A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

Protein tyrosine phosphatase receptor type O serves as a key regulator of insulin resistance-induced α-synuclein aggregation in Parkinson's disease.

Insulin resistance (IR) was found to be a critical element in the pathogenesis of Parkinson's disease (PD), facilitating abnormal α-synuclein (α-Syn) aggregation in neurons and thus promoting PD development. However, how IR contributes to abnormal α-Syn aggregation remains ill-defined. Here, we analyzed six PD postmortem brain transcriptome datasets to reveal module genes implicated in IR-mediated α-Syn aggregation. In addition, we induced IR in cultured dopaminergic (DA) neurons overexpressing α-Syn to identify IR-modulated differentially expressed genes (DEGs). Integrated analysis of data from PD patients and cultured neurons revealed 226 genes involved in α-Syn aggregation under IR conditions, of which 53 exhibited differential expression between PD patients and controls. Subsequently, we conducted an integrated analysis of the 53 IR-modulated genes employing transcriptome data from PD patients with different Braak stages and DA neuron subclasses with varying α-Syn aggregation scores. Protein tyrosine phosphatase receptor type O (PTPRO) was identified to be closely associated with PD progression and α-Syn aggregation. Experimental validation in a cultured PD cell model confirmed that both mRNA and protein of PTPRO were reduced under IR conditions, and the downregulation of PTPRO significantly facilitated α-Syn aggregation and cell death. Collectively, our findings identified PTPRO as a key regulator in IR-mediated α-Syn aggregation and uncovered its prospective utility as a therapeutic target in PD patients with IR.

Targeting undruggable phosphatase overcomes trastuzumab resistance by inhibiting multi-oncogenic kinases.

AIMS: Resistance to targeted therapy is one of the critical obstacles in cancer management. Resistance to trastuzumab frequently develops in the treatment for HER2+ cancers. The role of protein tyrosine phosphatases (PTPs) in trastuzumab resistance is not well understood. In this study, we aim to identify pivotal PTPs affecting trastuzumab resistance and devise a novel counteracting strategy. METHODS: Four public datasets were used to screen PTP candidates in relation to trastuzumab responsiveness in HER2+ breast cancer. Tyrosine kinase (TK) arrays were used to identify kinases that linked to protein tyrosine phosphate receptor type O (PTPRO)-enhanced trastuzumab sensitivity. The efficacy of small activating RNA (saRNA) in trastuzumab-conjugated silica nanoparticles was tested for PTPRO upregulation and resistance mitigation in cell models, a transgenic mouse model, and human cancer cell line-derived xenograft models. RESULTS: PTPRO was identified as the key PTP which influences trastuzumab responsiveness and patient survival. PTPRO de-phosphorated several TKs, including the previously overlooked substrate ERBB3, thereby inhibiting multiple oncogenic pathways associated with drug resistance. Notably, PTPRO, previously deemed "undruggable," was effectively upregulated by saRNA-loaded nanoparticles. The upregulated PTPRO simultaneously inhibited ERBB3, ERBB2, and downstream SRC signaling pathways, thereby counteracting trastuzumab resistance. CONCLUSIONS: Antibody-conjugated saRNA represents an innovative approach for targeting "undruggable" PTPs.

Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH.

The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. Through whole genome sequencing we have uncovered conserved V483M Ptprh mutations in PyMT induced tumors. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while rescue with catalytically dead PTPRH did not. A dose response curve illustrated that two human NSCLC lines with naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Osimertinib treatment of these tumors resulted in a reduction of tumor volume relative to vehicle controls. PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Together these data suggest mutations in PTPRH in NSCLC is inhibitory to PTPRH function, resulting in aberrant EGFR activity and ultimately may result in clinically actionable alterations using existing therapies.

PTPRO inhibits LPS-induced apoptosis in alveolar epithelial cells.

