SH2 Domain-containing Tyrosine Phosphatase Research Articles

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

C-X-C motif chemokine ligand 12 improves the developmental potential of bovine oocytes by activating SH2 domain-containing tyrosine phosphatase 2 during maturation†.

In vitro maturation of mammalian oocytes is an important means in assisted reproductive technology. Most bovine immature oocytes complete nuclear maturation, but less than half develop to the blastocyst stage after fertilization. Thus, inefficient in vitro production is mainly caused by a suboptimal in vitro culture process, in which oocyte quality appears to be the limiting factor. In our study, a potential maternal regulator, C-X-C motif chemokine ligand 12, was identified by analyzing transcriptome data. C-X-C motif chemokine ligand 12 supplementation promoted the developmental potential of oocytes by improving protein synthesis and reorganizing cortical granules and mitochondria during in vitro maturation, which eventually increased blastocyst formation efficiency and cell number after parthenogenesis, fertilization, and cloning. All these promoting effects by C-X-C motif chemokine ligand 12 were achieved by activating SH2 domain-containing tyrosine phosphatase 2, thereby promoting the mitogen-activated protein kinase signaling pathway. These findings provide an in vitro maturation system that closely resembles the maternal environment to provide high-quality oocytes for in vitro production.

LncRNA MEG8 sponging miR-181a-5p contributes to M1 macrophage polarization by regulating SHP2 expression in Henoch-Schonlein purpura rats

Background Long noncoding RNAs (LncRNAs) are regulatory molecules that play important roles in various biological and pathological processes. Herein, we aimed to explore whether maternally expressed gene 8 (MEG8) promotes M1 macrophage polarization among Henoch-Schonlein purpura (HSP) rats, and to investigate the underlying mechanism. Methods Relative mRNA expression of MEG8, miR-181a-5p and suppressor of SH2 domain-containing tyrosine phosphatase 2 (SHP2) were examined using quantitative reverse transcription polymerase chain reaction. Furthermore, expression of SHP2 and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway-related proteins was identified using western blot. Luciferase activity assay was conducted to evaluate whether miR-181a-5p could bind to MEG8 or SHP2. The macrophage phenotype was determined using flow cytometry and enzyme-linked immunosorbent assay. Results We observed macrophage polarization towards the M2 phenotype in the peripheral blood of HSP rats. Furthermore, MEG8 and SHP2 expression were down-regulated, while miR-181a-5p was up-regulated in monocyte-derived macrophages from the HSP rats compared to the control group. Furthermore, MEG8 functioned as a sponge for miR-181a-5p in order to facilitate SHP2 expression. Moreover, miR-181a-5p mimic and SHP2 knockdown significantly reversed the MEG8 overexpression-mediated suppression of JAK2/STAT3 signalling, and promotion of M1 polarization. Conclusions The lncRNA MEG8 sponged miR-181a-5p, which contributes to M1 macrophage polarization by regulating SHP2 expression in HSP rats. Key Messages LncRNA MEG8 downregulation and M2 polarization in Henoch Schonlein purpura rats. MEG8 upregulation enhances M1 polarization and suppresses JAK2/STAT3 pathway. MEG8 sponges miRNA-181a-5p to regulate SHP2 expression. MiRNA-181a-5p upregulation reverses lncRNA MEG8-mediated enhancement of M1 polarization and inhibition of JAK2/STAT3 pathway. SHP2 downregulation reverses lncRNA MEG8-mediated enhancement of M1 polarization and inhibition of JAK2/STAT3 pathway.

Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase.

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position −2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue −2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues −1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core β sheet and the loop that closes the pY binding pocket.

