scholarly journals A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2570-2578 ◽  
Author(s):  
Dafydd H. Thomas ◽  
Todd M. Getz ◽  
Tiffanny N. Newman ◽  
Carol A. Dangelmaier ◽  
Nick Carpino ◽  
...  
Keyword(s):  
T Cell ◽  
Phospholipase C ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Platelet Glycoprotein ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Downstream Effector

Abstract T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

scholarly journals TULA-2 Deficiency Enhances Platelet Functional Responses to CLEC-2 Agonists

TH Open ◽  
2018 ◽  
Vol 02 (04) ◽  
pp. e411-e419 ◽  
Author(s):  
John Kostyak ◽  
Benjamin Mauri ◽  
Carol Dangelmaier ◽  
Akruti Patel ◽  
Yuhang Zhou ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Surface Receptors ◽  
Downstream Signaling ◽  
Glycoprotein Vi ◽  
Thromboxane Production ◽  
Activation Motif

AbstractPlatelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-μg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2−/− platelets treated with 2.5-μg/mL CLEC-2 antibody. TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.

A Novel Histidine Tyrosine Phosphatase, TULA-2, Associates with Syk and Negatively Regulates GPVI Signaling in Platelets.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1843-1843
Author(s):  
Dafydd H Thomas ◽  
Carol Dangelmaier ◽  
Jianguo Jin ◽  
Alexander Tsygankov ◽  
Satya P. Kunapuli ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Tyrosine Kinase ◽  
Tyrosine Phosphatase ◽  
Dense Granule ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Signaling Cascade ◽  
Glycoprotein Vi ◽  
Platelet Receptor

Abstract Glycoprotein VI (GPVI) is the primary platelet receptor for collagen signaling. Following damage to the vascular endothelium, the GPVI receptor interacts with the exposed sub-endothelial collagen. This interaction initiates a signaling cascade involving phosphorylation of the dual ITAM motif of the FcRγ chain by Fyn and Lyn, followed by the recruitment, phosphorylation and activation of Syk. This leads to the eventual activation of PLCγ2 and the release of calcium from intracellular stores to cause platelet activation. While a lot is known about the activation processes involved in GPVI signaling less is known about its negative regulation. The T-cell ubiquitin ligand (TULA) family of proteins has been implicated in the negative regulation protein tyrosine kinase (PTK)-dependent signaling pathways. More recently, it has been shown the TULA family member, TULA-2, exhibits phosphatase activity towards PTKs, including Syk, and this activity is responsible for the negative regulation of T-cell receptor signaling (Mikhailik et. al. 2007, Agrawal et. al. 2008). Thus, we investigated the role of TULA-2 in the negative regulation of the GPVI signaling cascade. We show that TULA-2 is expressed in both human and murine platelets. Deletion of TULA-2 in murine platelets manifests itself functionally as enhanced aggregation in response to the GPVI agonist convulxin as well as enhanced dense granule secretion when compared to wild type platelets. No difference was witnessed in response to the PAR4 agonist AYPGKF. TULA-2-deficient platelets also exhibit sustained hyperphosphorylation of Syk at tyrosines 525 and 526 as well as hyperphosphorylation of PLCγ2 at tyrosines 753 and 759, indicative of enhanced kinase and phospholipase activity respectively. GST-pulldown experiments suggest that Syk and TULA- 2 are able to associate in resting and convulxin stimulated platelets and in-vitro phosphatase assays demonstrate that TULA-2 can dephosphorylate Syk at tyrosines 525 and 526. Taken together, these data suggest that TULA-2 is a negative regulator of GPVI signaling and this regulation is mediated by an association of TULA-2 with Syk, allowing the dephosphorylation of Syk at catalytically important tyrosine residues.

scholarly journals Themis-associated phosphatase activity controls signaling in T cell development

2018 ◽  
Vol 115 (48) ◽  
pp. E11331-E11340 ◽  
Author(s):  
Monika Mehta ◽  
Joanna Brzostek ◽  
Elijah W. Chen ◽  
Desmond W. H. Tung ◽  
Shuting Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Phosphatase Activity ◽  
Ex Vivo ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Conditional Knockout ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
Thymic Development ◽  
Double Deletion

Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.

