochemical mechanisms for SH-PTP2 regulation. The full-length protein is a weak catalyst compared with other PTPases(12, 13) . However, its catalytic activity can be altered. Deletion either of the SH2 domains or a 57-residue fragment from the C terminus of SH-PTP2 significantly increases activity(12, 13) . No further enhancement in activity is observed when both regions are deleted, suggesting that in a mechanistic sense the effects are related. When either SH2 domain of intact SH-PTP2 is occupied by a high affinity phosphopeptide, catalytic activity is also enhanced, although not to as great a degree as domain truncation(14, 15) . Addition of phospholipids leads to increased catalytic activity as well, possibly indicating that SH-PTP2 is activated in vivo by associating with the plasma membrane (13) . Phosphorylation of serine or threonine residues within SH-PTP2 by mitogen-activated protein kinase decreases catalytic efficiency(16) , while tyrosine phosphorylations may increase activity(3) .
We have now devised a tethered bisphosphotyrosyl peptide ligand that binds with high affinity to both SH2 domains of SH-PTP2. At low concentrations the tethered ligand potently activates the PTPase, whereas at higher concentrations profound inhibition was observed. These findings provide insights into potential mechanisms of SH-PTP2 regulation during cellular signaling.
The substrate
[P]RCM-lysozyme (2 µM) and
phosphopeptides or control compounds (varying concentrations) were
incubated in 25 mM Hepes buffer (pH 7.4) containing 150 mM NaCl, 125 µg/ml bovine serum albumin, 5 mM EDTA, and
10 mM dithiothreitol. PTPase reactions (30 µl) were
initiated by adding wild type or mutated SH-PTP2 to final enzyme
concentrations ranging from 5 to 30 nM. After 5 min at 30
°C, reactions were terminated by adding a suspension of activated
charcoal. Following centrifugation, product release was measured as
[
P]phosphate in the supernatant solutions.
Linear rates for phosphate release were
observed(12, 15) .
Figure 1:
Stimulation of SH-PTP2 PTPase activity.
PTPase velocities (pmol of P released/min/pmol of enzyme)
are plotted versus ligand concentrations for
pY1172(X
)pY1222 (
), the monophosphorylated
peptides pY1172 (
) and pY1222 (
), alone or in mixture
(
), the unphosphorylated bispeptide Y1172(X
)Y1222
(
), and the X
tetramer of 6-aminocaproic acid
(
).
Since previous results suggested that
IRS-1-derived pY1172 and pY1222 peptides interact with the N- and
C-terminal SH-PTP2 SH2 domains,
respectively(15, 17) , we tested the
combined effect of the peptides on catalytic activation (Fig. 1). An equimolar mixture yielded a maximal velocity of 7.5
pmol/min/pmol. The maximal effect was at 200 µM total
peptide concentration, the ED
was 45 µM, and
inhibition was seen at concentrations greater than 200 µM.
Therefore, at low concentrations the effect of the peptides in mixture
was greater than might be expected if the effects were simply additive.
Since these results suggested that simultaneous occupancy of both
SH2 domains of SH-PTP2 might potently stimulate catalytic activity, we
were interested in designing a single ligand that could bridge the N-
and C-terminal SH2 domains. In the absence of a solved structure of
full-length SH-PTP2 or its tandem SH2 domains, it is difficult to
predict the distance between peptide binding sites of the two domains.
Since the N- and C-terminal SH2 domains bind with high affinity to
peptides IRS-1 pY1172 and IRS-1 pY1222, respectively, we wanted to
incorporate these sequences. However, the corresponding tyrosines are
separated in IRS-1 by 49 residues, so we opted to tether the peptides
with a chemical linker. Tyr-740 and Tyr-751 within the PDGF receptor
act as docking sites for PI 3-kinase p85 recognition, and a
bisphosphorylated peptide with similar spacings stimulates PI 3-kinase
activity at low concentrations(22) . Moreover, ZAP-70 and Syk
bind to tyrosine-based activation motifs comprising paired
YXXL sequences in which tyrosines are separated by 9-11
residues(23) . Therefore, we designed a single peptide with
similar spacing between phosphotyrosines that was predicted to interact
simultaneously with both SH2 domains of SH-PTP2. This was designed to
have sufficient length based on estimates from the aforementioned
systems and to be flexible but not too hydrophobic. Four 6-aminocaproic
acid molecules were incorporated sequentially to provide peptide IRS-1
pY1172(X)pY1222.
The bisphosphopeptide
pY1172(X)pY1222 stimulated catalytic activity much
more potently (80-160-fold) than either monophosphorylated
peptide (Fig. 1). Maximal effects were at concentrations between
1.0 and 100 µM. The ED
was
600
nM, compared with 45 µM for the peptide mixture
and >50 µM for either peptide alone. Moreover, the
level of catalytic activation was significantly greater for
pY1172(X
)pY1222 than the monophosphopeptides. The
maximal velocity was 20 pmol/min/pmol compared with 4.1-7.8
pmol/min/pmol for the mixture or either monophosphopeptide alone. We
were unable to distinguish whether predominant effects were on V
or K
because the
bisphosphopeptide-stimulated PTPase did not follow Michaelis-Menten
kinetics with the RCM-lysozyme substrate. However, using p-nitrophenyl phosphate as a substrate the mono- and
bisphosphorylated peptides clearly influence V
. (
)The unphosphorylated peptide
Y1172(X
)Y1222 had no effect on catalytic activity,
and the X
aminocaproic acid tetramer itself had no
effect either alone (Fig. 1) or when combined with the
monophosphopeptides (data not shown). These findings indicate that
tethering two high affinity motifs facilitates the simultaneous
occupancy of both SH2 domains of SH-PTP2, and this leads to a potent
stimulation of catalytic activity.