Acute lung injury (ALI) and its severe manifestation, acute respiratory distress syndrome (ARDS), represent critical clinical syndromes with multifactorial origins, notably stemming from sepsis within intensive care units (ICUs). Despite their high mortality rates, no selective cure is available beside ventilation support. Apoptosis plays a complex and pivotal role in the pathophysiology of acute lung injury. Excessive apoptosis of alveolar epithelial and microvascular endothelial cells can lead to disruption of lung epithelial barrier integrity, impairing the body's ability to exchange blood and gas. At the same time, apoptosis of damaged or dysfunctional cells, including endothelial and epithelial cells, can help maintain tissue integrity and accelerate recovery from organ pro-inflammatory stress. The balance between pro-survival and pro-apoptotic signals in lung injury determines patient outcomes, making the modulation of apoptosis an area of intense research in the quest for more effective therapies. Here we found that protein tyrosine phosphatase receptor type O (PTPRO), a poorly understood receptor-like protein tyrosine phosphatase, is consistently upregulated in multiple tissue types of mice under septic conditions and in the lung alveolar epithelial cells. PTPRO reduction by its selective short-interfering RNA (siRNA) leads to excessive apoptosis in lung alveolar epithelial cells without affecting cell proliferation. Consistently PTPRO overexpression by a DNA construct attenuates apoptotic signaling induced by LPS. These effects of PTPTO on cellular apoptosis are dependent on an ErbB2/PI3K/Akt/NFκB signaling pathway. Here we revealed a novel regulatory pathway of cellular apoptosis by PTPRO in lung alveolar epithelial cells during sepsis.

Activation of Angiopoietin-Tie2 Signaling Protects the Kidney from Ischemic Injury by Modulation of Endothelial-Specific Pathways.

SIGNIFICANCE STATEMENT: Ischemia-reperfusion AKI (IR-AKI) is common and causes significant morbidity. Effective treatments are lacking. However, preclinical studies suggest that inhibition of angiopoietin-Tie2 vascular signaling promotes injury, whereas activation of Tie2 is protective. We show that kidney ischemia leads to increased levels of the endothelial-specific phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP; PTPRB), which inactivates Tie2. Activation of Tie2 through VE-PTP deletion, or delivery of a novel angiopoietin mimetic (Hepta-ANG1), abrogated IR-AKI in mice. Single-cell RNAseq analysis showed Tie2 activation promotes increased Entpd1 expression, downregulation of FOXO1 target genes in the kidney vasculature, and emergence of a new subpopulation of glomerular endothelial cells. Our data provide a molecular basis and identify a candidate therapeutic to improve endothelial integrity and kidney function after IR-AKI. BACKGROUND: Ischemia-reperfusion AKI (IR-AKI) is estimated to affect 2%-7% of all hospitalized patients. The significant morbidity and mortality associated with AKI indicates urgent need for effective treatments. Previous studies have shown activation of the vascular angiopoietin-Tie2 tyrosine kinase signaling pathway abrogates ischemia-reperfusion injury (IRI). We extended previous studies to (1) determine the molecular mechanism(s) underlying kidney injury and protection related to decreased or increased activation of Tie2, respectively, and (2) to test the hypothesis that deletion of the Tie2 inhibitory phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP) or injection of a new angiopoietin mimetic protects the kidney from IRI by common molecular mechanism(s). METHODS: Bilateral IR-AKI was performed in VE-PTP wild-type or knockout mice and in C57BL/6J mice treated with Hepta-ANG1 or vehicle. Histologic, immunostaining, and single-cell RNA sequencing analyses were performed. RESULTS: The phosphatase VE-PTP, which negatively regulates the angiopoietin-Tie2 pathway, was upregulated in kidney endothelial cells after IRI, and genetic deletion of VE-PTP in mice protected the kidney from IR-AKI. Injection of Hepta-ANG1 potently activated Tie2 and protected the mouse kidney from IRI. Single-cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function. Overlap was found between endothelial-specific genes upregulated by Hepta-ANG1 treatment and those downregulated in HUVECs with constitutive FOXO1 activation, including Entpd1 / ENTPD1 that modulates purinergic receptor signaling. CONCLUSIONS: Our data support a key role of the endothelium in the development of IR-AKI, introduce Hepta-ANG1 as a putative new therapeutic biologic, and report a model to explain how IRI reduces Tie2 signaling and how Tie2 activation protects the kidney. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_05_23_JSN_Ang_EP23_052323.mp3.

Delayed progressive sensorineural hearing loss due to a novel compound heterozygous PTPRQ mutation in a Chinese patient.

BACKGROUND: The Protein tyrosine phosphatase receptor Q (PTPRQ) gene encodes a member of the type III receptor-like protein tyrosine phosphatase family found in the stereocilium. Mutations in PTPRQ are mostly associated with deafness, autosomal recessive type 84 (DFNB 84), which usually results in progressive familial hearing loss. METHODS: A 25-year-old woman and her sister, both with postlingual-delayed progressive sensorineural hearing loss, were examined. They were from a nonconsanguineous marriage and had no family history of hearing loss. New compound heterozygous PTPRQ gene mutations, nonsense (c.90C > A, p.Y30X) and splice (c.5426 + 1G > A) mutations in two PTPRQ alleles, were identified in the two sisters and were presumably autosomal recessive. The c.90C > A (p.Y30X) mutation was mapped to exon 2 of PTPRQ (NM_001145026). RESULTS: The c.90C > A mutation leads to a premature stop codon and a truncated protein. The c.5426 + 1G > A mutation leads to a truncated protein lacking the extracellular domain. Hence, both mutations were predicted to be pathogenic, leading to a deficiency of the extracellular, transmembrane, and phosphatase domains because of nonsense-mediated mRNA degradation. CONCLUSIONS: This study increases the spectrum of PTPRQ gene mutations that might be involved in delayed progressive autosomal recessive non-syndromic hearing loss.

PNPT1, MYO15A, PTPRQ, and SLC12A2-associated genetic and phenotypic heterogeneity among hearing impaired assortative mating families in Southern India.

The study was conducted between 2018 and 2020. From a cohort of 113 hearing impaired (HI), five non-DFNB12 probands identified with heterozygous CDH23 variants were subjected to exome analysis. This resolved the etiology of hearing loss (HL) in four South Indian assortative mating families. Six variants, including three novel ones, were identified in four genes: PNPT1 p.(Ala46Gly) and p.(Asn540Ser), MYO15A p.(Leu1485Pro) and p.(Tyr1891Ter), PTPRQ p.(Gln1336Ter), and SLC12A2 p.(Pro988Ser). Compound heterozygous PNPT1 variants were associated with DFNB70 causing prelingual profound sensorineural hearing loss (SNHL), vestibular dysfunction, and unilateral progressive vision loss in one family. In the second family, MYO15A variants in the myosin motor domain, including a novel variant, causing DFNB3, were found to be associated with prelingual profound SNHL. A novel PTPRQ variant was associated with postlingual progressive sensorineural/mixed HL and vestibular dysfunction in the third family with DFNB84A. In the fourth family, the SLC12A2 novel variant was found to segregate with severe-to-profound HL causing DFNA78, across three generations. Our results suggest a high level of allelic, genotypic, and phenotypic heterogeneity of HL in these families. This study is the first to report the association of PNPT1, PTPRQ, and SLC12A2 variants with HL in the Indian population.

miR-4443 promotes radiation resistance of esophageal squamous cell carcinoma via targeting PTPRJ.

