Receptor Protein Tyrosine Phosphatase Alpha Research Articles

Clonal Evolution of a Mutation in PTPRA As a Potential Cause for Resistance Against Anti-CD19 Directed Chimeric Antigen Receptor T-Cell (CAR-T) Therapy with CTL019 in DLBCL Patients

Introduction:Anti-CD 19 directed CAR-T cell therapy has shown very promising effects in treatment of diffuse-large B-cell lymphoma (DLBCL). However, a substantial fraction of patients does not respond to treatment or relapses after approximately three to six months [Schuster, NEJM, 2017; Neelapu, NEJM, 2017]. Resistance mechanisms against anti-CD19 directed CAR-T cell therapy have so far only been described in B-cell acute lymphoblastic leukemia (B-ALL) and involve mutations, splice variants or even loss of CD19 [Sotillo, Cancer Discovery, 2015]. To further elucidate mechanisms of acquired resistance in DLBCL, we obtained matched pair tumor samples from four patients before and after treatment with CTL019 treated within the phase II JULIET trial.Methods:We performed whole exome sequencing in one patient (#UKK2) employing one matched normal sample that has been sequenced at a coverage of 130x and one pre-treatment as well as two post-treatment samples that were all sequenced at a coverage of 160x. Whole exome sequencing data were used to reconstruct clonal evolution of DLBCL after treatment with CTL019 as has been described previously [Herling, Nature Communications, 2018]. Furthermore, we obtained biopsies at relapse or disease progression including matched normal samples from three more patients. Whole exome sequencing from the three missing patients as well as 3´RNA sequencing from all four patients' samples are ongoing and results will be updated for presentation at the meeting.Results:The first analyzed patient, #UKK2, is a 67 year old male patient, who had chemotherapy refractory disease after treatment with R-CHOP, R-DHAP and Pixantrone. He showed predominantly pelvic lesions including infiltration of the iliopsoas muscle. The patient achieved a complete response at month 3 (Figure 1A) after CTL019 transfusion. At month 5 he developed new cutaneous lesions on his left thigh (Figure 1B, posttreatment sample 1) as well as new muscle infiltrations on his right quadriceps femoris muscle (posttreatment sample 2) and rectus abdominis muscle. Pathological examination confirmed relapse of CD19 positive DLBCL in both posttreatment samples. Whole exome sequencing from a biopsy obtained before study entry and the above mentioned posttreament biopsies showed clonal evolution of a single missense mutation in PTPRA (N297D). PTPRA encodes for the receptor protein-tyrosine phosphatase alpha, a receptor phosphatase involved in activation of c-Src via dephosphorylation of an inhibitory tyrosine at position 530 (Y530) [Mustelin, Science Signaling, 2002 and Gut, International Journal of Oncology, 2017]. On the other hand, c-Src is an onco-protein known to cause an invasive phenotype and metastasis in different tumor entities [Ishizawar, Cancer Cell, 2004]. All other mutations observed were either already clonal before CTL019 treatment or gained clonality in only one posttreatment biopsy (Figure 1C).Conclusion:To our knowledge, this is the first report identifying clonal evolution during CD19 redirected CAR-T cell therapy in r/r DLBCL involving PTPRA. We hypothesize that this PTPRA mutation contributes to an invasive phenotype and extranodal lymphoma growth pattern allowing the lymphoma to escape from the pattern allowing the lymphoma to escape from the CAR-T cell attack. Genomic workup of patients with pre- and posttreatment biopsies and functional investigation of this hypothesis is ongoing and will be presented. [Display omitted] DisclosuresBalke-Want:Novartis: Honoraria. Borchmann:Novartis: Consultancy, Honoraria.

Regulation of RPTPα-c-Src signalling pathway by miR-218.

Receptor protein tyrosine phosphatase alpha (RPTPα), an activator of Src family kinases, is found significantly overexpressed in human cancer tissues. However, little is known about the regulation of RPTPα expression. miRNAs target multiple genes and play important roles in many cancer processes. Here, we identified a miRNA, miR-218 that binds directly to the 3'-UTR of RPTPα. Ectopic overexpression of miR-218 decreased RPTPα protein leading to decreased dephosphorylation of c-Src and decreased tumour growth invitro and invivo. A feedback loop between c-Src and miR-218 was revealed where c-Src inhibits transcription of SLIT2, which intronically hosts miR-218. These results show a novel regulatory pathway for RPTPα-c-Src signalling.

Global Phosphotyrosine Proteomics Identifies PKCδ as a Marker of Responsiveness to Src Inhibition in Colorectal Cancer

Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.

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.

