Identification of protein tyrosine phosphatases with specificity for the ligand-activated growth hormone receptor

Protein tyrosine phosphatases (PTPs) play key roles in switching off tyrosine phosphorylation cascades, such as initiated by cytokine receptors. We have used substrate-trapping mutants of a large set of PTPs to identify members of the PTP family that have substrate specificity for the phosphorylated human GH receptor (GHR) intracellular domain. Among 31 PTPs tested, T cell (TC)-PTP, PTP-beta, PTP1B, stomach cancer-associated PTP 1 (SAP-1), Pyst-2, Meg-2, and PTP-H1 showed specificity for phosphorylated GHR that had been produced by coexpression with a kinase in bacteria. We then used GH-induced, phosphorylated GH receptor, purified from overexpressing mammalian cells, in a Far Western-based approach to test whether these seven PTPs were also capable of recognizing ligand-induced, physiologically phosphorylated GHR. In this assay, only TC-PTP, PTP1B, PTP-H1, and SAP-1 interacted with the mature form of the phosphorylated GHR. In parallel, we show that these PTPs recognize very different subsets of the seven GHR tyrosines that are potentially phosphorylated. Finally, mRNA tissue distribution of these PTPs by RT-PCR analysis and coexpression of the wild-type PTPs to test their ability to dephosphorylate ligand-activated GHR suggest PTP-H1 and PTP1B as potential candidates involved in GHR signaling.     

Regulation of insulin signaling through reversible oxidation of the protein-tyrosine phosphatases TC45 and PTP1B

Many studies have illustrated that the production of reactive oxygen species (ROS) is important for optimal tyrosine phosphorylation and signaling in response to diverse stimuli. Protein-tyrosine phosphatases (PTPs), which are important regulators of signal transduction, are exquisitely sensitive to inhibition after generation of ROS, and reversible oxidation is becoming recognized as a general physiological mechanism for regulation of PTP function. Thus, production of ROS facilitates a tyrosine phosphorylation-dependent cellular signaling response by transiently inactivating those PTPs that normally suppress the signal. In this study, we have explored the importance of reversible PTP oxidation in the signaling response to insulin. Using a modified ingel PTP assay, we show that stimulation of cells with insulin resulted in the rapid and transient oxidation and inhibition of two distinct PTPs, which we have identified as PTP1B and TC45, the 45-kDa spliced variant of the T cell protein-tyrosine phosphatase. We investigated further the role of TC45 as a regulator of insulin signaling by combining RNA interference and the use of substrate-trapping mutants. We have shown that TC45 is an inhibitor of insulin signaling, recognizing the beta-subunit of the insulin receptor as a substrate. The data also suggest that this strategy, using ligand-induced oxidation to tag specific PTPs and using interference RNA and substrate-trapping mutants to illustrate their role as regulators of particular signal transduction pathways, may be applied broadly across the PTP family to explore function.     

Insulin Modulation of Cloned Mouse NMDA Receptor Currents in Xenopus Oocytes

Modulation of recombinant N-methyl-D-aspartate receptor (NMDAR) currents by insulin was studied using the Xenopus oocyte expression system. Insulin (0.8 microM, 10 min) regulated NMDAR currents in a subunit-specific manner. Currents from epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 receptors were variably potentiated, whereas currents from epsilon3/zeta1 receptors were not. Protein tyrosine kinases (PTKs) and protein kinase C were found to be involved in insulin-mediated modulation in an NMDAR subtype-specific way. Pretreatment with a specific PTK inhibitor, lavendustin A, attenuated and blocked the insulin effect on epsilon2/zeta1 and epsilon4/zeta1, respectively. Preincubation with selective protein kinase C inhibitors, staurosporine or calphostin C, depressed the response of epsilon1/zeta1 and epsilon2/zeta1 receptors to insulin. Basal regulation of NMDAR currents by endogenous PTKs and protein tyrosine phosphatases (PTPs) was also investigated. Of the four receptor subtypes, only epsilon1/zeta1 receptor currents were affected by basal PTK inhibition via lavendustin A, whereas PTP inhibition by phenylarsine oxide or orthovanadate enhanced currents from epsilon1/zeta1 and epsilon2/zeta1 receptors. Surprisingly, a stimulatory PTP modulation was observed for epsilon4/zeta1. As NMDAR subunits are differentially expressed in the brain, the observed subtype-specific modulations of NMDAR currents by insulin, PTKs, and PTPs may provide important insights into certain NMDAR-dependent physiological and pathological processes.     

Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.     

