Transmembrane Receptor Protein Tyrosine Phosphatase Research Articles

Role of Receptor Protein Tyrosine Phosphatases (RPTPs) in Insulin Signaling and Secretion.

Changes in lifestyle in developed countries have triggered the prevalence of obesity and type 2 diabetes mellitus (T2DM) in the latest years. Consequently, these metabolic diseases associated to insulin resistance, and the morbidity associated with them, accounts for enormous costs for the health systems. The best way to face this problem is to identify potential therapeutic targets and/or early biomarkers to help in the treatment and in the early detection. In the insulin receptor signaling cascade, the activities of protein tyrosine kinases and phosphatases are coordinated, thus, protein tyrosine kinases amplify the insulin signaling response, whereas phosphatases are required for the regulation of the rate and duration of that response. The focus of this review is to summarize the impact of transmembrane receptor protein tyrosine phosphatase (RPTPs) in the insulin signaling cascade and secretion, and their implication in metabolic diseases such as obesity and T2DM.

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases

Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.

Pleiotrophin stimulates tyrosine phosphorylation of β-adducin through inactivation of the transmembrane receptor protein tyrosine phosphatase β/ζ

Pleiotrophin (PTN the protein, Ptn the gene) signals through a unique mechanism; it inactivates the tyrosine phosphatase activity of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)β/ζ, and increases tyrosine phosphorylation of the substrates of RPTPβ/ζ through the continued activity of a yet to be described protein tyrosine kinase(s) in PTN-stimulated cells. We have now found that the cytoskeletal protein β-adducin interacts with the intracellular domain of RPTPβ/ζ in a yeast two-hybrid system, that β-adducin is a substrate of RPTPβ/ζ, that β-adducin is phosphorylated in tyrosine in cells not stimulated by PTN, and that tyrosine phosphorylation of β-adducin is sharply increased in PTN-stimulated cells, suggesting that β-adducin is a downstream target of and regulated by the PTN/RPTPβ/ζ signaling pathway. β-Catenin was the first downstream target of the PTN/RPTPβ/ζ signaling pathway to be identified; these data thus also suggest that PTN coordinately regulates steady state levels of tyrosine phosphorylation of the important cytoskeletal proteins β-adducin and β-catenin and, through PTN-stimulated tyrosine phosphorylation, β-adducin may contribute to the disruption of cytoskeletal structure, increased plasticity, and loss of homophilic cell–cell adhesion that are the consequences of PTN stimulation of cells and a characteristic feature of different malignant cells with mutations that activate constitutive expression of the endogenous Ptn gene.

Cortical neurons express PSI, a novel isoform of phosphacan/RPTPbeta

Phosphacan is a chondroitin sulfate proteoglycan representing the secreted extracellular part of a transmembrane receptor protein tyrosine phosphatase (RPTP-beta). These isoforms have been implicated in cell-extracellular matrix signaling events associated with myelination, axon growth, and cell migration in the developing central nervous system and may play critical roles in the context of brain pathologies. Recently, we have reported the identification of a new isoform of phosphacan, the phosphacan short isoform (PSI), the expression of which peaks in the second postnatal week. PSI interacts with the neuronal receptors L1 and F3/contactin and can promote neurite growth of cortical neurons. In this study, we have assessed, by in situ hybridization, the expression profile of PSI in the rat brain at postnatal day 7. PSI is largely expressed in the gray matter of the developing cerebral cortex in which it colocalizes with phosphacan, whereas the expression of RPTPbeta receptor forms is restricted to the ventricular area in which PSI has not been observed. Neurons from all layers of the cortex express PSI. In the cerebellum, on the other hand, no expression of PSI has been detected, although the other phosphacan/RPTP-beta isoforms show strong PSI expression here. Overall, our study suggests that PSI is expressed during the postnatal period in differentiated neurons of the cortex but is absent from structures in which proliferation and migration occur. The significance of these observations is discussed in the context of previous models of phosphacan/RPTP-beta functions.

Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase β/ζ-signaling pathway: Regulation of tyrosine phosphorylation of Fyn by pleiotrophin

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)β/ζ, disrupting the balanced activity of RPTPβ/ζ and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPβ/ζ, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPβ/ζ in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPβ/ζ in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPβ/ζ, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that β-catenin and β-adducin are targets of the PTN/RPTPβ/ζ-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of β-catenin and β-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPβ/ζ-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of β-catenin, β-adducin, and Fyn through the PTN/RPTPβ/ζ-signaling pathway and that together Fyn, β-adducin, and β-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell–cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.

Receptor Protein Tyrosine Phosphatase μ Regulates the Paracellular Pathway in Human Lung Microvascular Endothelia

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

Inhibition of netrin-mediated axon attraction by a receptor protein tyrosine phosphatase.

During axon guidance, the ventral guidance of the Caenorhabditis elegans anterior ventral microtubule axon is controlled by two cues, the UNC-6/netrin attractant recognized by the UNC-40/DCC receptor and the SLT-1/slit repellent recognized by the SAX-3/robo receptor. We show here that loss-of-function mutations in clr-1 enhance netrin-dependent attraction, suppressing ventral guidance defects in slt-1 mutants. clr-1 encodes a transmembrane receptor protein tyrosine phosphatase (RPTP) that functions in AVM to inhibit signaling through the DCC family receptor UNC-40 and its effector, UNC-34/enabled. The known effects of other RPTPs in axon guidance could result from modulation of guidance receptors like UNC-40/DCC.

Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration

We have examined the role of chondroitin sulfate proteoglycans (CSPGs) and keratan sulfate proteoglycans (KSPGs) in directing mossy fiber (MF) outgrowth and regeneration in rat hippocampal slice cultures. MFs normally exhibit a very specific innervation pattern that is restricted to the stratum lucidum (SL). In addition, MFs in hippocampal slice cultures will regenerate this specific innervation pattern after transection. CSPGs are one of the best characterized inhibitory axon guidance molecules in the CNS and are widely expressed in all areas of the hippocampus except SL. KSPGs are also widely expressed in the hippocampus, but their role in axon outgrowth has not been extensively studied in the CNS where phosphacan is the only protein that appears to contain KS-GAGs. Cultured hippocampal slices were treated with either chondroitin ABC lyase or keratanases to reduce the inhibitory axon guidance properties of CS and KS proteoglycans, respectively. The ability of transected MFs to regenerate their normal innervation pattern after digestion of CS and KS-GAGS sugars with these enzymes was examined. Only keratanase treatment resulted in misrouting of MFs. Identifying the mechanism by which keratanase produced MF misrouting is complicated by the presence of splice variants of the phosphacan gene that include the extracellular form of phosphacan and the transmembrane receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). Both forms of phosphacan are made by astrocytes, suggesting that keratanase alters MF outgrowth by modifying astrocyte function.

Intra- and Intermolecular Interactions between Intracellular Domains of Receptor Protein-tyrosine Phosphatases

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The generally inactive membrane-distal PTP domains (RPTP-D2s) bind and are proposed to regulate the membrane-proximal PTP domains (RPTP-D1s). We set out to characterize the interactions between RPTP-D1s and RPTP-D2s in vivo by co-immunoprecipitation of hemagglutinin-tagged fusion proteins encoding the transmembrane domain and RPTP-D1 and myc-tagged RPTP-D2. Seven RPTPs from four different subfamilies were used: RPTPalpha, RPTPepsilon, LAR, RPTPvarsigma, RPTPdelta, CD45, and RPTP(mu). We found that RPTP-D2s bound to RPTPs with different affinities. The presence of intrinsic RPTP-D2 altered the binding specificity toward other RPTP-D2s positively or negatively, depending on the identity of the RPTPs. Furthermore, the C terminus of RPTP-D2s and the "wedge" in RPTP-D1s played a central role in binding specificity. Finally, full-length RPTPalpha and LAR heterodimerized in an oxidative stress-dependent manner. Like RPTPalpha-D2, the LAR-D2 conformation was affected by oxidative stress, suggesting a common regulatory mechanism for RPTP complex formation. Taken together, interactions between RPTP-D1s and RPTP-D2s are a common but specific mechanism that is likely to be regulated. The RPTP-D2s and the wedge structures are crucial determinants of binding specificity, thus regulating cross-talk between RPTPs.

The transmembrane receptor protein tyrosine phosphatase DEP1 interacts with p120(ctn).

The receptor-like protein tyrosine phosphatase DEP1, also known as CD148, is expressed predominantly in epithelial cells, in a variety of tumor cell lines, and in lymphocytes. Expression of DEP1 is enhanced at high cell density, and this observation suggests that DEP1 may function in the regulation of cell adhesion and possibly contact inhibition of cell growth. In order to investigate the function of DEP1, substrate-trapping mutants of the phosphatase were used to identify potential substrates. GST-fusion proteins containing the DEP1 catalytic domain with a substrate-trapping D/A mutation were found to interact with p120(ctn), a component of adherens junctions. DEP1 also interacted with other members of the catenin gene family including beta-catenin and gamma-catenin. The interaction with p120(ctn) is likely to be direct, as the interaction occurs in K562 cells lacking functional adherens junctions and E-cadherin expression. Catalytic domains of the tyrosine phosphatases PTP-PEST, CD45, and PTPbeta did not interact with proteins of the catenin family to detectable levels, suggesting that the interaction of DEP1 with these proteins is specific. DEP1 expression was concentrated at sites of cell-cell contact in A549 cells. p120(ctn) was found to colocalize with these structures. Together these data suggest an important role for DEP-1 in the function of cell-cell contacts and adherens junctions.

Regulation of receptor protein-tyrosine phosphatase alpha by oxidative stress

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPalpha prohibited intermolecular interactions. Interestingly, stress factors such as H(2)O(2), UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPalpha and RPTPalpha-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPalpha-SpD2, Cys723, was required for the H(2)O(2) effect on RPTPalpha. H(2)O(2) induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPalpha-SpD2 in a single chimeric protein. Importantly, H(2)O(2) treatment stabilized RPTPalpha dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPalpha-D2, leading to stabilization of RPTPalpha dimers, and thus to inhibition of RPTPalpha activity.

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP σ was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the Gq/11-protein-coupled bradykinin B2 receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR.