Tyrosine Phosphatase Receptor Research Articles

Presynaptic PTPσ regulates postsynaptic NMDA receptor function through direct adhesion-independent mechanisms.

Synaptic adhesion molecules regulate synapse development and function. However, whether and how presynaptic adhesion molecules regulate postsynaptic NMDAR function remains largely unclear. Presynaptic LAR family receptor tyrosine phosphatases (LAR-RPTPs) regulate synapse development through mechanisms that include trans-synaptic adhesion; however, whether they regulate postsynaptic receptor functions remains unknown. Here we report that presynaptic PTPσ, a LAR-RPTP, enhances postsynaptic NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity in the hippocampus. This regulation does not involve trans-synaptic adhesions of PTPσ, suggesting that the cytoplasmic domains of PTPσ, known to have tyrosine phosphatase activity and mediate protein-protein interactions, are important. In line with this, phosphotyrosine levels of presynaptic proteins, including neurexin-1, are strongly increased in PTPσ-mutant mice. Behaviorally, PTPσ-dependent NMDAR regulation is important for social and reward-related novelty recognition. These results suggest that presynaptic PTPσ regulates postsynaptic NMDAR function through trans-synaptic and direct adhesion-independent mechanisms and novelty recognition in social and reward contexts.

PTPRS drives adaptive resistance to MEK/ERK inhibitors through SRC.

PTPRS is the most commonly mutated receptor tyrosine phosphatase in colorectal cancer (CRC). PTPRS has been shown to directly affect ERK and regulate its activation and nuclear localization. Here we identify that PTPRS may play a significant role in developing adaptive resistance to MEK/ERK inhibitors (MEKi/ERKi) through SRC activation. Moreover, we demonstrate a new clinical approach to averting adaptive resistance through the use of the SRC inhibitor, dasatinib. Our data suggest the potential for dasatinib to enhance the efficacy of MEKi and ERKi by preventing adaptive resistance pathways operating through SRC.

Vascular Endothelial Receptor Tyrosine Phosphatase: Identification of Novel Substrates Related to Junctions and a Ternary Complex with EPHB4 and TIE2*[S

Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling.

Identification of four Drosophila Toll-related proteins as ligands for the PTP69D receptor tyrosine phosphatase.

Identification of four Drosophila Toll-related proteins as ligands for the PTP69D receptor tyrosine phosphatase.

NGL-3 in the regulation of brain development, Akt/GSK3b signaling, long-term depression, and locomotive and cognitive behaviors.

Netrin-G ligand-3 (NGL-3) is a postsynaptic adhesion molecule known to directly interact with the excitatory postsynaptic scaffolding protein postsynaptic density-95 (PSD-95) and trans-synaptically with leukocyte common antigen-related (LAR) family receptor tyrosine phosphatases to regulate presynaptic differentiation. Although NGL-3 has been implicated in the regulation of excitatory synapse development by in vitro studies, whether it regulates synapse development or function, or any other features of brain development and function, is not known. Here, we report that mice lacking NGL-3 (Ngl3−/− mice) show markedly suppressed normal brain development and postnatal survival and growth. A change of the genetic background of mice from pure to hybrid minimized these developmental effects but modestly suppressed N-methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated synaptic transmission in the hippocampus without affecting synapse development, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated basal transmission, and presynaptic release. Intriguingly, long-term depression (LTD) was near-completely abolished in Ngl3−/− mice, and the Akt/glycogen synthase kinase 3β (GSK3β) signaling pathway, known to suppress LTD, was abnormally enhanced. In addition, pharmacological inhibition of Akt, but not activation of NMDARs, normalized the suppressed LTD in Ngl3−/− mice, suggesting that Akt hyperactivity suppresses LTD. Ngl3−/− mice displayed several behavioral abnormalities, including hyperactivity, anxiolytic-like behavior, impaired spatial memory, and enhanced seizure susceptibility. Among them, the hyperactivity was rapidly improved by pharmacological NMDAR activation. These results suggest that NGL-3 regulates brain development, Akt/GSK3β signaling, LTD, and locomotive and cognitive behaviors.

WASH phosphorylation balances endosomal versus cortical actin network integrities during epithelial morphogenesis

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.

Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol-3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho-associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline-inducible system in PTEN-null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non-phosphorylatable ED mutant exerted growth-suppressive and radiation-sensitizing effects, while the pseudo-phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein-protein interactions using co-immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS-associated proteins, including importin-β and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

Intracellular protein complexes involved in synapse assembly in presynaptic neurons.

The presynaptic active zone, composed of evolutionarily conserved protein complexes, is a specialized area that serves to orchestrate precise and efficient neurotransmitter release by organizing various presynaptic proteins involved in mediating docking and priming of synaptic vesicles, recruiting voltage-gated calcium channels, and modulating presynaptic nerve terminals with aligned postsynaptic structures. Among membrane proteins localized to active zone, presynaptic neurexins and LAR-RPTPs (leukocyte common antigen-related receptor tyrosine phosphatase) have emerged as hubs that orchestrate both shared and distinct extracellular synaptic adhesion pathways. In this chapter, we discuss intracellular signaling cascades involved in recruiting various intracellular proteins at both excitatory and inhibitory synaptic sites. In particular, we highlight recent studies on key active zone proteins that physically and functionally link these cascades with neurexins and LAR-RPTPs in both vertebrate and invertebrate model systems. These studies allow us to build a general, universal view of how presynaptic active zones operate together with postsynaptic structures in neural circuits.

In Silico and in Vitro Studies of Fluorinated Chroman-2-Carboxilic Acid Derivatives as an Anti-tubercular Agent.

Despite the use of traditional method, Ugi reaction currently is a well-established multicomponent reaction. Chromane motif itself possesses a variety of biological functions. In order to improve its anti-tubercular activity, it is necessary to modify it accordingly. To ensure relation between in silico and in vitro study, we have carried out in vitro screening against H37Rv anti-tubercular agent. Ugi four-component condensation (U-4CCRs) between 6-fluorochroman-2-carboxylic acid, various aryl aldehyde, 3,4,5-trimethoxy amine and tert-butyl isocyanide, gave N-((tert-butylcarbamoyl)(4-substitutedphenyl) methyl)-6-fluoro-N-(3,4,5-trimethoxyphenyl) chroman-2-carboxamide. The molecular level insight of all compounds was carried out by molecular docking study against the receptor tyrosine phosphatase PtpB. All these newly synthesized compounds were screened for their anti-microbial activity against Mycobacterium tuberculosis H37Rv to determine the MIC, IC50 and IC90 of the compound. The compound 5d also shows large hydrophobic surface contact on the face of the α7-α8 (Ile 207, Phe 211, Met 206, Ile203, Phe161, Phe80, Met126, Tyr130, Val231 and Leu101) that lines one side of the entrance to the active site of the receptor. The compound 5d bind with tyrosine phosphatase PtpB with predicted docking geometric score of 4664, whereas a score of rifampicin was 6586 determined. From the docking studies, compound 5d, was considered to be the potent inhibitor, which gave strong supportive coordinate with the in vitro study. It is highly active against H37Rv, having MIC and IC50 value of was 70 μM and 53 μM respectively in in vitro study.

CRISPR-Cas9 Mediated CD45 Knockout Inactivates Src Family Kinases and Impairs Cell Migration in Multiple Myeloma