Receptor-type protein tyrosine phosphatase epsilon (PTPepsilonM) is a negative regulator of insulin signaling in primary hepatocytes and liver

Impaired insulin receptor (IR) signaling leads to insulin resistance and type 2 diabetes mellitus. Several inhibitors of the IR tyrosine kinase activity have recently been described and associated with human insulin resistance. Among these negative regulators, protein tyrosine phosphatases (PTPs) are likely to play a pivotal role in IR signaling. Transgenic studies revealed that PTP1B and TCPTP are primary candidates but IR of these animals can be finally dephosphorylated, suggesting that other PTPs are also involved in the dephosphorylation of IR. In this study, we showed that receptor-type PTPepsilon (PTP epsilonM) dephosphorylated IR in rat primary hepatocytes and tyrosines 972, 1158, 1162 and 1163 were primary targets of PTP epsilonM. Wild type as well as substrate-trapping DA forms of PTPepsilonM suppressed phosphorylation of IR downstream enzymes such as Akt, extracellular regulated kinase (ERK) and glycogen synthase kinase 3 (GSK3). It was also demonstrated that PTPepsilonM suppressed insulin-induced glycogen synthesis and inhibited insulin-induced suppression of phosphoenol pyruvate carboxykinase (PEPCK) expression in primary hepatocytes. Furthermore, adenovirally introduced PTPepsilonM also exhibited inhibitory activity against suppression of PEPCK expression in mouse liver. These results suggest that PTPepsilonM is a negative regulator of IR signaling and involved in insulin-induced glucose metabolism mainly through direct dephosphorylation and inactivation of IR in hepatocytes and liver.     

Generation of inhibitor-sensitive protein tyrosine phosphatases via active-site mutations

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of phosphotyrosine, a central control element in mammalian signal transduction. Small-molecule inhibitors that are specific for each cellular PTP would be valuable tools in dissecting phosphorylation networks and for validating PTPs as therapeutic targets. However, the common architecture of PTP active sites impedes the discovery of selective PTP inhibitors. Our laboratory has recently used enzyme/inhibitor-interface engineering to generate selective PTP inhibitors. The crux of the strategy resides in the design of "inhibitor-sensitized" PTPs through protein engineering of a novel binding pocket in the target PTP. "Allele-specific" inhibitors that selectively target the sensitized PTP can be synthesized by modifying broad-specificity inhibitors with bulky chemical groups that are incompatible with wild-type PTP active sites; alternatively, specific inhibitors that serendipitously recognize the sensitized PTP's non-natural pocket may be discovered from panels of "non-rationally" designed compounds. In this review, we describe the current state of the PTP-sensitization strategy, with emphases on the methodology of identifying PTP-sensitizing mutations and synthesizing the compounds that have been found to target PTPs in an allele-specific manner. Moreover, we discuss the scope of PTP sensitization in regard to the potential application of the approach across the family of classical PTPs.     

Computational analysis of the regulation of EGFR by protein tyrosine phosphatases

The tyrosine phosphorylated epidermal growth factor receptor (EGFR) initiates numerous cell signaling pathways. Although EGFR phosphorylation levels are ultimately determined by the balance of receptor kinase and protein tyrosine phosphatase (PTP) activities, the kinetics of EGFR dephosphorylation are not well understood. Previous models of EGFR signaling have generally neglected PTP activity or computed PTP activity by considering data that do not fully reveal the kinetics and compartmentalization of EGFR dephosphorylation. We developed a compartmentalized, mechanistic model to elucidate the kinetics of EGFR dephosphorylation and the coupling of this process to phosphorylation-dependent EGFR endocytosis. Model regression against data from HeLa cells for EGFR phosphorylation response to EGFR activation, PTP inhibition, and EGFR kinase inhibition led to the conclusion that EGFR dephosphorylation occurs at the plasma membrane and in the cell interior with a timescale that is smaller than that for ligand-mediated EGFR endocytosis. The model further predicted that sufficiently rapid dephosphorylation of EGFR at the plasma membrane could potentially impede EGFR endocytosis, consistent with recent experimental findings. Overall, our results suggest that PTPs regulate multiple receptor-level phenomena via their action at the plasma membrane and cell interior and point to new possibilities for targeting PTPs for modulation of EGFR dynamics.     

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.     

Modulation of protein tyrosine phosphatase 1B by erythropoietin in UT-7 cell line.

BACKGROUND/ AIMS: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. METHODS: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. RESULTS: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). CONCLUSION: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.     

Protein tyrosine phosphatase receptor type Z dephosphorylates TrkA receptors and attenuates NGF-dependent neurite outgrowth of PC12 cells

Protein tyrosine phosphatase receptor type Z (Ptprz/Ptpzeta / RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.     