The Persistent Survival of MPN Cells to JAK2 Inhibition Is Dependent on SHP2 Activity, Which May Provide a Therapeutic Target to Enhance Current Anti-MPN Therapies

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis, are hematopoietic stem cell disorders that affect about 300,000 people in the U.S. PV and ET can progress to myelofibrosis where patients are susceptible to bone marrow failure, increased incidence of acute leukemia, and poor survival. MPNs are driven by aberrant JAK2 activity that is a consequence of mutations in JAK2, Mpl, or CalR. The JAK1/2 inhibitor ruxolitinib, which is FDA-approved for a subset of MPN patients, is effective in reducing patient symptomology but fails to induce remission, as it has little effect on the neoplastic cell burden. However, a modest increase in survival and decrease of allele burden has been suggested following long-term ruxolitinib treatment. The quality of life benefit afforded by ruxolitinib suggests it will likely remain a mainstay of future MPN therapies. Identifying the mechanism(s) by which cells persistently survive in the presence of ruxolitinib could prove pivotal to improving JAK2 inhibitor therapies in MPN patients.In order to identify mechanisms by which JAK2-driven MPN cells survive ruxolitinib treatment, we utilized global phospho-tyrosine proteomics to compare ruxolitinib-sensitive and persistent cells, the latter of which are drug persistent by nature due to their reacquisition of JAK2 inhibitor sensitivity upon removal of ruxolitinib. We utilized the JAK2-V617F-driven MPN model cell lines UKE1 and SET2 for this analysis. Pathway analysis from the datasets obtained indicated changes in phosphorylation of proteins involved in growth factor and cytokine signaling. Notably, we identified changes in phosphorylation of several tyrosine phosphatases, including SHP1 and SHP2. The SH2 domain-containing tyrosine phosphatase SHP2 (encoded by PTPN11) plays important roles in relaying signals from cell surface receptors, notably mediating activation of ERKs, and was the first phosphatase identified as leukemogenic. SHP2 regulates JAK2 signaling and is found mutated in ~8% of post-MPN leukemia, suggesting it may play a role in the biology of MPNs. In addition, SHP2 has been identified as a potential mediator of targeted therapy resistance in cancer. Therefore, we hypothesized that SHP2 activity may contribute to the persistent survival of MPN cells in ruxolitinib and provide a therapeutic target in MPNs. To test our hypothesis, we treated ruxolitinib-sensitive (drug naïve) and persistent cells with a specific allosteric inhibitor of SHP2, SHP099. Ruxolitinib-persistent cell growth was more sensitive to SHP2 inhibition, and SHP099 enhanced the growth inhibitory effects of ruxolitinib on ruxolitinib-sensitive cells. Treatment of UKE1 cells with a high concentration (~10X GI50) of ruxolitinib leads to the persistent survival of a small population of cells and this was prevented by SHP099. SHP2 inhibition was also deleterious to the maintenance of an existing drug persistent state in a high concentration of ruxolitinib. Interestingly, signaling pathways in cells persistently growing in ruxolitinib differentially respond to SHP2 inhibition. In both ruxolitinib-sensitive and persistent cells activation of ERK was inhibited by SHP099. However, in ruxolitinib-persistent UKE1 cells, this inhibition was more rapid, suggesting the kinetic regulation of ERK activation by SHP2 is altered in these cells. Also, SHP099 led to an increase in pSTAT3 in both ruxolitinib-sensitive and persistent cells. However, SHP099 led to an increase in pSTAT5 in ruxolitinib sensitive UKE1 cells, but not in cells persistently growing in ruxolitinib. Re-activation of signaling pathways following ruxolitinib treatment likely contributes to cell survival. We observed that ERK becomes re-activated shortly after ruxolitinib treatment, and this re-activation depends on SHP2 activity. Finally, we observed dose-dependent inhibition of the neoplastic growth of primary MPN patient cells by SHP099 and synergistic inhibition with ruxolitinib, suggesting co-targeting SHP2 may improve JAK2-targeted MPN therapies.In conclusion, our data suggest that re-wiring of signaling in cells challenged with ruxolitinib may create greater dependence on SHP2 activity that contributes to cell survival to JAK2 inhibition. Thus, targeting SHP2 may improve the efficacy of ruxolitinib as an anti-MPN therapy. DisclosuresHaura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding.

