Receptor-like Tyrosine Phosphatase Research Articles

Insights into olfactory ensheathing cell development from a laser-microdissection and transcriptome-profiling approach.

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.

Spatial confinement of receptor activity by tyrosine phosphatase during directional cell migration

Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in Caenorhabditis elegans, the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1-dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.

An extracatalytic function of CD45 in B cells is mediated by CD22

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal "ignorance."

The Structural Wedge Domain of the Receptor-like Tyrosine Phosphatase CD45 Enforces B Cell Tolerance by Regulating Substrate Specificity

CD45 is a receptor-like tyrosine phosphatase that positively regulates BCR signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases. We showed previously that a single point mutation, E613R, introduced into the cytoplasmic membrane-proximal "wedge" domain of CD45 is sufficient to drive a lupus-like autoimmune disease on a susceptible genetic background. To clarify the molecular mechanism of this disease, we took advantage of a unique allelic series of mice in which the expression of CD45 is varied across a broad range. Although both E613R B cells and those with supraphysiologic CD45 expression exhibited hyperresponsive BCR signaling, they did so by opposite regulation of the Src family kinase Lyn. We demonstrated that the E613R allele of CD45 does not function as a hyper- or hypomorphic allele but rather alters the substrate specificity of CD45 for Lyn. Despite similarly enhancing BCR signaling, only B cells with supraphysiologic CD45 expression became anergic, whereas only mice harboring the E613R mutation developed frank autoimmunity on a susceptible genetic background. We showed that selective impairment of a Lyn-dependent negative-regulatory circuit in E613R B cells drove autoimmunity in E613R mice. This demonstrates that relaxing negative regulation of BCR signaling, rather than enhancing positive regulation, is critical for driving autoimmunity in this system.

Novel Tools to Dissect the Dynamic Regulation of TCR Signaling by the Kinase Csk and the Phosphatase CD45

Although the biochemical events induced by T-cell receptor (TCR) triggering have been well studied, both the mediators and function of basal signaling in T cells remain poorly understood. Furthermore, the precise mechanisms by which MHC-peptide interaction with the TCR disrupt the basal equilibrium to induce downstream signaling are also unclear. Here we describe novel approaches to understand the basal state of T cells and the mechanisms of TCR triggering by perturbing regulation of the Src family kinases (SFKs). The SFKs are critical proximal mediators of TCR signaling that are in turn tightly regulated by the tyrosine kinase Csk and the receptor-like tyrosine phosphatase CD45. We have developed a small-molecule analog-sensitive allele of Csk and an allelic series of mice in which expression of CD45 is varied across a broad range. Our studies have unmasked contributions of Csk and CD45 to maintain the basal state of T cells and also suggest that dynamic regulation of Csk may be involved in TCR triggering.

Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.

Phosphatases Fight over fMLF

Receptor-like tyrosine phosphatases have opposing roles in regulating chemoattractant receptor signaling in mouse neutrophils.

Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival

The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor signaling by dephosphorylating the inhibitory tyrosine of the src family kinases. CD45-deficient mice fail to fully unmask the role of CD45 in B cells because of the expression of a partially redundant tyrosine phosphatase, CD148. However, mice that are doubly deficient in CD45 and CD148 exhibit a very early block in B-cell development, thereby obscuring later roles for CD45. To overcome these limitations, here we take advantage of an allelic series of mice in which CD45 expression is titrated broadly (0-180%). Although high expression of CD45 inhibits T-cell receptor (TCR) signaling, we show that CD45 plays a purely positive regulatory role during B-cell receptor (BCR) signaling. In concert with exaggerated BCR signaling, increasing CD45 expression drives enhanced receptor editing in the bone marrow and profound loss of follicular and marginal zone B cells in the spleen. In the context of the IgHEL/sHEL model of B-cell tolerance, such high CD45 expression transforms anergy into deletion. Unexpectedly, elimination of the autoantigen sHEL in this model system in order to block clonal deletion fails to rescue survival of mature B cells. Rather, high CD45 expression reduces B-cell activating factor receptor (BAFFR) expression and inhibits B-cell activating factor (BAFF)-induced B-cell survival in a cell-intrinsic manner. Taken together, our findings reveal how CD45 function diverges in T cells and B cells, as well as how autoreactive B cells are censored as they transit development.

Receptor-like Tyrosine Phosphatases CD45 and CD148 Have Distinct Functions in Chemoattractant-Mediated Neutrophil Migration and Response to S. aureus

Neutrophils, critical innate immune effectors, use bacterial-derived chemoattractant-induced G protein-coupled receptor (GPCR) signaling for their pursuit of bacteria. Tyrosine phosphorylation pathways and receptor-like tyrosine phosphatases (RPTPs) are rarely considered in chemoattractant-mediated GPCR signaling. Here, we report that two RPTPs, CD45 and CD148, previously shown to share redundant roles in positively regulating Src family kinases (SFKs) in immunoreceptor signaling pathways in B cells and macrophages, are critical in the neutrophil response to S. aureus infection and, surprisingly, in chemoattractant-mediated chemotaxis. Remarkably, deficiency in either of these RPTPs influenced neutrophil GPCR responses in unique ways. Our results reveal that CD45 positively while CD148 positively and negatively regulate GPCR function and proximal signals including Ca(2+), phosphatidylinositol 3'OH kinase (PI3K), and phospho-extracellular regulated kinase (pERK) activity. Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways.

Titration of CD45 expression regulates sequential tolerance checkpoints during B cell development (45.2)

Abstract The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor (AgR) signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases (SFKs). Here we take advantage of an allelic series of mice in which CD45 expression is titrated across a broad range (0-180%). Although high expression of CD45 inhibits TCR signaling, here we show that CD45 plays a purely positive regulatory role during BCR signaling. In concert with enhanced BCR signaling, mice expressing very high levels of CD45 exhibit marked loss of follicular and marginal zone B cells in a cell intrinsic manner. Although we do not see B cell loss at earlier stages of bone marrow development, we do find increasing lambda light chain usage across the CD45 allelic series suggesting that receptor editing is triggered by high CD45 expression. In the context of the IgHEL/sHEL model of B cell tolerance, such high CD45 expression transforms anergy into deletion. However, elimination of autoreactive ligand in this system fails to fully rescue survival of mature B cells in these mice. Indeed, we find that B cell survival upon exposure to BAFF is severely impaired by high CD45 expression, while low CD45 expression promotes response to BAFF. Titration of BAFF responsiveness by CD45 is cell intrinsic and correlates with BAFFR surface expression. This constitutes a novel mechanism by which B cells exhibiting hyper-responsive AgR signaling are kept in check during splenic B cell development.

An allelic series of CD45 dosages uncouples basal and inducible TCR signaling during thymic development (84.13)

Abstract CD45 is a receptor-like tyrosine phosphatase expressed at very high levels on all nucleated hematopoietic cells. CD45 serves as a critical positive regulator of TCR signal transduction by dephosphorylating the inhibitory tyrosine of the Src family kinases, Lck and Fyn. Previously, CD45 deficient animals have revealed a critical positive regulatory role in beta-selection and positive selection during thymic development. Here we describe a novel allele of CD45, lightning, in which a single residue substitution in the extracellular domain produces low protein expression levels but normal isoform splicing. An allelic series of CD45 dosages reveals differential requirements for CD45 in basal and inducible TCR signaling during thymic development. While low levels of CD45 are sufficient to rescue T cell development and TCR inducible signaling, high doses of CD45 are required to fully reconstitute beta-selection and basal signaling. Furthermore, a negative regulatory role for CD45 is identified during inducible but not basal signaling. Hyperphosphorylation of the activation loop tyrosine of Lck but not Fyn at intermediate doses of CD45 correlates with hyper-responsive inducible signaling and demonstrates differential regulation of the src family kinases. Further, distinct thymic subsets exhibit differential requirements for CD45, Fyn and co-receptor-associated Lck in TCR signaling.

A novel mechanism for inhibition of Hyperpolarization-activated Pacemaker Channels by Receptor-like Tyrosine Phosphatase alpha

We have previously reported an important role of increased tyrosine phosphorylation activity by Src in the modulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. Using a combination of whole-cell patch clamp technique, Western blot, and confocal fluorescence imaging, we assessed the hypothesis that decreased tyrosine dephosphorylation may enhance HCN channel activity as well. We discovered that the receptor-like protein tyrosine phosphatase alpha (RPTPα) significantly inhibited or even eliminated HCN2 currents expressed in HEK293 cells.

Structurally Distinct Phosphatases CD45 and CD148 Both Regulate B Cell and Macrophage Immunoreceptor Signaling

The receptor-type protein tyrosine phosphatase (RPTP) CD148 is thought to have an inhibitory function in signaling and proliferation in nonhematopoietic cells. However, its role in the immune system has not been thoroughly studied. Our analysis of CD148 loss-of-function mice showed that CD148 has a positive regulatory function in B cells and macrophages, similar to the role of CD45 as a positive regulator of Src family kinases (SFKs). Analysis of CD148 and CD45 doubly deficient B cells and macrophages revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SFKs accompanied by substantial alterations in B and myeloid lineage development and defective immunoreceptor signaling. Because these findings suggest the C-terminal tyrosine of SFKs is a common substrate for both CD148 and CD45 phosphatases and imply a level of redundancy not previously appreciated, a reassessment of the function of CD45 in the B and myeloid lineages based on prior data from the CD45-deficient mouse is warranted.

Reciprocal Regulation of DAPK

The tumor suppressor death-associated protein kinase (DAPK) functions by inhibiting cell adhesion and migration and promoting apoptosis. It is positively regulated by calmodulin and is thought to inactivate integrins, thus preventing cell adhesion-induced survival signals. Wang et al . sought to determine the mechanism of regulation of DAPK. Yeast two-hybrid screening identified leukocyte common antigen-related protein (LAR), a receptor-like tyrosine phosphatase, as a binding partner for the ankyrin repeat domain of DAPK. This interaction was confirmed by coimmunoprecipitation studies in 293T cells. Western blotting showed that DAPK was a substrate for LAR and, using DAPK mutants, the authors identified Tyr 491 and Tyr 492 as the target dephosphorylation sites. In vitro kinase assays demonstrated that the kinase activity of DAPK was increased in 293T transfectants coexpressing LAR but not in cells expressing a phosphatase-deficient mutant LAR. 293T cells cotransfected with DAPK and LAR performed poorly in adhesion assays compared with 293T cells transfected with DAPK alone. Adhesion of cells to fibronectin resulted in the phosphorylation of DAPK, which was reduced by inhibitors of Src tyrosine kinases. Western blotting revealed that Src phosphorylated DAPK at the same residues targeted by LAR, and Src-mediated phosphorylation of DAPK promoted cell adhesion and migration. Epidermal growth factor (EGF) activates Src and decreases the abundance of LAR at the membrane. Treatment of epithelial carcinoma cells with EGF resulted in DAPK inactivation and increased EGF-stimulated migration. Immunohistochemistry demonstrated a correlation between the incidence of hyperphosphorylated, inactivated DAPK and increased Src activity in tumor cells. Together, these data establish a possible link between EGF-induced tumorigenesis and DAPK inactivation through the reciprocal regulation of DAPK by LAR and Src. W.-J. Wang, J.-C. Kuo, W. Ku, Y.-R. Lee, F.-C. Lin, Y.-L. Chang, Y.-M. Lin, C.-H. Chen, Y.-P. Huang, M.-J. Chiang, S.-W. Yeh, P.-R. Wu, C.-H. Shen, C.-T. Wu, R.-H. Chen, The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR. Mol. Cell 27 , 701-716 (2007). [PubMed]

A monoclonal antibody against CD148, a receptor-like tyrosine phosphatase, inhibits endothelial-cell growth and angiogenesis