IntroductionMultiple myeloma (MM) is a plasma cell malignancy that manifests continuous cell dissemination to multiple bone marrow (BM) niches and extramedullary (EM) sites. However, the molecular mechanisms behind this phenomenon remain elusive. CD45, a receptor tyrosine phosphatase, is an important regulator for T-cell and B-cell signaling pathways. In MM, the loss of CD45 expression has been correlated with earlier disease progression and inferior treatment outcomes. The downstream targets of CD45, Src family kinases (SFK), are associated with cell migration in many malignancies. It is also known to interact with proline-rich kinase (Pyk2) that binds to cytoskeleton-regulating proteins. Our previous in vitro studies demonstrated a ‘metastatic’ phenotype for CD45 negative MM. We hypothesise that CD45 acts as a marker for disease progression and mediates cell mobility through SFKs.MethodCD45 expression in human myeloma cell lines (HMCL) was assessed by flow cytometry. To investigate the functions of CD45 in MM, CRISPR-Cas9 mediated CD45 knockout (CD45KO) models were established from a HMCL, OCI-MY1. The resulting phenotypic and transcriptional changes were identified by immunoblotting, modified Boyden chamber assays, RNA sequencing and Phospho Explorer Antibody Array. SFK inhibitor (Saracatinib), Pyk2 inhibitor (PF573228) and siRNAs were used to validate the role of SFK (predominately Lyn and Fyn) and Pyk2 in migration.ResultsWe first compared 2 pairs of HMCL contemporaneously derived from BM and extramedullary disease (EM) in the same patient: TK1 (BM) and TK2 (EM), and TK17 (EM) and TK18 (BM). Both TK1 and TK18 had higher CD45 expression than their paired HMCL as expected.To avoid confounding intercellular genetic heterogeneity, we generated CD45KO models from OCI-MY1 and the loss of extracellular and intracellular portion of CD45 was confirmed by flow cytometry and immunoblotting. OCI-MY1 CD45KO cells showed significant SFK (Lyn and Fyn) and Pyk2 inactivation as compared to CD45 wild-type (CD45WT) cells. These cells demonstrated a significant reduction in homing capacity towards healthy and MM-patient derived BM stromal cells (reduced to 11.5% and 2.7%, p<0.0001, respectively) compared with the CD45WT cells. Treatment of CD45WT cells with Saracatinib and PF573228 similarly inhibited the homing capacity (47.3%, p<0.0001 and 71.3%, p<0.01 respectively). Moreover, silencing of Lyn and Fyn with siRNAs in CD45WT cells recapitulated the findings seen with the CD45KO cells (76%, p<0.01 and 55%, p<0.001, reduction in homing, respectively).The inactivation of SFK prompted us to investigate the transcriptional changes in the CD45KO cells. RNA sequencing identified differentially expressed migration-related genes in the CD45KO cell, in particular ADAM19, PARVB, AFAP1L2 and ITGAL, thus raising the possibility that dysregulation of these genes led to the observed reduction of homing potential of the CD45KO cells. To further understand the role of CD45 phosphatase activity, we analysed protein phosphorylation profiles in the CD45KO and CD45WT cells. In addition to the recognised inactivation of SFK and Pyk2, the Phospho Explorer Antibody Array also demonstrated reduced phosphorylation of JAK1 (Y1022), TYK2 (Y1054), c-Raf (S259) and MDM2 (S166), and increased phosphorylation of IRS-1 (S794), SHP-2 (Y580), c-Jun (Y170) and STAT6 (T645). We are currently investigating the correlation of these phosphorylation and MM homing mechanisms.ConclusionOur data demonstrate that CD45 plays an important role in regulating MM homing towards BM stroma by modulating SFK and Pyk2 activity with the loss of CD45 expression also resulting in the dysregulation of migration-related genes and widespread phosphorylation changes. Further in vivo studies evaluating the ‘metastatic’ impact of loss of CD45 expression are in progress. DisclosuresNo relevant conflicts of interest to declare.

PO-181 Anaplastic lymphoma kinase addictive neuroblastoma cell lines are associated with growth upon treatment with MEK inhibitor trametinib

Introduction The RAS-MAPK pathway is a major player in initiation and progression of multiple cancers where it can be hyper-activated by upstream regulatory proteins, such as receptor tyrosine kinases, phosphatases and GTPase’s. Several inhibitors targeting the RAS-MAPK pathway exhibit anti-cancer activity and are approved as a single agent in specific cancers. One such is the MEK inhibitor trametinib, which is included as a rational polytherapy strategy for treating EML4-ALK, EGFR and K-RAS mutant lung cancer and neuroblastoma containing hyper activating RAS-MAPK pathway mutations. In neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations of ALK, NRAS, NF1 genes, leading to hyperactivation of RAS-MAPK signalling. Targeting the RAS-MAPK pathways offers attractive options for combinatorial treatment together with ALK inhibitors since mono-treatment has not yet produced strong clinical results in ALK-positive neuroblastoma patients. Material and methods In order to determine which signalling core should be blocked for combinatorial treatment, we treated a panel of neuroblastoma cell lines using Trametinib, a MEK inhibitor. Resazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting. Results and discussions Here we investigate the response of ALK-positive neuroblastoma cell lines to MEK inhibition, employing trametinib. We show that pharmacological inhibition of MEK-ERK pathway results in increased levels of activation/phosphorylation of AKT and ERK5. This feedback response is regulated by SIN1 of mTORC2. Taken together, our results indicate that blocking MEK-ERK leads to ‘super-activation’ of AKT signalling core in ALK-positive neuroblastoma, increasing survival signals of these cells. Conclusion Our results contraindicate the use of MEK inhibitors as an effective therapeutic strategy in ALK-positive neuroblastoma.

Phosphoinositide 3-Kinase/Akt Signaling and Redox Metabolism in Cancer.

Metabolic rewiring and the consequent production of reactive oxygen species (ROS) are necessary to promote tumorigenesis. At the nexus of these cellular processes is the aberrant regulation of oncogenic signaling cascades such as the phosphoinositide 3-kinase and AKT (PI3K/Akt) pathway, which is one of the most frequently dysregulated pathways in cancer. In this review, we examine the regulation of ROS metabolism in the context of PI3K-driven tumors with particular emphasis on four main areas of research. (1) Stimulation of ROS production through direct modulation of mitochondrial bioenergetics, activation of NADPH oxidases (NOXs), and metabolic byproducts associated with hyperactive PI3K/Akt signaling. (2) The induction of pro-tumorigenic signaling cascades by ROS as a consequence of phosphatase and tensin homolog and receptor tyrosine phosphatase redox-dependent inactivation. (3) The mechanisms through which PI3K/Akt activation confers a selective advantage to cancer cells by maintaining redox homeostasis. (4) Opportunities for therapeutically exploiting redox metabolism in PIK3CA mutant tumors and the potential for implementing novel combinatorial therapies to suppress tumor growth and overcome drug resistance. Further research focusing on the multi-faceted interactions between PI3K/Akt signaling and ROS metabolism will undoubtedly contribute to novel insights into the extensive pro-oncogenic effects of this pathway, and the identification of exploitable vulnerabilities for the treatment of hyperactive PI3K/Akt tumors.

Molecular rationale for ERK and EGFR inhibition in colorectal cancer.

679 Background: Mutational activation of the RAS pathway occurs in &gt;50% of human CRC tumors, and to date has been difficult to overcome. We recently identified receptor tyrosine phosphatase-S (PTPRS) as an important regulator of the RAS pathway, which is mutated in ~11% of CRC. Methods: Global gene expression and DNA sequencing data (1321 cancer genes) were integrated across 468 CRC tumors to identify PTPRS as one of the most commonly mutated genes with a high correlation to a RAS activation score. Apoptotic responses to inhibitors of ERK, EGFR and their combination were tested in isogenic wild-type and mutant RAS cell lines where PTPRS had been knocked out by CRISPR. Results: PTPRS was in the top 15 genes correlating with RAS pathway activation scores; as expected, KRAS, BRAF were the most correlated genes. In order to determine the effect of the loss of PTPRS on RAS/ERK signaling, we used CRISPR to knockout PTPRS in an isogenic pair of CRC cell lines (HCT116) with mutant or wild type KRAS. Cell lines lacking PTPRS expression had elevated phospho-ERK activity. Interestingly, cell lines with PTPRS knockout and elevated ERK phosphorylation had an increased sensitivity to ERK inhibitors. Moreover, the loss of PTPRS allowed ERK to localize to the nucleus. Apoptosis increased dramatically from 13.1% in control cells to 53.8% in PTPRS KO cells treated with both ERK and EGFR inhibitors (Table), a synergistic effect not seen in wild type KRAS cells, where only ERK inhibitors were active. Conclusions: The loss of PTPRS allows increased activation of ERK (and the RAS pathway) which is associated with ERK addiction and increased sensitivity to ERK inhibitors. Surprisingly, ERK inhibitors synergize with EGFRi in wild type KRAS cell lines absent PTPRS. These data suggest the potential for mutant PTPRS to serve as a biomarker for ERK/EGFR inhibition. [Table: see text]

A dual-strategy expression screen for candidate connectivity labels in the developing thalamus.

The thalamus or “inner chamber” of the brain is divided into ~30 discrete nuclei, with highly specific patterns of afferent and efferent connectivity. To identify genes that may direct these patterns of connectivity, we used two strategies. First, we used a bioinformatics pipeline to survey the predicted proteomes of nematode, fruitfly, mouse and human for extracellular proteins containing any of a list of motifs found in known guidance or connectivity molecules. Second, we performed clustering analyses on the Allen Developing Mouse Brain Atlas data to identify genes encoding surface proteins expressed with temporal profiles similar to known guidance or connectivity molecules. In both cases, we then screened the resultant genes for selective expression patterns in the developing thalamus. These approaches identified 82 candidate connectivity labels in the developing thalamus. These molecules include many members of the Ephrin, Eph-receptor, cadherin, protocadherin, semaphorin, plexin, Odz/teneurin, Neto, cerebellin, calsyntenin and Netrin-G families, as well as diverse members of the immunoglobulin (Ig) and leucine-rich receptor (LRR) superfamilies, receptor tyrosine kinases and phosphatases, a variety of growth factors and receptors, and a large number of miscellaneous membrane-associated or secreted proteins not previously implicated in axonal guidance or neuronal connectivity. The diversity of their expression patterns indicates that thalamic nuclei are highly differentiated from each other, with each one displaying a unique repertoire of these molecules, consistent with a combinatorial logic to the specification of thalamic connectivity.

Identification of function-regulating antibodies targeting the receptor protein tyrosine phosphatase sigma ectodomain.

Receptor tyrosine phosphatase sigma (RPTPσ) plays an important role in the regulation of axonal outgrowth and neural regeneration. Recent studies have identified two RPTPσ ligands, chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPG), which can modulate RPTPσ activity by affecting its dimerization status. Here, we developed a split luciferase assay to monitor RPTPσ dimerization in living cells. Using this system, we demonstrate that heparin, an analog of heparan sulfate, induced the dimerization of RPTPσ, whereas chondroitin sulfate increased RPTPσ activity by inhibiting RPTPσ dimerization. Also, we generated several novel RPTPσ IgG monoclonal antibodies, to identify one that modulates its activity by inducing/stabilizing dimerization in living cells. Lastly, we demonstrate that this antibody promotes neurite outgrowth in SH-SY5Y cells. In summary, we demonstrated that the split luciferase RPTPσ activity assay is a novel high-throughput approach for discovering novel RPTPσ modulators that can promote axonal outgrowth and neural regeneration.

Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature.

Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.

Fat2 and Lar Define a Basally Localized Planar Signaling System Controlling Collective Cell Migration

Collective migration of epithelial cells underlies diverse tissue-remodeling events, but the mechanisms that coordinate individual cell migratory behaviors for collective movement are largely unknown. Studying the Drosophila follicular epithelium, we show that the cadherin Fat2 and the receptor tyrosine phosphatase Lar function in a planar signaling system that coordinates leading and trailing edge dynamics between neighboring cells. Fat2 signals from each cell's trailing edge to induce leading edge protrusions in the cell behind, in part by stabilizing Lar's localization in these cells. Conversely, Lar signals from each cell's leading edge to stimulate trailing edge retraction in the cell ahead. Fat2/Lar signaling is similar to planar cell polarity signaling in terms of sub-cellular protein localization; however, Fat2/Lar signaling mediates short-range communication between neighboring cells instead of transmitting long-range information across a tissue. This work defines a key mechanism promoting epithelial migration and establishes a different paradigm for planar cell-cell signaling.

A Small Molecule Inhibitor of Protein Tyrosine Phosphatase-Sigma (PTPσ) Promotes Hematopoietic Stem Cell (HSC) Regeneration

Receptor tyrosine kinases (RTKs), such as c-kit, Flt-3 and Tie2, regulate hematopoietic stem cell (HSC) proliferation, differentiation and maintenance. Substantially less is known regarding the function of receptor protein tyrosine phosphatases (PTPs) in regulating HSC fate. We recently discovered that receptor protein tyrosine phosphatase sigma (PTPσ) is highly expressed by murine and human HSCs. Interestingly, constitutive deletion of PTPσ caused a marked increase in HSC repopulating capacity as measured in primary and secondary competitive repopulation assays (J Clin Invest 2015;125:177-182). Further, negative selection of human cord blood (CB) HSCs for PTPσ surface expression (PTPσ - negative CB HSCs) conferred more than 10-fold increased human CB hematopoietic engraftment through 20 weeks in transplanted NSG mice. Additionally, PTPσ-deficient mice displayed significantly augmented recovery of phenotypic bone marrow (BM) HSCs and colony forming cells at day +10 following 550 cGy total body irradiation. Based on these observations, we hypothesized that PTPσ may function as a negative regulator of HSC self-renewal and regeneration. We sought to develop pharmacologic strategies to inhibit PTPσ function as a means to augment HSC functional capacity. Based on structure-activity-relationship analysis of PTPσ, we screened candidate small molecules for ability to modulate PTPσ function on BM HSCs. We identified a small molecule, 5483071 (Chembridge), as a candidate PTPσ inhibitor. In silico simulation indicated that 5483071 rigidly docked into the binding site of PTPσ intracellular domain through hydrogen bonding and electrostatic interactions. In a model of HSC injury and regeneration, we irradiated C57Bl6 mice with 700 cGy TBI and treated mice systemically with either 10 μcg of 5483071 or water subcutaneously every other day from day +1 to day +14. Irradiated mice treated with 5483071 displayed significantly increased 60 day survival compared to controls (P=0.0007). Irradiated mice treated with 5483071 showed accelerated recovery of BM SLAM+kit+sca-1+lin- HSCs (P=0.02), BM KSL cells (P=0.01), and colony forming cells (CFCs) (P=0.0003). In vitro culture of BM KSL cells with 5483071 significantly increased the levels of activated Rac1 (P=0.0004), which recapitulated the effects of PTPσ deletion on Rac1 activation in HSCs. Importantly, treatment of BM KSL cells from PTPσ -/- mice with 5483071 caused no change in Rac1 activation, suggesting that 5483071 acted specifically on PTPσ and was not mediating effects via inhibition of other phosphatases. Systemic administration of 5483071 to irradiated mice caused an increase in BM KSL cell cycling at 72 hours compared to irradiated control mice (p=0.02), while also decreasing BM KSL cell apoptosis at 24 hours after TBI (p=0.02). Subsequent to these findings, we have generated several new small molecule inhibitors of PTPσ with novel composition of matter and have taken a lead compound into pre-clinical studies for investigational new drug (IND) development. PTPσ represents a novel receptor tyrosine phosphatase that regulates HSC self-renewal and regeneration. Targeted inhibition of PTPσ has high therapeutic potential to promote hematopoietic regeneration in patients receiving myelosuppressive chemotherapy and/or radiotherapy or undergoing myeloablative hematopoietic cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Targeting of Ras-mediated FGF signaling suppresses Pten-deficient skin tumor

Deficiency in PTEN (phosphatase and tensin homolog deleted on chromosome 10) is the underlying cause of PTEN hamartoma tumor syndrome and a wide variety of human cancers. In skin epidermis, we have previously identified an autocrine FGF signaling induced by loss of Pten in keratinocytes. In this study, we demonstrate that skin hyperplasia requires FGF receptor adaptor protein Frs2α and tyrosine phosphatase Shp2, two upstream regulators of Ras signaling. Although the PI3-kinase regulatory subunits p85α and p85β are dispensable, the PI3-kinase catalytic subunit p110α requires interaction with Ras to promote hyperplasia in Pten-deficient skin, thus demonstrating an important cross-talk between Ras and PI3K pathways. Furthermore, genetic and pharmacological inhibition of Ras-MAPK pathway impeded epidermal hyperplasia in Pten animals. These results reveal a positive feedback loop connecting Pten and Ras pathways and suggest that FGF-activated Ras-MAPK pathway is an effective therapeutic target for preventing skin tumor induced by aberrant Pten signaling.

SALM4 suppresses excitatory synapse development by cis-inhibiting trans-synaptic SALM3-LAR adhesion.

Synaptic adhesion molecules regulate various aspects of synapse development, function and plasticity. These functions mainly involve trans-synaptic interactions and positive regulations, whereas cis-interactions and negative regulation are less understood. Here we report that SALM4, a member of the SALM/Lrfn family of synaptic adhesion molecules, suppresses excitatory synapse development through cis inhibition of SALM3, another SALM family protein with synaptogenic activity. Salm4-mutant (Salm4−/−) mice show increased excitatory synapse numbers in the hippocampus. SALM4 cis-interacts with SALM3, inhibits trans-synaptic SALM3 interaction with presynaptic LAR family receptor tyrosine phosphatases and suppresses SALM3-dependent presynaptic differentiation. Importantly, deletion of Salm3 in Salm4−/− mice (Salm3−/−; Salm4−/−) normalizes the increased excitatory synapse number. These results suggest that SALM4 negatively regulates excitatory synapses via cis inhibition of the trans-synaptic SALM3–LAR adhesion.