Abstract 1633: Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas

Abstract The discovery of mutations in the ALK oncogene as the genetic etiology of familial neuroblastoma (NB) and as somatically acquired in 14% of patients with the most aggressive form of the disease has positioned ALK as the major tractable oncogene product for targeted therapy in NB. ALK tyrosine kinase domain mutations are found mostly at three hotspots (F1174, F1245 and R1275), with the F1174* and F1245* mutations conferring broad resistance to crizotinib. We have recently demonstrated that lorlatinib, a novel ATP-competitive macrocyclic ALK inhibitor, overcomes de novo resistance and exerts unprecedented activity as a single agent in patient-derived xenografts (PDXs) harboring F1174L or F1245C mutations, while also inducing durable responses in R1275Q xenografts. The objective of our work was to identify differences in reprogramming of the kinome upon ALK inhibition with crizotinib and lorlatinib. We hypothesize that identification of the targets through which lorlatinib mediates its superior anti-tumor effects will provide insights into the mechanisms by which full-length ALK inhibition abrogates tumorigenesis, activates compensatory pathways, and uncover novel targets to overcome resistance to ALK inhibition. We used Multiplexed Inhibitor Beads coupled with Mass Spectrometry (MIB/MS) to quantitatively measure kinase activity dynamics on a proteomic scale. Three PDX models harboring F1174L (COG-N-453x) or the other two most-common mutations, R1275Q (NB1643) and R1245C (Felix) were treated with either crizotinib (100 mg/kg/day) or lorlatinib (10 mg/kg/day) for 2.5 days or 6.5 days prior to MIB/MS analysis. Lorlatinib more potently inhibited ALK and preferentially inhibited a series of other tyrosine kinases, correlating with the superior efficacy of lorlatinib in pre-clinical studies. Moreover, a number of kinases involved in G2/M cell cycle transition including PLK1, CHEK1 and aurora kinases were significantly inhibited by lorlatinib, proposing that this multi-kinase inhibition is responsible for the robust anti-tumor activity observed in vivo with lorlatinib. We interrogated the role of SHP2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase recently implicated in the proliferation of receptor-tyrosine kinase driven cancers and shown to have a direct role in the G2/M checkpoint. Inhibition of ALK by treatment with lorlatinib as well as shRNA knockdown of ALK in SY5Y (ALK F1174L, PTPN11 T507K) and NB1643 NB cell lines showed decreased levels of pSHP2. Depletion of SHP2 using CRISPR constructs significantly inhibited cell proliferation in SY5Y, suggesting that SHP2 plays a crucial role in ALK driven NB. Additionally, knockdown of ALK in SY5Y downregulated PLK1 expression and arrested cells in G2/M phase. Whether SHP2 is regulated primarily downstream of ALK or jointly through alternative or compensatory signaling networks warrants further investigation. Citation Format: Smita Matkar, Renata Sano, Colleen Larmour, Kateryna Krytska, Gabriela M. Witek, Mark Gerelus, Tim J. Stuhlmiller, Gary L. Johnson, Yael P. Mosse. Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1633.

Regulation of Brain-Derived Neurotrophic Factor and Growth Factor Signaling Pathways by Tyrosine Phosphatase Shp2 in the Retina: A Brief Review.

SH2 domain-containing tyrosine phosphatase-2 (PTPN11 or Shp2) is a ubiquitously expressed protein that plays a key regulatory role in cell proliferation, differentiation and growth factor (GF) signaling. This enzyme is well expressed in various retinal neurons and has emerged as an important player in regulating survival signaling networks in the neuronal tissues. The non-receptor phosphatase can translocate to lipid rafts in the membrane and has been implicated to regulate several signaling modules including PI3K/Akt, JAK-STAT and Mitogen Activated Protein Kinase (MAPK) pathways in a wide range of biochemical processes in healthy and diseased states. This review focuses on the roles of Shp2 phosphatase in regulating brain-derived neurotrophic factor (BDNF) neurotrophin signaling pathways and discusses its cross-talk with various GF and downstream signaling pathways in the retina.