Regulatory Effects of Nitric Oxide on Src Kinase, FAK, p130Cas, and Receptor Protein Tyrosine Phosphatase Alpha (PTP-α): A Role for the Cellular Redox Environment

The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.

Catalytically active membrane‐distal phosphatase domain of receptor protein‐tyrosine phosphatase α is required for Src activation

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein with tandem cytoplasmic phosphatase domains. Most of the catalytic activity is contained by the membrane-proximal catalytic domain (D1). We found a spontaneous Arg554 to His mutation in the pTyr recognition loop of the membrane-distal phosphatase domain (D2) of a human patient. This mutation was not linked to the disease. Here, we report that the R554H mutation abolished RPTPalpha-D2 catalytic activity. The R554H mutation impaired Src binding to RPTPalpha. RPTPalpha, with a catalytic site cysteine to serine mutation in D2, also displayed diminished binding to Src. Concomitant with decreased Src binding of the R554H and C723S mutants compared with wild-type RPTPalpha, enhanced phosphorylation of the inhibitory Src Tyr527 site was observed, as well as reduced Src activation. To confirm that catalytic activity of RPTPalpha-D2 was required for these effects, we analyzed a third mutant, RPTPalpha-R729K, which had an inactive D2. Again, Src binding was reduced and Tyr527 phosphorylation was enhanced. Our results suggest that a catalytically active D2 is required for RPTPalpha to bind and dephosphorylate its well-characterized substrate, Src.

H2O2-induced Intermolecular Disulfide Bond Formation between Receptor Protein-tyrosine Phosphatases

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTPalpha catalytic activity by controlling rotational coupling within RPTPalpha dimers. RPTPalpha-D2 changes conformation and thereby rotational coupling within RPTPalpha dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTPalpha from cells treated with H2O2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTPalpha dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTPalpha and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTPalpha fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H2O2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation.

Receptor protein tyrosine phosphatase alpha signaling is involved in androgen depletion-induced neuroendocrine differentiation of androgen-sensitive LNCaP human prostate cancer cells.

The neuroendocrine (NE) cells represent the third cell population in the normal prostate. Results of several clinical studies strongly indicate that the NE cell population is greatly increased in prostate carcinomas during androgen ablation therapy that correlates with hormone-refractory growth and poor prognosis. However, the mechanism of NE cell enrichment in prostate carcinoma remains an enigma. We investigated the molecular mechanism by which androgen-sensitive C-33 LNCaP human prostate cancer cells become NE-like cells in an androgen-reduced environment, mimicking clinical phenomenon. In the androgen-depleted condition, androgen-sensitive C-33 LNCaP cells gradually acquired the NE-like morphology and expressed an increased level of neuron-specific enolase (NSE), a classical marker of neuronal cells. Several NE-like subclone cells were established. Biochemical characterizations of these subclone cells showed that receptor-type protein-tyrosine phosphatase alpha (RPTPalpha) is elevated and ERK is constitutively activated, several folds higher than that in parental cells. In androgen-depleted condition, PD98059, an MEK inhibitor, could efficiently block not only the activation of ERK, but also the acquisition of the NE-like morphology and the elevation of NSE in C-33 LNCaP cells. In RPTPalpha cDNA-transfected C-33 LNCaP cells, ERK was activated and NSE was elevated. In those cells in the presence of PD98059, the ERK activation and NSE elevation were abolished, following a dose-response fashion. Additionally, in constitutively active MEK mutant cDNA-transfected C-33 LNCaP cells, ERK was activated and NSE level was elevated, and cells obtained the NE-like phenotype. Our data collectively indicated that RPTPalpha signaling via ERK is involved in the NE transdifferentiation of androgen-sensitive C-33 LNCaP human prostate cancer cells in the androgen-depleted condition.

Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation.

Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra(-/-) mice. However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPalpha also underperform in the radial-arm water-maze test. These studies identify RPTPalpha as a key mediator of neuronal migration and synaptic plasticity.

Redox-regulated Rotational Coupling of Receptor Protein-tyrosine Phosphatase α Dimers

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTP alpha revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTP alpha and of constitutively dimeric, active RPTP alpha-F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTP alpha-P137C was. All three proteins were expressed on the plasma membrane to a similar extent, and the accessibility of their extracellular domains did not differ as determined by biotinylation studies. Dimerization was required for masking the HA tag, and we identified a region in the N terminus of RPTP alpha that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTP alpha-D2). Here we report that H(2)O(2) treatment of cells induced a change in rotational coupling in RPTP alpha dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys(723) in RPTP alpha-D2, which was required for the conformational change of RPTP alpha-D2 upon H(2)O(2) treatment, was essential for the H(2)O(2)-induced increase in accessibility. These results show for the first time that a conformational change in the intracellular domain of RPTP alpha led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling.