BACKGROUND: Radiotherapy is one of the main treatments for esophageal squamous cell carcinoma (ESCC), but its efficacy is limited by radioresistance. MicroRNAs play a crucial role in posttranscriptional regulation, which is linked to the cancer response to radiation. METHODS: We successfully established a radioresistant cell line model by using fractionated irradiation. qRT-PCR was adopted to detect the expression of miR-4443 in human normal esophageal cell lines, tumor cells, and radioresistant cells. Next, CCK-8, colony formation, apoptosis, and cell cycle assays were used to assess the biological effect of miR-4443. Weighted gene coexpression network analysis (WGCNA) was performed to identify potential radiosensitivity-related genes. Additionally, we predicted the probable targets of the miRNA using bioinformatic methods and confirmed them using Western blot. RESULTS: miR-4443 was significantly upregulated in radioresistant ESCC cells. Enhancement of miR-4443 further decreased the radiosensitivity of ESCC cells, while inhibition of miR-4443 increased the radiosensitivity of ESCC cells. Notably, miR-4443 modulated radiosensitivity by influencing DNA damage repair, apoptosis, and G2 cycle arrest. By using WGCNA and experimental validation, we identified PTPRJ as a key target for miRNA-4443 to regulate radiosensitivity. The effects of miR-4443 overexpression or inhibition could be reversed by increasing or decreasing PTPRJ expression. CONCLUSION: In this study, miR-4443 is found to promote radiotherapy resistance in ESCC cells by regulating PTPRJ expression, which provides a new perspective and clue to alleviate radioresistance.

Interplay between the tyrosine kinases Chk and Csk and phosphatase PTPRJ is critical for regulating platelets in mice.

The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.

Mutational and transcriptional alterations and clinicopathological factors predict the prognosis of stage I hepatocellular carcinoma : Prediction of stage I HCC prognosis.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) has been extensively studied. However, the impact on prognosis of stage I HCC has not been well studied at clincopathological, mutational and transcriptional levels. METHODS: Here we first characterized the influencing factors of prognosis of stage I HCC patients by downloading and analyzing the whole-exome somatic mutation data, messenger ribonucleic acid (mRNA) transcription data, along with demographic and clinical information of 163 stage I HCC patients from the TCGA database. The relationship between the influencing factors and HCC prognosis was studied in detail, and a prediction Nomogram model was established. Figures and tables were plotted using the R software. RESULTS: TP53, CTNNB1, TTN, MUC16 and ALB were the top mutated genes in stage I HCC. A series of co-mutations and mutually exclusive mutations were identified. Twenty-nine genes with significant stratification on prognosis were identified, including highly mutated LRP1B, ARID1A and PTPRQ. Patients with wild type (WT) genes unanimously exhibited significantly better overall survival rate than those with mutants. Patients with the top 10% tumor mutational burden (TMB) exhibited significantly worse prognosis than the rest 90%. Further characterization of transcriptional profile revealed that membrane functions, cell skeleton proteins, ion channels, receptor function and cell cycle were comprehensively altered in stage I HCC. Univariate and multivariate analyses were performed at clinicopathological, mutational and transcriptional levels. The combined analysis revealed sex, race, TMB, neoplasm histologic grade, Child-Pugh grade, MMRN1, OXT and COX6A2 transcription as independent risk factors. These factors were used to establish a Nomogram model to predict the prognosis of individual HCC patients. CONCLUSIONS: The influencing factors of prognosis of stage I HCC have been characterized for the first time at clinicopathological, mutational and transcriptional levels. A Nomogram model has been established to predict the prognosis. Further validation is needed to confirm the effectiveness and reliability of the model.

CD148 Deficiency in Fibroblasts Promotes the Development of Pulmonary Fibrosis.