Protein Tyrosine Phosphatase SHP-2 Is Involved in the Interleukin-21-Induced Activation of Extracellular Signal-Regulated Kinase 1/2

The cytokine interleukin-21 (IL-21) is mainly produced from activated CD4+ T cells and natural killer T (NKT) cells. IL-21 enhances the proliferation and differentiation of T cells and B cells and also increases cytotoxicity of CD8+ T cells and NK cells through the IL-21 receptor and its downstream signaling molecules such as signal transducers and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2). SH2 domain-containing tyrosine phosphatase (SHP-2) is ubiquitously expressed, including hematopoietic cells. SHP-2 has been implicated in the regulation of IL-6 and IL-3 signaling, but its function in IL-21 signaling has not been investigated. Therefore, we studied the role of SHP-2 in IL-21 signaling by SHP-2 overexpression and knockdown experiments. For the SHP-2 overexpression, we used 293T human embryonic kidney cells, in which the IL-21 receptor system were easily reconstituted and high amounts of exogenous SHP-2 were expressed by vector transfection. In 293T cells, overexpressed SHP-2 caused the increase in the degree of the IL-21-induced ERK1/2 activation. Subsequently, SHP-2 knockdown experiments were performed in the mouse pro-B cell line, BAF21RWT-1, which constitutively expresses human IL-21 receptor and proliferates in an IL-21-dependent manner. SHP-2 knockdown reduced the degree of the IL-21-induced ERK1/2 activation and suppressed cell proliferation. These results suggest that SHP-2 may augment the ERK1/2 activity and cell proliferation activity in IL-21 signaling. We propose that SHP-2 is involved in the IL-21-mediated ERK1/2 activation and cell proliferation.

Role of SHP2 in hematopoiesis and leukemogenesis.

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11 plays an important role in regulating signaling from cell surface receptor tyrosine kinases during normal development as well as oncogenesis. Herein we review recently discovered roles of SHP2 in normal and aberrant hematopoiesis along with novel strategies to target it. Cell autonomous role of SHP2 in normal hematopoiesis and leukemogenesis has long been recognized. The review will discuss the newly discovered role of SHP2 in lineage specific differentiation. Recently, a noncell autonomous role of oncogenic SHP2 has been reported in which activated SHP2 was shown to alter the bone marrow microenvironment resulting in transformation of donor derived normal hematopoietic cells and development of myeloid malignancy. From being considered as an 'undruggable' target, recent development of allosteric inhibitor has made it possible to specifically target SHP2 in receptor tyrosine kinase driven malignancies. SHP2 has emerged as an attractive target for therapeutic targeting in hematological malignancies for its cell autonomous and microenvironmental effects. However a better understanding of the role of SHP2 in different hematopoietic lineages and its crosstalk with signaling pathways activated by other genetic lesions is required before the promise is realized in the clinic.

The role of SHP-2 on NK cell development and functions

Abstract The SH2 domain-containing tyrosine phosphatase SHP-2 (PTPN11) has been shown to play essential roles in immune cell signaling. Several inhibitory receptors expressed on natural killer (NK) cells associate with SHP-2 to exert downstream functions. Here we examined the influence of SHP-2 on NK cell development and effector activity. Because SHP-2 is required for embryonic development in mice, we generated mice conditionally deficient for SHP-2 in NK cells. SHP-2fl/fl mice were crossed to NKp46-Cre mice in order to inactivate Ptpn11 in NKp46+ cells. These mice were also crossed to R26R-EYFP reporter mice to track cells with active Cre recombinase. We found an increase in NK cell frequency and number in these mice compared to littermate controls. The frequency of mature NK cells (CD11b+CD27−) was also increased in the periphery while decreased in the bone marrow. In addition, the homeostatic proliferation of SHP-2-deficient NK cells was impaired. However, effector functions were not affected significantly. These included IFN-γ production at the peak of the NK cell response during MCMV infection, rejection of β2-microglobulin deficient splenocytes, and degranulation via Ly49H using M157-transfected cells as targets. Taken together, our data suggest that SHP-2 is dispensable for NK cell effector functions and education, but is important for NK cell maturation.

