©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Potent Stimulation of SH-PTP2 Phosphatase Activity by Simultaneous Occupancy of Both SH2 Domains (*)

(Received for publication, December 15, 1994)

Scott Pluskey (1) Thomas J. Wandless (2)(§) Christopher T. Walsh (2) Steven E. Shoelson (1)(¶)

From the  (1)Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 and the (2)Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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, (^1)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.


MATERIALS AND METHODS

Recombinant Proteins

Escherichia coli BL21 cells were transformed with pET vectors encoding either wild-type or mutated forms of SH-PTP2, including SH-PTP2(R32K), SH-PTP2(R138K), and SH-PTP(R32K/R138K). Following induction, harvesting, and cell lysis the proteins were purified to >90% homogeneity by chromatography on Q-Sepharose, phenyl-Sepharose, and Mono S HR columns (12, 15) . To generate the N + C SH2 domain as a fusion protein, cDNA encoding SH-PTP2 residues 1-267 was amplified and EcoRI sites were introduced at the 5` and 3` ends(7) . The polymerase chain reaction product was digested with EcoRI and ligated into a pGEX-2T vector. Glutathione S-transferase fusion proteins were expressed and purified as described(17) .

Phosphopeptide Synthesis

Phosphopeptides were synthesized following a modified N-Fmoc protecting group strategy(18) . Due to difficulties encountered in synthesizing peptides containing two phosphotyrosines, a polyethylene glycol-polystyrene resin (Millipore) was used, and problematic couplings were with HATU (O-(7-azabenzotriazolyl)-N,N,N`,N`-tetramethyluronium hexafluorophosphate) rather than BOP (benzotriazolyloxytris-(dimethylamino)-phosphonium hexafluorophosphate). 6-Aminocaproic acid was incorporated as its Fmoc derivative. The peptides were purified by reversed-phase high pressure liquid chromatography and characterized by amino acid composition and mass spectrometric analyses. Sequences of peptide and related polymers for these studies include (pY is phosphotyrosine; X is 6-aminocaproic acid): IRS-1 pY1172(X(4))pY1222, LNpYIDLDLVXXXXLSTpYASINFQK-NH(2); IRS-1 pY1172, SLNpYIDLDLVK-OH; IRS-1 pY1222, LSTpYASINFQK-OH; IRS-1 Y1172(X(4))1222, LNYIDLDLVXXXXLSTYASINFQK-NH(2); X(4), acetyl-XXXX-NH(2).

Phosphatase Assays

[P]RCM-lysozyme was prepared by phosphorylating RCM-lysozyme (Sigma) on its only tyrosine with recombinant Lck kinase (provided by M. Eck, Harvard Medical School, Boston, MA). The kinase (200 nM) and RCM-lysozyme (200 µM) were incubated with 500 µM [-P]ATP (700 µCi/ml) in 50 mM Hepes buffer, pH 7.5, containing 10 mM MgCl(2) and 2 mM sodium vanadate for 14 h at 30 °C. The product was precipitated with trichloroacetic acid (19) to yield [P]RCM-lysozyme having a specific activity of approx2000 cpm/pmol.

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) .

Phosphopeptide/N + C SH2 Domain Binding Assay

Binding assays with the tandem SH2 domain and bisphosphotyrosyl peptides were conducted as described previously for assays with single SH2 domains and monophosphopeptides(17, 18) . A version of IRS-1 pY1172(X(4)) pY1222 with an additional tyrosine at its N terminus was I-iodinated using sodium [I]iodide and lactoperoxidase(20) . Glutathione S-transferase/N + C SH2 domain fusion protein (0.5-1.0 µM), approx35 fmol of I-phosphopeptide, and varying concentrations of unlabeled peptides were combined in 150 µl of a pH 7.4 buffer containing 20 mM Tris-HCl, 250 mM NaCl, 0.1% bovine serum albumin, and 10 mM dithiothreitol. Glutathione-agarose (50 µl of a 1:10 slurry) was added, and the samples were incubated overnight at 22 °C with constant mixing. Radioactivity associated with the glutathione pellets was used to determine binding(17, 18) .