Figure 2:
Stimulation of wild type SH-PTP2 and
variants in which either or both SH2 domains were mutated. The arginine
B5 residues within the N- (R32K) and/or C-terminal (R138K) SH2
domains were mutated to lysine to reduce phosphopeptide
binding(15) . Results are shown for
pY1172(X
)pY1222 stimulation of wild type SH-PTP2
activity (
), SH-PTP2 variants mutated in either the N- (
)
or C-terminal (
) SH2 domain, or SH-PTP2 in which both domains were
mutated (
). Equivalent amounts of enzyme were used in each
case.
Figure 3:
N
+ C SH2 domain binding assays. Competition assays were conducted
with the tandem N- plus C-terminal SH2 domains and a radiolabeled
version of the pY1172(X)pY1222. Data are shown for
competition with pY1172(X
)pY1222 (
), the
corresponding monophosphorylated peptides IRS-1 pY1172 (
) and
IRS-1 pY1222 (
), an equimolar mixture of the mono-phosphorylated
peptides (
), and a tethered but unphosphorylated ligand
Y1172(X
)Y1222
(
).
Binding assays were also performed with a mixture of
peptides IRS-1 pY1172 and IRS-1 pY1222, and a tethered but
unphosphorylated peptide. The ED value for competition by
the equimolar mixture was 63 µM (total peptide
concentration). Therefore, a mixture of monophosphorylated peptides
acts in this assay essentially like the individual peptides. At the
highest concentrations the unphosphorylated, tethered sequence also
competes for binding. Since unphosphorylated sequences corresponding to
monophosphopeptides rarely compete at these concentrations for binding
with single SH2 domains (17, 18, 25) , the
additive effects of interactions that are not mediated by phosphate may
play a proportionately greater role for tandem SH2 domain interactions.
The following model may help to explain how the tethered ligand stimulates catalysis at low concentrations while inhibiting PTPase activity at higher concentrations. Under basal conditions SH-PTP2 exists in an inactivated, ``closed'' configuration (Fig. 4A). Removal of either its SH2 domains or its C-terminal tail leads to PTPase activation(15) . Since removal of either region leads to activation and simultaneous removal of both regions has no greater effect, it is likely that these truncations activate the PTPase by similar mechanisms (i.e. induction of an open configuration). Monophosphopeptide occupancy of the SH2 domains also stimulates catalytic rates, presumably by inducing a related open state (Fig. 4B). The tethered ligand binds simultaneously with both SH2 domains and in so doing stimulates catalytic activity (Fig. 4C). Since it binds to the SH2 domains with higher affinity, the tethered ligand exerts its stimulatory effects at much lower concentrations than either monophosphopeptide.
Figure 4:
Models for activation and inhibition of
the SH-PTP2 PTPase. A, the ``closed'' state. Under
basal, unstimulated conditions the unoccupied SH2 domains and
C-terminal tail region interact with one another or the PTPase domain
to inhibit PTPase activity. B, PTPase activation by
monophosphotyrosyl peptides. Peptide binding at high concentrations to
either SH2 domain stimulates catalysis by stabilizing an
``open,'' active form of the catalytic domain. C,
PTPase activation by the bisphosphopeptide. The tethered peptide binds
at low concentrations to both SH2 domains to stimulate catalysis. D, PTPase inhibition by the tethered ligand. At higher
bisphosphopeptide concentrations distinct ligands occupy the two SH2
domains. The unbound phosphotyrosine of each ligand is thus free to
interact with the PTPase domain to inhibit dephosphorylation of
[P]RCM-lysozyme.
At concentrations higher than those required for
activation the tethered ligand inhibited PTPase activity. In fact at
the highest concentrations there was no apparent dephosphorylation of
[P]RCM-lysozyme. PTPase inhibition was also
observed at the highest concentrations of monophosphorylated peptide
IRS-1 pY1172, suggesting that phosphopeptides can compete with
[
P]RCM-lysozyme at the PTPase active site.
Peptide IRS-1 pY1222 was not inhibitory, suggesting that it is a weaker
PTPase substrate (K
values for peptide inhibition
of [
P]RCM-lysozyme dephosphorylation should
parallel K
values for the peptides as direct
substrates). The tethered ligand may inhibit the PTPase at lower
concentrations than the monophosphopeptide because it is bound to one
of the SH2 domains (Fig. 4D). At higher concentrations
two tethered ligands can bind independently to two SH2 domains, and
this could bring the unligated phosphotyrosine of either bound
bisphosphopeptide into the proximity of the catalytic active site.
Since peptide IRS-1 pY1172 is a better substrate of the PTPase, we
predict that the tethered ligand might best inhibit PTPase action when
ligated to the C-terminal SH2 domain via its pY1222 sequence. This
model also provides a molecular ruler for estimating distances between
the SH2 domain binding sites and the PTPase active site. Since the
tethered ligand inhibits the PTPase when ligated to at least one of the
SH2 domains, the maximal distance between this SH2 and the PTPase
domains must be 74 Å as well.