Role of antioxidant defenses in the tolerance of severe dehydration by anurans. The case of the leopard frog Rana pipiens

Protein tyrosine phosphatases (PTPs) play a critical role in regulating insulin action in part through dephosphorylation of the active (autophosphorylated) form of the insulin receptor (IRK) and attenuation of its tyrosine kinase activity. Following insulin binding the activated IRK is rapidly internalized into the endosomal apparatus, a major site at which the IRK is dephosphorylated in vivo. Studies in rat liver suggest a complex regulatory process whereby PTPs may act, via selective IRK tyrosine dephosphorylation, to modulate IRK activity in both a positive and negative manner. Use of peroxovanadium (pV) compounds, shown to be powerful PTP inhibitors, has been critical in delineating a close relationship between the IRK and its associated PTP(s) in vivo. Indeed the in vivo administration of pV compounds effected activation of IRK in parallel with an inhibition of IRK-associated PTP activity. This process was accompanied by a lowering of blood glucose levels in both normal and diabetic rats thus implicating the IRK-associated PTP(s) as a suitable target for defining a novel class of insulin mimetic agents. Identification of the physiologically relevant IRK-associated PTP(s) should facilitate the development of drugs suitable for managing diabetes mellitus.     

Engineered inhibitor sensitivity in the WPD loop of a protein tyrosine phosphatase

Small molecules that can be used to turn off the activities of specific cellular proteins are essential tools for chemical biology. Few such compounds are known, however, and they are particularly difficult to identify for members of large protein families. Here, we present a method for insertion of a chemical "off switch" into a catalytically essential loop region (the "WPD loop") of a protein tyrosine phosphatase (PTP). Using a combination of point mutations and amino acid insertions, we have engineered variants of T-cell PTP (TCPTP) that possess cysteine-rich WPD loops. The engineered WPD loops, which contain sequences that appear in no wild-type PTP, confer upon TCPTP the ability to bind a cell-permeable small molecule (the biarsenical fluorescein derivative, FlAsH) that is not an inhibitor of wild-type PTPs. We have identified sites in TCPTP's WPD loop that can be modified to display FlAsH-binding cysteine residues without disrupting TCPTP's inherent PTP activity, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. Upon addition of the FlAsH ligand, however, the activities of the mutants drop dramatically. Inhibition of the FlAsH-sensitized TCPTP mutants is rapid and specific; and strong FlAsH sensitivity was observed in mutants that contain as few as two cysteine point mutations in their engineered WPD loops. Our results show that relatively conservative substitutions can be used to engineer precise small-molecule control of PTP activity. Moreover, since all known classical PTPs utilize the WPD-loop mechanism targeted in this study, it is likely that a substantial fraction of the PTP superfamily can be sensitized through an analogous approach.     

Protein tyrosine phosphatase profiling analysis of HIB-1B cells during brown adipogenesis

A number of evidence have been accumulated that the regulation of reversible tyrosine phosphorylation, which can be regulated by the combinatorial activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays crucial roles in various biological processes including differentiation. There are a total of 107 PTP genes in the human genome, collectively referred to as the "PTPome." In this study, we performed PTP profiling analysis of the HIB-1B cell line, a brown preadipocyte cell line, during brown adipogenesis. Through RT-PCR and real-time PCR, several PTPs showing differential expression pattern during brown adipogenesis were identified. In the case of PTP-RE, it was shown to decrease significantly until 4 days after brown adipogenic differentiation, followed by a dramatic increase at 6 days. The overexpression of PTP-RE led to decreased brown adipogenic differentiation via reducing the tyrosine phosphorylation of the insulin receptor, indicating that PTP-RE functions as a negative regulator at the early stage of brown adipogenesis.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Proteomic approaches to studying protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that play key roles in cell signaling. Deregulation of PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. Since phosphate removal by the PTPs can both enhance and antagonize cellular signaling, it is essential to elucidate the physiological context in which PTPs operate. Two powerful proteomic approaches have been developed to rapidly establish the exact functional roles for every PTP, both in normal cellular physiology and in pathogenic conditions. In the first, an affinity-based substrate-trapping approach has been employed for PTP substrate identification. Identification and characterization of specific PTP-substrate interactions will associate functions with PTP as well as implicate PTP to specific signaling pathways. In the second, a number of activity-based PTP probes have been developed that can provide a direct readout of the functional state of the PTPs in complex proteomes. The ability to profile the entire PTP family on the basis of changes in their activity is expected to yield new functional insights into pathways regulated by the PTPs and contribute to the discovery of PTPs as novel therapeutic targets. Effective application of these proteomic techniques will accelerate the functional characterization of PTPs, thereby facilitating our understanding of PTPs in cell signaling and in diseases.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Regulation of Signaling by Protein-Tyrosine Phosphatases: Potential Roles in the Nervous System

During neuronal development, cells respond to a variety of environmental cues through cell surface receptors that are coupled to a signaling transduction machinery based on protein tyrosine phosphorylation and dephosphorylation. Receptor and non-receptor tyrosine kinases have received a great deal of attention; however, in the last few years, receptor (plasma membrane associated) and non-receptor protein-tyrosine phosphatases (PTPs) have also been shown to play important roles in development of the nervous system. In many cases PTPs have provocative distribution patterns or have been shown to be associated with specific cell adhesion and growth factor receptors. Additionally, altering PTP expression levels or activity impairs neuronal behavior. In this review we outline what is currently known about the role of PTPs in development, differentiation and neuronal physiology.     