RESULTS AND DISCUSSION

PTPase Assays with Intact SH-PTP2

In the absence of an SH2 domain ligand, the velocity for SH-PTP2-catalyzed release of [P]phosphate was 0.48 pmol/min/pmol ([P]RCM-lysozyme concentrations were at or greater than K(m)). Tyrosines 1172 and 1222 of IRS-1 are phosphorylated by the insulin receptor and may provide docking sites for SH-PTP2 in cells(21) . The corresponding phosphopeptides IRS-1 pY1172 and IRS-1 pY1222 bind with high affinity to the N- and C-terminal SH2 domains of SH-PTP2, respectively (17) , (^2)and stimulate its catalytic activity (15) (Fig. 1). The maximal rate of P release was increased in the presence of IRS-1 pY1172 to 4.1 pmol/min/pmol. The half-maximal effect (ED) was at a peptide concentration of approx50 µM, the maximal effect was observed at concentrations from 100-500 µM, and inhibition of the maximal effect was seen at a higher peptide concentration (1.0 mM). Increasing concentrations of peptide IRS-1 pY1222 led to greater P release. The maximal rate was 7.8 pmol/min/pmol, and it appears that peptide concentrations greater than 1.0 mM might yield even higher rates as saturation was not obtained.


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(4))pY1222 (bullet), the monophosphorylated peptides pY1172 () and pY1222 (), alone or in mixture (), the unphosphorylated bispeptide Y1172(X(4))Y1222 (circle), and the X(4) 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) ,^2 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(4))pY1222.

The bisphosphopeptide pY1172(X(4))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 approx600 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(4))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(max) or K(m) 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(max). (^3)The unphosphorylated peptide Y1172(X(4))Y1222 had no effect on catalytic activity, and the X(4) 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.

PTPase Assays with SH-PTP2 Having Mutations in Its SH2 Domains

To test whether both SH2 domains were indeed involved in catalytic activation, the SH2 domains of SH-PTP2 were mutated to alter phosphopeptide binding. It had been suggested previously that mutation of the betaB5 arginine within the conserved FLVR sequence of the Abl SH2 domain to lysine abolished binding(24) . Therefore, the corresponding betaB5 arginines within either or both SH2 domains of intact SH-PTP2 were mutated to lysine(15) . The effects of these mutations on catalytic activation were assessed with the tethered peptide pY1172(X(4))pY1222 (Fig. 2). Using identical enzyme concentrations the highest level of stimulation was observed with wild-type SH-PTP2 (maximal velocity was 10.5 pmol/min/pmol at 5 µM peptide; the ED value was 600-700 nM). Point mutations in either SH2 domain decreased the effect. When the C-terminal domain was mutated, the maximal velocity was 6.2 pmol/min/pmol at 10 µM peptide; the ED value was 1.5 µM. When the N-terminal domain was mutated the maximal velocity was 4.6 pmol/min/pmol at 23 µM peptide, and the ED value was 1.7 µM. Point mutations in both SH2 domains had a greater effect, leading to a maximal velocity of 4.2 pmol/min/pmol at 50 µM peptide and an ED value of 2.2 µM. Therefore, RbetaB5K mutations lead to 2-3-fold reduced levels of activity, and up to an 8-fold increase in tethered peptide concentration was needed to elicit a maximal effect. However, the RbetaB5K mutations do not abolish these SH2 domain-mediated effects, as might be expected if these mutations really abolished binding. The effects seen here on catalytic rates are consistent with effects seen recently in binding assays with SH2 domains of SH-PTP1 and SH-PTP2 having corresponding RbetaB5K mutations, where a range of 3-10-fold reductions in relative affinity was observed. The progressive loss in maximal velocities as one or two SH2 domains were mutated, coupled with the increasing concentrations of peptide pY1172(X(4))pY1222 required for maximal effects, confirm the participation of both SH2 domains in activation by the tethered peptide.


Figure 2: Stimulation of wild type SH-PTP2 and variants in which either or both SH2 domains were mutated. The arginine betaB5 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(4))pY1222 stimulation of wild type SH-PTP2 activity (bullet), SH-PTP2 variants mutated in either the N- () or C-terminal (circle) SH2 domain, or SH-PTP2 in which both domains were mutated (). Equivalent amounts of enzyme were used in each case.



