scholarly journals Shp-2 Tyrosine Phosphatase Functions as a Negative Regulator of the Interferon-Stimulated Jak/STAT Pathway

1999 ◽  
Vol 19 (3) ◽  
pp. 2416-2424 ◽  
Author(s):  
Min You ◽  
De-Hua Yu ◽  
Gen-Sheng Feng
Keyword(s):  
Cell Growth ◽  
Cytotoxic Effect ◽  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Wild Type ◽  
Mobility Shift ◽  
Ifn Γ ◽  
Mutant Cells ◽  
Stat Pathway

ABSTRACT Shp-2 is an SH2 domain-containing protein tyrosine phosphatase. Although the mechanism remains to be defined, substantial experimental data suggest that Shp-2 is primarily a positive regulator in cell growth and development. We present evidence here that Shp-2, while acting to promote mitogenic signals, also functions as a negative effector in interferon (IFN)-induced growth-inhibitory and apoptotic pathways. Treatment of mouse fibroblast cells lacking a functional Shp-2 with IFN-α or IFN-γ resulted in an augmented suppression of cell viability compared to that of wild-type cells. To dissect the molecular mechanism, we examined IFN-induced activation of signal transducers and activators of transcription (STATs) by electrophoretic mobility shift assay, using a specific DNA probe (hSIE). The amounts of STAT proteins bound to hSIE upon IFN-α or IFN-γ stimulation were significantly increased in Shp-2−/− cells. Consistently, tyrosine phosphorylation levels of Stat1 upon IFN-γ treatment and, to a lesser extent, upon IFN-α stimulation were markedly elevated in mutant cells. Furthermore, IFN-γ induced a higher level of caspase 1 expression in Shp-2−/− cells than in wild-type cells. Reintroduction of wild-type Shp-2 protein reversed the hypersensitivity of Shp-2−/− fibroblasts to the cytotoxic effect of IFN-α and IFN-γ. Excessive activation of STATs by IFNs was also diminished in mutant cells in which Shp-2 had been reintroduced. Together, these results establish that Shp-2 functions as a negative regulator of the Jak/STAT pathway. We propose that Shp-2 acts to promote cell growth and survival through two mechanisms, i.e., the stimulation of growth factor-initiated mitogenic pathways and the suppression of cytotoxic effect elicited by cytokines, such as IFNs.

scholarly journals Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 345 ◽  
Author(s):  
Klebanovych ◽  
Sládková ◽  
Sulimenko ◽  
Vosecká ◽  
Čapek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Mast Cell Activation ◽  
Mouse Bone Marrow ◽  
Microtubule Nucleation ◽  
Protein Tyrosine

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

scholarly journals Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

2007 ◽  
Vol 6 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Dana Schaefer ◽  
Pierre Côte ◽  
Malcolm Whiteway ◽  
Richard J. Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Cell Growth ◽  
Cell Fusion ◽  
Aspartyl Protease ◽  
Wild Type ◽  
Pheromone Activity ◽  
Important Regulator ◽  
Mutant Cells ◽  
Type Strains

ABSTRACT Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Δbar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in α cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Δbar1 a and α cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.

Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2213-2213
Author(s):  
Saskia Gueller ◽  
Sigal Gery ◽  
H. Phillip Koeffler
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
Nih3t3 Cells ◽  
Sh2 Domains ◽  
Juxtamembrane Region

Abstract PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including: a mutation in the SH2 domain (R392E); deletion of the SH2 domain; deletion of the PH and SH2 domains and a construct only containing the DD domain. 293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.

scholarly journals Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3360-3364 ◽  
Author(s):  
Sigal Gery ◽  
Saskia Gueller ◽  
Katya Chumakova ◽  
Norihiko Kawamata ◽  
Liqin Liu ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Adaptor Protein ◽  
Myeloproliferative Disorders ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Downstream Signaling ◽  
Cellular Pathways ◽  
Insight Into

Abstract Recently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhances proliferation. Lnk blocks the activation of Jak2, Stat3, Erk, and Akt in these cells. Furthermore, MPLW515L-expressing cells are more susceptible to Lnk inhibitory functions than their MPL wild-type (MPLWT)–expressing counterparts. Lnk associates with activated MPLWT and MPLW515L and colocalizes with the receptors at the plasma membrane. The SH2 domain of Lnk is essential for its binding and for its down-regulation of MPLWT and MPLW515L. Lnk itself is tyrosine-phosphorylated following thrombopoietin stimulation. Further elucidating the cellular pathways that attenuate MPLW515L will provide insight into the pathogenesis of MPD and could help develop specific therapeutic approaches.

scholarly journals Regulation of Exocyst Function in Pollen Tube Growth by Phosphorylation of Exocyst Subunit EXO70C2

2021 ◽  
Vol 11 ◽  
Author(s):  
Antonietta Saccomanno ◽  
Martin Potocký ◽  
Přemysl Pejchar ◽  
Michal Hála ◽  
Hiromasa Shikata ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Negative Regulator ◽  
Tube Growth ◽  
Wild Type ◽  
Functional Studies ◽  
Tobacco Pollen ◽  
Pollen Tube Growth Rate ◽  
Mutant Cells ◽  
Dose Dependent

Exocyst is a heterooctameric protein complex crucial for the tethering of secretory vesicles to the plasma membrane during exocytosis. Compared to other eukaryotes, exocyst subunit EXO70 is represented by many isoforms in land plants whose cell biological and biological roles, as well as modes of regulation remain largely unknown. Here, we present data on the phospho-regulation of exocyst isoform EXO70C2, which we previously identified as a putative negative regulator of exocyst function in pollen tube growth. A comprehensive phosphoproteomic analysis revealed phosphorylation of EXO70C2 at multiple sites. We have now performed localization and functional studies of phospho-dead and phospho-mimetic variants of Arabidopsis EXO70C2 in transiently transformed tobacco pollen tubes and stably transformed Arabidopsis wild type and exo70C2 mutant plants. Our data reveal a dose-dependent effect of AtEXO70C2 overexpression on pollen tube growth rate and cellular architecture. We show that changes of the AtEXO70C2 phosphorylation status lead to distinct outcomes in wild type and exo70c2 mutant cells, suggesting a complex regulatory pattern. On the other side, phosphorylation does not affect the cytoplasmic localization of AtEXO70C2 or its interaction with putative secretion inhibitor ROH1 in the yeast two-hybrid system.

scholarly journals p45NFE2 Is a Negative Regulator of Erythroid Proliferation Which Contributes to the Progression of Friend Virus-Induced Erythroleukemias

2001 ◽  
Vol 21 (1) ◽  
pp. 73-80 ◽  
Author(s):  
You-Jun Li ◽  
Rachel R. Higgins ◽  
Brian J. Pak ◽  
Ramesh A. Shivdasani ◽  
Paul A. Ney ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Globin Gene ◽  
Negative Regulator ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Globin Genes ◽  
Friend Virus ◽  
Wild Type

ABSTRACT In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific genep45 NFE2 . Frequent disruption ofp45 NFE2 due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV inp45 NFE2 mutant mice. Sincep45 NFE2 homozygous mice mostly die at birth, erythroleukemia was induced in +/− and +/+ mice. We demonstrate that +/− mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/− mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/− and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/− mice. Establishment in culture was associated with the loss of the remaining wild-typep45 NFE2 allele in 9 of 10 of these cell lines. The loss of a functional p45NFE2 in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-typep45 NFE2 in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression ofp45 NFE2 in these cells also slows tumor growth in vivo. These results indicate thatp45 NFE2 functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

scholarly journals Sorafenib Inhibits Non–Small Cell Lung Cancer Cell Growth by Targeting B-RAF in KRAS Wild-Type Cells and C-RAF in KRAS Mutant Cells

2009 ◽  
Vol 69 (16) ◽  
pp. 6515-6521 ◽  
Author(s):  
Ken Takezawa ◽  
Isamu Okamoto ◽  
Kimio Yonesaka ◽  
Erina Hatashita ◽  
Yuki Yamada ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Growth ◽  
Small Cell Lung Cancer ◽  
Cancer Cell ◽  
Small Cell ◽  
Wild Type ◽  
Lung Cancer Cell ◽  
Small Cell Lung ◽  
Cancer Cell Growth ◽  
Mutant Cells

scholarly journals The Drosophila Ortholog of MLL3 and MLL4 , trithorax related , Functions as a Negative Regulator of Tissue Growth

2013 ◽  
Vol 33 (9) ◽  
pp. 1702-1710 ◽  
Author(s):  
Hiroshi Kanda ◽  
Alexander Nguyen ◽  
Leslie Chen ◽  
Hideyuki Okano ◽  
Iswar K. Hariharan
Keyword(s):  
Histone H3 ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Cyclin B ◽  
Wild Type ◽  
H3k4 Methylation ◽  
Content Type ◽  
Promote Cell Survival ◽  
Low Levels ◽  
Mutant Cells

The human MLL genes ( MLL1 to MLL4 ) and their Drosophila orthologs, trithorax ( trx ) and trithorax related ( trr ), encode proteins capable of methylating histone H3 on lysine 4. MLL1 and MLL2 are most similar to trx , while MLL3 and MLL4 are more closely related to trr . Several MLL genes are mutated in human cancers, but how these proteins regulate cell proliferation is not known. Here we show that trr mutant cells have a growth advantage over their wild-type neighbors and display changes in the levels of multiple proteins that regulate growth and cell division, including Notch, Capicua, and cyclin B. trr mutant clones display markedly reduced levels of H3K4 monomethylation without obvious changes in the levels of H3K4 di- and trimethylation. The trr mutant phenotype resembles that of Utx , which encodes a H3K27 demethylase, consistent with the observation that Trr and Utx are found in the same protein complex. In contrast to the overgrowth displayed by trr mutant tissue, trx clones are underrepresented, express low levels of the antiapoptotic protein Diap1, and exhibit only modest changes in global levels of H3K4 methylation. Thus, in Drosophila eye imaginal discs, Trr, likely functioning together with Utx, restricts tissue growth. In contrast, Trx appears to promote cell survival.

scholarly journals Two Dictyostelium tyrosine kinase–like kinases function in parallel, stress-induced STAT activation pathways

2014 ◽  
Vol 25 (20) ◽  
pp. 3222-3233 ◽  
Author(s):  
Tsuyoshi Araki ◽  
Linh Hai Vu ◽  
Norimitsu Sasaki ◽  
Takefumi Kawata ◽  
Ludwig Eichinger ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Serine Phosphorylation ◽  
Negative Regulator ◽  
Structural Basis ◽  
Phosphorylation Event ◽  
Activation Pathways

When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.

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.

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