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.

Eye defects in receptor protein-tyrosine phosphatase alpha knock-down zebrafish.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is highly expressed in the developing retina of different species, but little is known about its function there. Here, we report that injection of antisense morpholinos in zebrafish embryos reduced RPTP alpha expression to almost nondetectable levels up to 3 days postfertilization (dpf). RPTP alpha was detectable again from 4 dpf onward. RPTP alpha knock-down resulted in smaller eyes. Examination of sections of the retina at different developmental stages demonstrated that already at 28 hours postfertilization (hpf) fewer cells were present in the retina of RPTP alpha-morpholino-injected embryos. At 3 dpf, the layered organization of the retina was absent. In addition, the morphology and labeling with an axon specific antibody, acetylated tubulin, demonstrated that most cells appeared to be undifferentiated. Strikingly, at 5 dpf the lamination of the retina was partially restored, concomitant with re-expression of RPTP alpha protein. Although cells in the retina were now differentiated, the layering of the retina remained disrupted and significant gaps were observed in the amacrine cell layer. Therefore, knock-down of RPTP alpha protein provides evidence that RPTP alpha is essential for normal retinal development.

Expression of receptor protein-tyrosine phosphatase alpha, sigma and LAR during development of the zebrafish embryo.

Receptor protein-tyrosine phosphatases (RPTPs) are key players in Drosophila development. To study the role of RPTPs in vertebrate development, we have cloned zebrafish (zf) RPTPs, including RPTP alpha (RPTPalpha), RPTP sigma (RPTPsigma) and LAR. These three RPTPs are broadly transcribed in early development. At 24h post fertilisation (hpf), all three genes are expressed in the nervous system in partially overlapping patterns. At 3 days post fertilisation zf-RPTPalpha and zf-LAR show similar expression patterns in the central nervous system (CNS), the pharyngeal arches, the pectoral fins and the spinal cord. Interestingly, zf-LAR is uniquely expressed in the neuromast cells, whereas zf-RPTPsigma expression is confined to the central nervous system.

Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.

BackgroundDimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.ResultsIn order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.ConclusionsWe demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Role of receptor protein tyrosine phosphatase alpha (RPTPalpha) and tyrosine phosphorylation in the serotonergic inhibition of voltage-dependent potassium channels.

The activity of voltage-gated potassium (Kv) channels can be dynamically modulated by several events, including neurotransmitter-stimulated biochemical cascades mediated by G-protein-coupled receptors. By using a heterologous expression system, we show that activating the 5-HT2C receptor inhibits both Kv1.1 and Kv1.2 channels through a tyrosine phosphorylation mechanism. The major molecular determinants of channel inhibition were identified as two tyrosine residues located in the N-terminal region of the Kv channel subunit. Furthermore, we demonstrate that receptor protein tyrosine phosphatase alpha (RPTPalpha), a receptor protein tyrosine phosphatase, co-ordinates the inhibition process mediated via 5-HT2C receptors. We therefore propose that the serotonergic regulation of human Kv1.1 and Kv1.2 channel activity by the 5-HT2C receptor involves the dual coordination of both RPTPalpha and specific tyrosine kinases coupled to this receptor.

Identification of p130 cas as an in VivoSubstrate of Receptor Protein-tyrosine Phosphatase α

We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase alpha (RPTPalpha). Here we report that a substrate-trapping mutant of the RPTPalpha membrane proximal catalytic domain (D1), RPTPalpha-D1-C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTPalpha (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTPalpha-D1-C433S varied between cell lines, but a protein of approximately 130 kDa was pulled down from every cell line. This protein was identified as p130(cas). Tyrosine-phosphorylated p130(cas) from fibronectin-stimulated NIH3T3 cells bound to RPTPalpha-D1-C433S as well, suggesting that p130(cas) is a physiological substrate of RPTPalpha. RPTPalpha dephosphorylated p130(cas) in vitro, and RPTPalpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130(cas), and RPTPalpha or inactive, mutant RPTPalpha indicated that RPTPalpha dephosphorylated p130(cas) in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTPalpha under these conditions, suggesting that p130(cas) is a specific substrate of RPTPalpha in living cells. In conclusion, our results provide evidence that p130(cas) is a physiological substrate of RPTPalpha in vivo.