Binding Assays between Phosphopeptides and the Tandem SH2 Domains

Additional experiments provided a mechanism for understanding how the tethered pY1172(X(4))pY1222 peptide could stimulate higher levels of PTPase activation at much lower concentrations than the single peptides. A binding assay was developed to determine relative affinities of the tandem SH2 domains of SH-PTP2 for various phosphopeptides, analogous to previously described single SH2 domain assays(17, 18, 25) . A variant of peptide pY1172(X(4))pY1222 was radiolabeled for use as a tracer, and a protein containing the two SH2 domains of SH-PTP2 was expressed as a glutathione S-transferase fusion protein. The radiolabeled peptide interacted specifically with the double SH2 domain, and unlabeled peptide pY1172(X(4))pY1222 effectively competed for binding with an ED value of 3.3 µM (Fig. 3). In contrast, ED values for the corresponding peptides IRS-1 pY 1172 and IRS-1 pY1222 were 300 and 220 µM, respectively. Therefore, the monophosphorylated peptides bind to the tandem SH2 domain with 50-100fold lower affinity than peptide pY1172(X(4))pY1222. The binding data correlate well with the concentration dependences for catalytic activation; pY1172(X(4))pY1222 stimulates PTPase activity at 40-100-fold lower concentrations than either monophosphorylated peptide. Since it is likely that catalytic activation results directly from SH2 domain occupancy and the bisphosphoryl peptide binds with significantly higher affinity than either monophosphorylated peptide, it follows that catalysis is stimulated by the tethered peptide at correspondingly lower concentrations.


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(4))pY1222. Data are shown for competition with pY1172(X(4))pY1222 (), the corresponding monophosphorylated peptides IRS-1 pY1172 (circle) and IRS-1 pY1222 (), an equimolar mixture of the mono-phosphorylated peptides (bullet), and a tethered but unphosphorylated ligand Y1172(X(4))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.

Mechanisms for PTPase Activation and Inhibition

We have identified phosphopeptide sequences that stimulate the PTPase activity of SH-PTP2 by binding with high affinity to its N- or C-terminal SH2 domains(15, 17) . These phosphotyrosyl sequences were chemically tethered to one another, and the resulting compound binds to both SH2 domains of SH-PTP2 to stimulate PTPase activity at much lower concentrations than either peptide alone. Since the tethered bisphosphotyrosyl ligand binds simultaneously to both SH2 domains, we can effectively use it as a molecular ruler to begin to measure the distance between phosphopeptide binding sites on the two SH2 domains. In a fully extended configuration the distance between phosphotyrosines of peptide pY1172(X(4))pY1222 is approx74 Å, indicating that this is the maximum possible distance between phosphotyrosine binding sites of the N- and C-SH2 domains of intact SH-PTP2 when the ligand is bound. Depending on their orientations the SH2 domains could be significantly closer to one another. For comparison, the nominal diameter of an SH2 domain is 35-40 Å.

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(i) values for peptide inhibition of [P]RCM-lysozyme dephosphorylation should parallel K(m) 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.

Conclusion

By linking two peptides that bind with the SH2 domains of SH-PTP2, we have created a single ligand that interacts simultaneously with both SH2 domains. In binding with high affinity to both SH2 domains, the tethered ligand stimulates PTPase activity at much lower concentrations than previously described peptides. The tethered ligand also appears to interact simultaneously with an SH2 domain and the PTPase to suggest that the binding sites of all three domains are in physical proximity. These findings indicate that in cells, SH-PTP2 binding via its SH2 domains to juxtaposed phosphorylation sites within one or two phosphoproteins will potently regulate catalysis to effect downstream signals. Similar effects may be observed with additional proteins that have two SH2 domains, including PI 3-kinase, ZAP-70 and Syk, PLC-, and RasGAP. These findings may also provide potential leads for the development of pharmacologic agonists and antagonists of the actions of SH-PTP2 and additional proteins with two SH2 domains.


FOOTNOTES

*
These studies were funded by National Science Foundation Grant MCB 93-04469 and a grant from the Juvenile Diabetes Foundation, International (to S. E. S.) and National Institutes of Health Grant GM 20011 and a grant from Hoffmann-LaRoche, Ltd. (to C. T. W.). The Biochemistry Facility at the Joslin Diabetes Center is supported by National Institutes of Health Diabetes and Endocrinology Research Center Grant DK36836. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Science Foundation postdoctoral fellowship.

To whom correspondence should be addressed: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2528; Fax: 617-732-2593; Shoelson{at}Joslab.Harvard.edu.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; IRS-1, insulin receptor substrate 1; PTPase, phosphotyrosine phosphatase; pY, phosphotyrosine; RCM, reduced, carboxymethylated, and maleylated; SH2, Src homology 2; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PI, phosphatidylinositol.

(^2)
G. Wolf, A. Lynch, and S. E. Shoelson, manuscript in preparation.

(^3)
T. J. Wandless and C. T. Walsh, unpublished observation.


ACKNOWLEDGEMENTS

We sincerely thank Seiji Sugimoto and Ben Neel for providing selected constructs, helpful discussions, and critical reading of this manuscript.


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