SH2 Domain-Containing Phosphatase-2 Is a Novel Antifibrotic Regulator in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease with dismal prognosis and no cure. The potential role of the ubiquitously expressed SH2 domain-containing tyrosine phosphatase-2 (SHP2) as a therapeutic target has not been studied in IPF. To determine the expression, mechanistic role, and potential therapeutic usefulness of SHP2 in pulmonary fibrosis. The effects of SHP2 overexpression and inhibition on fibroblast response to profibrotic stimuli were analyzed in vitro in primary human and mouse lung fibroblasts. In vivo therapeutic effects were assessed in the bleomycin model of lung fibrosis by SHP2-lentiviral administration and transgenic mice carrying a constitutively active SHP2 mutation. SHP2 was down-regulated in lungs and lung fibroblasts obtained from patients with IPF. Immunolocalization studies revealed that SHP2 was absent within fibroblastic foci. Loss of SHP2 expression or activity was sufficient to induce fibroblast-to-myofibroblast differentiation in primary human lung fibroblasts. Overexpression of constitutively active SHP2 reduced the responsiveness of fibroblasts to profibrotic stimuli, including significant reductions in cell survival and myofibroblast differentiation. SHP2 effects were mediated through deactivation of fibrosis-relevant tyrosine kinase and serine/threonine kinase signaling pathways. Mice carrying the Noonan syndrome-associated gain-of-function SHP2 mutation (SHP2D61G/+) were resistant to bleomycin-induced pulmonary fibrosis. Restoration of SHP2 levels in vivo through lentiviral delivery blunted bleomycin-induced pulmonary fibrosis. Our data suggest that SHP2 is an important regulator of fibroblast differentiation, and its loss as observed in IPF facilitates profibrotic phenotypic changes. Augmentation of SHP2 activity or expression should be investigated as a novel therapeutic strategy for IPF.

Hippocalcin Promotes Neuronal Differentiation and Inhibits Astrocytic Differentiation in Neural Stem Cells.

SummaryHippocalcin (HPCA) is a calcium-binding protein that is restricted to nervous tissue and contributes to neuronal activity. Here we report that, in addition to inducing neurogenesis, HPCA inhibits astrocytic differentiation of neural stem cells. It promotes neurogenesis by regulating protein kinase Cα (PKCα) activation by translocating to the membrane and binding to phosphoinositide-dependent protein kinase 1 (PDK1), which induces PKCα phosphorylation. We also found that phospholipase D1 (PLD1) is implicated in the HPCA-mediated neurogenesis pathway; this enzyme promotes dephosphorylation of signal transducer and activator of transcription 3 (STAT3[Y705]), which is necessary for astrocytic differentiation. Moreover, we found that the SH2-domain-containing tyrosine phosphatase 1 (SHP-1) acts upstream of STAT3. Importantly, this SHP-1-dependent STAT3-inhibitory mechanism is closely involved in neurogenesis and suppression of gliogenesis by HPCA. Taken together, these observations suggest that HPCA promotes neuronal differentiation through activation of the PKCα/PLD1 cascade followed by activation of SHP-1, which dephosphorylates STAT3(Y705), leading to inhibition of astrocytic differentiation.

New and Unexpected Biological Functions for the Src-Homology 2 Domain-Containing Phosphatase SHP-2 in the Gastrointestinal Tract.

SHP-2 is a tyrosine phosphatase expressed in most embryonic and adult tissues. SHP-2 regulates many cellular functions including growth, differentiation, migration, and survival. Genetic and biochemical evidence show that SHP-2 is required for rat sarcoma viral oncogene/extracellular signal-regulated kinases mitogen-activated protein kinase pathway activation by most tyrosine kinase receptors, as well as by G-protein–coupled and cytokine receptors. In addition, SHP-2 can regulate the Janus kinase/signal transducers and activators of transcription, nuclear factor-κB, phosphatidyl-inositol 3-kinase/Akt, RhoA, Hippo, and Wnt/β-catenin signaling pathways. Emerging evidence has shown that SHP-2 dysfunction represents a key factor in the pathogenesis of gastrointestinal diseases, in particular in chronic inflammation and cancer. Variations within the gene locus encoding SHP-2 have been associated with increased susceptibility to develop ulcerative colitis and gastric atrophy. Furthermore, mice with conditional deletion of SHP-2 in intestinal epithelial cells rapidly develop severe colitis. Similarly, hepatocyte-specific deletion of SHP-2 induces hepatic inflammation, resulting in regenerative hyperplasia and development of tumors in aged mice. However, the SHP-2 gene initially was suggested to be a proto-oncogene because activating mutations of this gene were found in pediatric leukemias and certain forms of liver and colon cancers. Moreover, SHP-2 expression is up-regulated in gastric and hepatocellular cancers. Notably, SHP-2 functions downstream of cytotoxin-associated antigen A (CagA), the major virulence factor of Helicobacter pylori, and is associated with increased risks of gastric cancer. Further compounding this complexity, most recent findings suggest that SHP-2 also coordinates carbohydrate, lipid, and bile acid synthesis in the liver and pancreas. This review aims to summarize current knowledge and recent data regarding the biological functions of SHP-2 in the gastrointestinal tract.

Peroxiredoxin II Is an Antioxidant Enzyme That Negatively Regulates Collagen-stimulated Platelet Function

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis.

Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM)-Mediated Inhibitory Signaling Is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing nonreceptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton's tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals.

Advanced glycation end product Nε‐carboxymethyllysine induces endothelial cell injury: the involvement of SHP‐1‐regulated VEGFR‐2 dephosphorylation

N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications.

Distinct Roles for Neutrophils and Dendritic Cells in Inflammation and Autoimmunity in motheaten Mice

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

An investigation of hierachical protein recruitment to the inhibitory platelet receptor, G6B-b.

Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen.

The Helicobacter pylori Virulence Effector CagA Abrogates Human β-Defensin 3 Expression via Inactivation of EGFR Signaling

Antimicrobial peptides are constituents of the first-line innate mucosal defense system that acts as a barrier to establishment of infection. The highly successful human gastric pathogen, Helicobacter pylori, is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including human β-defensin 3 (hBD3). We find that hBD3 is highly active against H.pylori invitro and is rapidly induced during early infection via EGFR-dependent activation of MAP kinase and JAK/STAT signaling. However, during prolonged infection, hBD3 was subsequently downregulated by the H.pylori virulence determinant CagA. Upon translocation into host cells, CagA activated the cellular tyrosine phosphatase, SHP-2, terminating EGFR activation and downstream signaling and increasing bacterial viability. Chemical inhibition and knockdown of SHP-2 expression rescued hBD3 synthesis and bactericidal activity. Thus, we reveal how cagPAI-positive H.pylori strains use CagA to evade a key innate mucosal defense pathway to support the establishment of persistent infection.

Evolution of JAK-STAT pathway components: mechanisms and role in immune system development.

BackgroundLying downstream of a myriad of cytokine receptors, the Janus kinase (JAK) – Signal transducer and activator of transcription (STAT) pathway is pivotal for the development and function of the immune system, with additional important roles in other biological systems. To gain further insight into immune system evolution, we have performed a comprehensive bioinformatic analysis of the JAK-STAT pathway components, including the key negative regulators of this pathway, the SH2-domain containing tyrosine phosphatase (SHP), Protein inhibitors against Stats (PIAS), and Suppressor of cytokine signaling (SOCS) proteins across a diverse range of organisms.ResultsOur analysis has demonstrated significant expansion of JAK-STAT pathway components co-incident with the emergence of adaptive immunity, with whole genome duplication being the principal mechanism for generating this additional diversity. In contrast, expansion of upstream cytokine receptors appears to be a pivotal driver for the differential diversification of specific pathway components.ConclusionDiversification of JAK-STAT pathway components during early vertebrate development occurred concurrently with a major expansion of upstream cytokine receptors and two rounds of whole genome duplications. This produced an intricate cell-cell communication system that has made a significant contribution to the evolution of the immune system, particularly the emergence of adaptive immunity.