Rationale: CD148/PTRJ (receptor-like protein tyrosine phosphatase η) exerts antifibrotic effects in experimental pulmonary fibrosis via interactions with its ligand syndecan-2; however, the role of CD148 in human pulmonary fibrosis remains incompletely characterized.Objectives: We investigated the role of CD148 in the profibrotic phenotype of fibroblasts in idiopathic pulmonary fibrosis (IPF).Methods: Conditional CD148 fibroblast-specific knockout mice were generated and exposed to bleomycin and then assessed for pulmonary fibrosis. Lung fibroblasts (mouse lung and human IPF lung), and precision-cut lung slices from human patients with IPF were isolated and subjected to experimental treatments. A CD148-activating 18-aa mimetic peptide (SDC2-pep) derived from syndecan-2 was evaluated for its therapeutic potential.Measurements and Main Results: CD148 expression was downregulated in IPF lungs and fibroblasts. In human IPF lung fibroblasts, silencing of CD148 increased extracellular matrix production and resistance to apoptosis, whereas overexpression of CD148 reversed the profibrotic phenotype. CD148 fibroblast-specific knockout mice displayed increased pulmonary fibrosis after bleomycin challenge compared with control mice. CD148-deficient fibroblasts exhibited hyperactivated PI3K/Akt/mTOR signaling, reduced autophagy, and increased p62 accumulation, which induced NF-κB activation and profibrotic gene expression. SDC2-pep reduced pulmonary fibrosis in vivo and inhibited IPF-derived fibroblast activation. In precision-cut lung slices from patients with IPF and control patients, SDC2-pep attenuated profibrotic gene expression in IPF and normal lungs stimulated with profibrotic stimuli.Conclusions: Lung fibroblast CD148 activation reduces p62 accumulation, which exerts antifibrotic effects by inhibiting NF-κB-mediated profibrotic gene expression. Targeting the CD148 phosphatase with activating ligands such as SDC2-pep may represent a potential therapeutic strategy in IPF.

Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ . This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Mutational landscape of TRPC6, WT1, LMX1B, APOL1, PTPRO, PMM2, LAMB2 and WT1 genes associated with Steroid resistant nephrotic syndrome.

BACKGROUND: Nephrotic syndrome appears as a group of symptoms like proteinuria, edema and hyperlipidemia. Identification of monogenic forms revealed the physiology and pathogenesis of the SRNS. METHODS AND RESULTS: We performed Illumina panel sequencing of seven genes in 90 Indian patients to determine the role of these genetic mutations in nephrotic syndrome prognosis. Samtool was used for variants calling, and SnpEff and Snpsift did variants annotation. Clinical significance and variant classification were performed by the ClinVar database. In SSNS and SRNS patients, we found 0.78% pathogenic and 3.41% likely pathogenic mutations. Pathogenic mutations were found in LAMB2, LMX1B and WT1 genes, while likely pathogenic mutations were found in (6/13) LAMB2, (2/13) LMX1B, (2/13) TRPC6, (2/13) PTPRO and (1/13) PMM2 genes. Approximately 46% likely pathogenic mutations were contributed to the LAMB2 gene in SSNS and SRNS patients. We also detect 30 VUS (variants of uncertain significance), which were found (17/30) pathogenic and (13/30) likely pathogenic by different prediction tools. CONCLUSIONS: Multigene panels were used for genetic screening of heterogeneous disorders like nephrotic syndrome in the Indian population. We found pathogenic, likely pathogenic and certain VUS, which were responsible for the pathogenesis of the disease. Therefore, mutational analysis of SSNS and SRNS is necessary to avoid adverse effects of corticosteroids, modify the intensity of immunosuppressing agents, and prevent the disease's progression.

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

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.

Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP.

The angiopoietin (ANGPT)-TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.

Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine malignant melanoma by integrated comparative genomic analysis.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Candidate genes for hereditary colorectal cancer: Mutational screening and systematic review.