Multiple Interactions between Receptor Protein-tyrosine Phosphatase (RPTP) α and Membrane-distal Protein-tyrosine Phosphatase Domains of Various RPTPs

Receptor protein-tyrosine phosphatase (RPTP) alpha belongs to the large family of receptor protein-tyrosine phosphatases containing two tandem phosphatase domains. Most of the catalytic activity is retained in the first, membrane-proximal domain (RPTPalpha-D1), and little is known about the function of the second, membrane-distal domain (RPTPalpha-D2). We investigated whether proteins bound to RPTPalpha using the two-hybrid system and found that the second domain of RPTPsigma interacted with the juxtamembrane domain of RPTPalpha. We confirmed this interaction by co-immunoprecipitation experiments. Furthermore, RPTPalpha not only interacted with RPTPsigma-D2 but also with RPTPalpha-D2, LAR-D2, RPTPdelta-D2, and RPTPmu-D2, members of various RPTP subfamilies, although with different affinities. In the yeast two-hybrid system and in glutathione S-transferase pull-down assays, we show that the RPTP-D2s interacted directly with the wedge structure of RPTPalpha-D1 that has been demonstrated to be involved in inactivation of the RPTPalpha-D1/RPTPalpha-D1 homodimer. The interaction was specific because the equivalent wedge structure in LAR was unable to interact with RPTPalpha-D2 or LAR-D2. In vivo, we show that other interaction sites exist as well, including the C terminus of RPTPalpha-D2. The observation that RPTPalpha, but not LAR, bound to multiple RPTP-D2s with varying affinities suggests a specific mechanism of cross-talk between RPTPs that may regulate their biological function.

The Carboxyl-terminal Tyrosine Residue of Protein-tyrosine Phosphatase α Mediates Association with Focal Adhesion Plaques

The receptor protein-tyrosine phosphatase alpha (PTPalpha) is involved in the activation of c-Src kinase as well as in down-regulation of the insulin signal. To investigate the role of PTPalpha in activation of the Src kinase in more detail we tried to overexpress this phosphatase in NIH3T3 fibroblasts. Although PTPalpha has been overexpressed in rat embryonic fibroblasts and in embryonic carcinoma cells and should increase mitogenic responses we were not able to achieve a detectable overexpression. In contrast, expression of partially (C442S) or completely inactive (C442S,C732S) PTPalpha or of phosphatase active PTPalpha containing mutation Y781F or Y798F was possible. The level of expression, however, was reduced to background after several passages of lines expressing PTPalphaC442S,C732S and PTPalphaY781F. When employed in a focus formation assay, only infection with virus encoding PTPalphaY798F induced Src-dependent formation of foci. In immunofluorescence studies, PTPalphaC442S and PTPalphaY781F but not PTPalphaY798F colocalized with proteins found in focal adhesion plaques. Treatment of PTPalphaC442S-overexpressing cells with vanadate abolished this colocalization and led to proteolytic processing of the phosphatase. We conclude that tyrosine 798 in PTPalpha is important for localization at focal adhesion plaques. Inhibition of phosphatases by vanadate treatment releases PTPalpha from focal adhesions.

Expression of receptor protein tyrosine phosphatase alpha mRNA in human prostate cancer cell lines.

Receptor protein tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein phosphatase, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTPalpha mRNA in several normal human tissues. We further investigated the regulation of expression of RPTPalpha mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5alpha-dihydrotestosterone (DHT) down-regulates RPTPalpha mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTPalpha mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTPalpha as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTPalpha mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTPalpha is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.

Receptor protein tyrosine phosphatase alpha participates in the m1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity.

The phosphorylation state of a given tyrosine residue is determined by both protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activities. However, little is known about the functional interaction of these opposing activities at the level of an identified effector molecule. G protein-coupled receptors (GPCRs), including the m1 muscarinic acetylcholine receptor (mAChR), regulate a tyrosine kinase activity that phosphorylates and suppresses current generated by the Kv1.2 potassium channel. We examined the possibility that PTPs also participate in this signaling pathway since the tyrosine phosphatase inhibitor vanadate increases the extent of both Kv1.2 phosphorylation and suppression. We show that an endogenous transmembrane tyrosine phosphatase, receptor tyrosine phosphatase alpha (RPTPalpha), becomes tyrosine phosphorylated and co-immunoprecipitates with Kv1.2 in a manner dependent on m1 receptor activation. The N- and C-termini of Kv1.2 are shown to bind RPTPalpha in vitro. Overexpression of RPTPalpha in Xenopus oocytes increases resting Kv1.2 current. Biochemical and electrophysiological analysis reveals that recruiting RPTPalpha to Kv1.2 functionally reverses the tyrosine kinase-induced phosphorylation and suppression of Kv1.2 current in mammalian cells. Taken together, these results identify RPTPalpha as a new target of m1 mAChR signaling and reveal a novel regulatory mechanism whereby GPCR-mediated suppression of a potassium channel depends on the coordinate and parallel regulation of PTK and PTP activities.