AbstractAngiogenesis contributes to a wide range of neoplastic, ischemic, and inflammatory disorders. Definition of the intrinsic molecular controls in angiogenic vessel growth promises novel therapeutic approaches for angiogenesis-related diseases. CD148 (also named DEP-1/PTPη) is a receptor-like protein tyrosine phosphatase that is abundantly expressed in vascular endothelial cells. To explore a role of CD148 in endothelial vessel formation, we generated a monoclonal antibody, Ab1, against the ectodomain sequence of CD148 and examined its effects on endothelial-cell growth and vessel formation. Here we report that a bivalent, but not a monovalent, form of the Ab1 antibody inhibits endothelial-cell growth and blocks angiogenesis in mouse cornea in vivo. We further demonstrate that (1) bivalent Ab1 arrests cell-cycle progression of CD148-transfected CHO cells at G0/G1 phase, (2) coexpression of catalytically inactive CD148 mutants attenuates the Ab1-cell growth inhibition, and (3) bivalent Ab1 suppresses phosphorylation of ERK1/2 kinases and Met tyrosine kinase as activated CD148 does, with an increase in CD148-associated tyrosine phosphatase activity. Taken together, these findings demonstrate that Ab1-induced ectodomain oligomerization arrests endothelial-cell growth through catalytic activity of the CD148 cytoplasmic domain. The present study defines CD148 as a valuable molecular target for antiangiogenesis therapy.

Regulated expression of the receptor-like tyrosine phosphatase CD148 on hemopoietic cells.

CD148 is a receptor-like protein tyrosine phosphatase expressed on a wide variety of cell types. Through the use flow cytometry and immunofluorescence microscopy on tissue sections, we examined the expression of CD148 on multiple murine hemopoietic cell lineages. We found that CD148 is moderately expressed during all stages of B cell development in the bone marrow, as well as peripheral mature B cells. In contrast, CD148 expression on thymocytes and mature T cells is substantially lower. However, stimulation of peripheral T cells through the TCR leads to an increase of CD148 expression. This up-regulation on T cells can be partially inhibited by reagents that block the activity of src family kinases, calcineurin, MEK, or PI3K. Interestingly, CD148 levels are elevated on freshly isolated T cells from MRL lpr/lpr and CTLA-4-deficient mice, two murine models of autoimmunity. Together, these expression data along with previous biochemical data suggest that CD148 may play an important regulatory role to control an immune response.

RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages.

Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of αv/β3-integrin–mediated cell–matrix connections requires the receptor-like tyrosine phosphatase α (RPTPα). RPTPα colocalizes with αv-integrins at the leading edge during early spreading, and coimmunoprecipitates with αv-integrins during spreading on fibronectin and vitronectin. RPTPα-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of αv/β3-integrin–cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPα is an early component in force-dependent signal transduction pathways leading to the assembly of focal complexes on both fibronectin and vitronectin.

The receptor-like tyrosine phosphatase lar is required for epithelial planar polarity and for axis determination within drosophila ovarian follicles.

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. We have identified mutations in the receptor-like transmembrane tyrosine phosphatase Lar that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells, but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. We show that Lar acts somatically early in oogenesis, during follicle formation, and postulate that it functions in germarium intercyst cells that are required for polar cell specification and differentiation. Our studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals.

Identification of a receptor-like protein tyrosine phosphatase expressed during Xenopus development.

To begin to determine the role of receptor-like tyrosine phosphatases during Xenopus development, we have isolated a cDNA predicted to encode receptor-like tyrosine phosphatase with significant amino acid sequence identity to mouse and human protein tyrosine phosphatase alpha (PTPalpha). Xenopus PTPalpha (XPTPalpha) exists as a maternally expressed mRNA that decreases in expression during gastrulation and then maintains a constant lower level of expression through early tadpole stages. In situ hybridization reveals that XPTPalpha mRNA is expressed throughout the gastrula stage embryo. During subsequent development, XPTPalpha mRNA becomes restricted in its expression to various regions of the brain and the visceral arches. XPTPalpha mRNA is also expressed in several adult tissues and in Xenopus XTC cells. Immunoblot analysis demonstrates that XPTPalpha protein is expressed at relatively uniform levels throughout development. Expression of XPTPalpha protein in insect cells with a recombinant baculovirus results in a glycosylated polypeptide of 110-130 kDa with intrinsic phosphotyrosine phosphatase activity. The spatial and temporal patterns of expression of XPTPalpha indicate that it may play multiple roles during early development including development of the brain.

Structural analysis of a dimeric state of the first intracellular domain of PTPa, a protein receptor-like tyrosine phosphatase

Structural analysis of a dimeric state of the first intracellular domain of PTPa, a protein receptor-like tyrosine phosphatase