Peroxovanadium compounds: Biolgoical actions and mechanism of insulin-mimesis

When used alone, both vanadate and hydrogen peroxide (H₂O₂) are weakly insulin-mimetic, while in combination they are strongly synergistic due to the formation of aqueous peroxovanadium species pV_(aq). Administration of these pV_(aq) species leads to activation of the insulin receptor tyrosine kinase (IRK), autophosphorylation at tyrosine residues and inhibition of phosphotyrosine phosphatases (PTPs). We therefore undertook to synthesize a series of peroxovanadium (pV) compounds containing one or two peroxo anions, an oxo anion and an ancillary ligand in the inner co-ordination sphere of vanadium, whose properties and insulin-mimetic potencies could be assessed. These pV compounds were shown to be the most potent inhibitors of PTPs yet described. Their PTP inhibitory potency correlated with their capacity to stimulate IRK activity. Some pV compounds showed much greater potency as inhibitors of insulin receptor (IR) dephosphorylation than epidermal growth factor receptor (EGFR) dephosphorylation, implying relative specificity as PTP inhibitors. Replacement of vanadium with either molybdenum or tungsten resulted in equally potent inhibition of IR dephosphorylation. However IRK activation was reduced by greater than 80% suggesting that these compounds did not access intracellular PTPs. The insulin-like activity of these pV compounds were demonstrablein vivo. Intra venous (i.v.) administration of bpV(pic) and bpV(phen) resulted in the lowaring of plasma glucose concentrations in normal rats in a dose dependent manner. The greater potency of bpV(pic) compared to bpV(phen) was explicable, in part, by the capacity of the former but not the latter to act on skeletal muscle as well as liver. Finally administration of bpV(phen) and insulin led to a synergism, where tyrosine phosphorylation of the IR -subunit increased by 20-fold and led to the appearance of four insulin-dependentin vivo substrates. The insulin-mimetic properties of they pV compounds raises the possibility for their use as insulin replacements in the management of diabetes mellitus.     

The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities

The activity of protein tyrosine phosphatases (PTPs) is restricted by their substrate specificities. The analysis of PTP specificity was greatly helped by the discovery that "substrate-trapping" PTP mutants, such as PTP-1B D181A, stably and specifically bind their substrates. We have set up a PTP substrate specificity assay based on the SPOT technique, which involves the microsynthesis of (phospho)peptides on membranes. To validate this approach, substrate trapping PTP-1B was tested on its cognate ligand, the autophosphorylated insulin receptor (IR). On SPOT membranes, IR peptides with phosphotyrosine 1163 were efficiently bound by PTP1B D181A, and dephosphorylated by PTP-1B. Phosphotyrosine 1163 was preferred over the neighboring 1158 and 1162 phosphotyrosines. PTP-1B also recognized IR-like motifs in Trk autophosphorylation domains, and STAT 5 phosphopeptides. Using a gridded 20-by-20 SPOT library, we show that peptides with the YZM motif (Z: phosphotyrosine) are the strongest ligands for PTP-1B D181A, but not the optimal substrates for dephosphorylation by wild-type PTP1B. In addition we show that PTP-1B and PTP-beta dephosphorylation efficiency is strongly modulated by the introduction of phospho-serine or phospho-threonine in their cognate phospho-tyrosine substrates. Altogether our data illustrate that the SPOT technique is a highly efficient tool for the study of PTP substrate specificity.     

Crystal structure of a complex between protein tyrosine phosphatase 1B and the insulin receptor tyrosine kinase

Protein tyrosine phosphatase 1B (PTP1B) is a highly specific negative regulator of insulin receptor signaling in vivo. The determinants of PTP1B specificity for the insulin receptor versus other receptor tyrosine kinases are largely unknown. Here, we report a crystal structure at 2.3 A resolution of the catalytic domain of PTP1B (trapping mutant) in complex with the phosphorylated tyrosine kinase domain of the insulin receptor (IRK). The crystallographic asymmetric unit contains two PTP1B-IRK complexes that interact through an IRK dimer interface. Rather than binding to a phosphotyrosine in the IRK activation loop, PTP1B binds instead to the opposite side of the kinase domain, with the phosphorylated activation loops sequestered within the IRK dimer. The crystal structure provides evidence for a noncatalytic mode of interaction between PTP1B and IRK, which could be important for the selective recruitment of PTP1B to the insulin receptor.