Deletion of the Protein Tyrosine Phosphatase Gene PTPN2 in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 141-141
Author(s):  
Maria Kleppe ◽  
Idoya Lahortiga ◽  
Tiama El Chaar ◽  
Kim De Keersmaecker ◽  
Nicole Mentens ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Protein Tyrosine Phosphatase ◽  
Lymphoblastic Leukemia ◽  
Tyrosine Phosphatase ◽  
Janus Kinase ◽  
Negative Regulator ◽  
Aberrant Expression ◽  
Protein Tyrosine ◽  
Knock Down

Abstract Abstract 141 Introduction: T-cell lymphoblastic leukemia (T-ALL) arises from clonal expansion of a lymphoid progenitor that has undergone stepwise alteration at distinct stages of differentiation. It is suggested that a set of cooperative mutations that affect different pathways are required before thymocytes become fully malignant. Despite major improvements in our understanding of the molecular genetics of T-ALL, the underlying mechanisms that lead to the abnormal proliferation and enhanced survival of the leukemic cells remain largely unknown. Results: Array CGH analysis revealed an acquired homozygous microdeletion at chromosome 18p11 in 6 % of T-ALL cases. The deleted region was only 125 kb in size and restricted to the PTPN2 (protein tyrosine phosphatase, non-receptor type 2) locus. PTPN2 encodes an intracellular non-transmembrane tyrosine-specific phosphatase that functions as a negative regulator of a variety of signaling proteins including several members of the janus kinase (JAK) and of signal transducer and activator of transcription (STAT) families, growth factor receptors and SRC family kinases. Homozygous deletion of PTPN2 was specifically found in cases with aberrant expression of the TLX1 transcription factor, with two cases also harboring the NUP214-ABL1 fusion. Analysis of additional TLX1 positive cases by quantitative PCR identified loss of one copy of PTPN2 in 5 out of 20 cases. No mutations were detected in the coding region of PTPN2. To determine the effect of loss of PTPN2 in T-cells, we downregulated the expression of PTPN2 using RNAi technology. siRNA mediated knock-down of PTPN2 affected activation of JAK1 associated cytokine receptors implicated in T-cell development. Ligand stimulation of IL7 and interferon gamma receptor resulted in an augmented and prolonged phosphorylation of JAK1 as well as downstream targets STAT1 and STAT5 in T-ALL cell lines with knock-down of PTPN2. In addition, knock- down of Ptpn2 sensitized the pro B-cell line Ba/F3 to transformation by wild type JAK1 confirming a clear relationship between loss of PTPN2 and JAK1 activation. Knock-down of PTPN2 expression also provided a proliferative advantage and reduced sensitivity to kinase inhibitors in lymphoblastic leukemia cell lines HSB-2 and ALL-SIL. Conclusion: In conclusion, our data provide genetic and functional evidence for a tumor suppressor role of PTPN2 in T-ALL and warrant testing of JAK inhibitors for the treatment of this specific subset of T-ALLs as well as further analysis of a potential negative impact of loss of PTPN2 on responsiveness to anti-cancer treatments. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

scholarly journals T-Cell Protein Tyrosine Phosphatase (Tcptp) Is a Negative Regulator of Colony-Stimulating Factor 1 Signaling and Macrophage Differentiation

2006 ◽  
Vol 26 (11) ◽  
pp. 4149-4160 ◽  
Author(s):  
Paul D. Simoncic ◽  
Annie Bourdeau ◽  
Ailsa Lee-Loy ◽  
Larry R. Rohrschneider ◽  
Michel L. Tremblay ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Mononuclear Phagocyte ◽  
Negative Regulator ◽  
Cell Protein ◽  
Colony Stimulating Factor ◽  
Protein Tyrosine ◽  
Systemic Inflammatory Disease ◽  
Stimulating Factor

ABSTRACT Mice null for the T-cell protein tyrosine phosphatase (Tcptp−/−) die shortly after birth due to complications arising from the development of a systemic inflammatory disease. It was originally reported that Tcptp−/− mice have increased numbers of macrophages in the spleen; however, the mechanism underlying the aberrant growth and differentiation of macrophages in Tcptp−/− mice is not known. We have identified Tcptp as an important regulator of colony-stimulating factor 1 (CSF-1) signaling and mononuclear phagocyte development. The number of CSF-1-dependent CFU is increased in Tcptp−/− bone marrow. Tcptp−/− mice also have increased numbers of granulocyte-macrophage precursors (GMP), and these Tcptp−/− GMP yield more macrophage colonies in response to CSF-1 relative to wild-type cells. Furthermore, we have identified the CSF-1 receptor (CSF-1R) as a physiological target of Tcptp through substrate-trapping experiments and its hyperphosphorylation in Tcptp−/− macrophages. Tcptp−/− macrophages also have increased tyrosine phosphorylation and recruitment of a Grb2/Gab2/Shp2 complex to the CSF-1R and enhanced activation of Erk after CSF-1 stimulation, which are important molecular events in CSF-1-induced differentiation. These data implicate Tcptp as a critical regulator of CSF-1 signaling and mononuclear phagocyte development in hematopoiesis.

scholarly journals The T Cell Protein Tyrosine Phosphatase Is a Negative Regulator of Janus Family Kinases 1 and 3

2002 ◽  
Vol 12 (6) ◽  
pp. 446-453 ◽  
Author(s):  
Paul D. Simoncic ◽  
Ailsa Lee-Loy ◽  
Dwayne L. Barber ◽  
Michel L. Tremblay ◽  
C.Jane McGlade
Keyword(s):  
T Cell ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Cell Protein ◽  
Protein Tyrosine

scholarly journals Impaired GPVI Mediated Platelet Functional Responses and Signaling in CD45 Knockout Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1355-1355
Author(s):  
Vaishali Inamdar ◽  
John Kostyak ◽  
Rachit Badolia ◽  
Carol Dangelmaier ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Dense Granule ◽  
Receptor Protein ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Terminal Domain ◽  
Deficient Mice

Abstract Background: CD45 is a Receptor Protein Tyrosine Phosphatase C (PTPRC) and regulates Src Family Kinases activation in Lymphocytes. Although it is known to be absent from the platelet surface, proteomics studies prove that the CD45 c-terminal catalytic domain is present in platelets. Thus the aim of this study is to identify presence of CD45 c-terminal domain in platelets and characterize the functional implications of CD45 deficiency in platelets using a global CD45 knockout mouse. Results: Platelets from CD45-deficient mice displayed a selective impairment of aggregation and dense granule secretion mediated by the collagen receptor Glycoprotein VI. CD45 deficient mice show increased bleeding times, indicating an important role for CD45 in hemostasis. However, there was no difference observed in thrombus generation and thrombus stability using the ferric chloride-induced carotid artery injury model. Signaling downstream of the GPVI receptor, indicated by Src Family Kianse (SFK), Syk and Phospholipase C_2 (PLCg2) tyrosine phosphorylation, was also impaired. In order to establish the presence of CD45 in platelets we used an established primary antibody that recognizes the c-terminal domain of CD45. We observed that this antibody recognized a protein of approximately 65 kDa, which is the expected size of the c-terminal 1 and 2 domains of CD45, in wild type (WT) mice but not in knockout (KO)mice. Conclusion: Thus we conclude that CD45 is expressed in platelets as a truncated form, possibly generated by proteolytic cleavage, and regulates GPVI signaling, through regulation of Src Family Kinase activation. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Shp1 regulates T cell homeostasis by limiting IL-4 signals

2013 ◽  
Vol 210 (7) ◽  
pp. 1419-1431 ◽  
Author(s):  
Dylan J. Johnson ◽  
Lily I. Pao ◽  
Salim Dhanji ◽  
Kiichi Murakami ◽  
Pamela S. Ohashi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Biology ◽  
Tyrosine Phosphatase ◽  
Elevated Serum ◽  
Premature Death ◽  
Negative Regulator ◽  
T Cell Homeostasis ◽  
Tcr Signaling ◽  
Regulatory Molecule

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell–intrinsic role of Shp1, we characterized mice with a T cell–specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1fl/fl CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4+ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1-deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.

scholarly journals CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1818-1828 ◽  
Author(s):  
Cyndi Wong ◽  
Yong Liu ◽  
Jana Yip ◽  
Rochna Chand ◽  
Janet L. Wee ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Surface Glycoprotein ◽  
Type I ◽  
Wild Type ◽  
Glycoprotein Vi ◽  
Selective Ligand

Abstract Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)–γ-chain. ceacam1−/− platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1−/− mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1−/− mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1−/− mice showed a reversal in the more stable thrombus growth phenotype. ceacam1−/− mice were more susceptible to type I collagen–induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function

2021 ◽  
Author(s):  
Tony J. Zheng ◽  
Elizabeth R. Lofurno ◽  
Alexander R. Melrose ◽  
Hari Hara Sudhan Lakshmanan ◽  
Jiaqing Pang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Glycoprotein ◽  
Critical Systems ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Platelet Signaling ◽  
Granule Secretion ◽  
And Function ◽  
Btk Inhibitors

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

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