Genome-wide approaches applied for the identification of new hereditary colorectal cancer (CRC) genes, identified several potential causal genes, including RPS20, IL12RB1, LIMK2, POLE2, MRE11, POT1, FAN1, WIF1, HNRNPA0, SEMA4A, FOCAD, PTPN12, LRP6, POLQ, BLM, MCM9, and the epigenetic inactivation of PTPRJ. Here we attempted to validate the association between variants in these genes and nonpolyposis CRC by performing a mutational screening of the genes and PTPRJ promoter methylation analysis in 473 familial/early-onset CRC cases, a systematic review of the published cases, and assessment of allele frequencies in control population. In the studied cohort, 24 (5%) carriers of (predicted) deleterious variants in the studied genes and no constitutional PTPRJ epimutations were identified. Assessment of allele frequencies in controls compared with familial/early-onset patients with CRC showed association with increased nonpolyposis CRC risk of disruptive variants in RPS20, IL12RB1, POLE2, MRE11 and POT1, and of FAN1 c.149T>G (p.Met50Arg). Lack of association was demonstrated for LIMK2, PTPN12, LRP6, PTPRJ, POLQ, BLM, MCM9 and FOCAD variants. Additional studies are required to provide conclusive evidence for SEMA4A, WIF1, HNRNPA0 c.-110G>C, and FOCAD large deletions.

PTPRO exaggerates inflammation in ulcerative colitis through TLR4/NF-κB pathway.

Previous studies have implicated protein tyrosine phosphatase receptor type O (PTPRO) as a key regulator in inflammation-associated diseases; however, its role in ulcerative colitis (UC) remains largely unknown. Thus, we aim to elucidate the potential role and underlying mechanism of PTPRO in UC. In this study, increased expression of PTPRO, toll-like receptor (TLR4) and inflammatory cytokines were observed in mucosal tissues (MTs) from inflamed areas and lamina propria mononuclear cells (LPMCs) of patients with UC compared with those from healthy controls. Then, it was manifested that PTPRO promoted the expression of TLR4 and proinflammatory cytokines in lipopolysaccharide-induced (LPS-induced) inflammatory macrophage model. Besides, PTPRO inhibited the proliferation of intestinal epithelial cells (IECs) but enhanced the apoptosis of IECs in macrophages. Moreover, levels of phosphorylated nuclear factor κB (NF-κB)/p65 and inhibitor of NF-κB α (IκBα) were more significantly increased in PTPRO overexpressed macrophages. In addition, the area under receiver operating characteristic curve was 0.807 (95%CI = 0.686-0.958, P < .001) suggesting PTPRO as an ideal diagnostic marker for UC. Taken these, the present study shows strong evidence that PTPRO exaggerates inflammation in UC via TLR4/NF-κB signaling pathway.

Agonistic anti-CD148 monoclonal antibody attenuates diabetic nephropathy in mice.

CD148 is a transmembrane protein tyrosine phosphatase (PTP) that is expressed in the renal vasculature, including the glomerulus. Previous studies have shown that CD148 plays a role in the negative regulation of growth factor signals (including epidermal growth factor and vascular endothelial growth factor), suppressing cell proliferation and transformation. However, the role of CD148 in kidney disease remains unknown. Here, we generated an agonistic anti-CD148 antibody and evaluated its effects in murine diabetic nephropathy (DN). Monoclonal antibodies (mAbs) against the mouse CD148 ectodomain sequence were generated by immunizing CD148 knockout (CD148KO) mice. The mAbs that increased CD148 activity were selected by biological (proliferation) and biochemical (PTP activity) assays. The mAb (18E1) that showed strong agonistic activity was injected (10 mg/kg ip) in streptozotocin-induced wild-type and CD148KO diabetic mice for 6 wk, and the renal phenotype was then assessed. The effects of 18E1 mAb in podocyte growth factor signals were also assessed in culture. Compared with control IgG, 18E1 mAb significantly decreased albuminuria and mesangial expansion without altering hyperglycemia and blood pressure in wild-type diabetic mice. Immunohistochemical evaluation showed that 18E1 mAb significantly prevented the reduction of podocyte number and nephrin expression and decreased glomerular fibronectin expression and renal macrophage infiltration. The 18E1 mAb showed no effects in CD148KO diabetic mice. Furthermore, we demonstrated that 18E1 mAb reduces podocyte epidermal growth factor receptor signals in culture and in diabetic mice. These findings suggest that agonistic anti-CD148 mAb attenuates DN in mice, in part by reducing epidermal growth factor receptor signals in podocytes. This antibody may be used for the treatment of early DN.