Regulation of FcεRI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2138-2144 ◽  
Author(s):  
Kiyonao Sada ◽  
S. M. Shahjahan Miah ◽  
Koichiro Maeno ◽  
Shinkou Kyo ◽  
Xiujuan Qu ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Calcium Influx ◽  
Adaptor Protein ◽  
Cellular Protein ◽  
Sh2 Domain ◽  
External Sources ◽  
High Affinity Ige Receptor ◽  
Basophilic Leukemia

Abstract Aggregation of high-affinity IgE receptor FcεRI induces sequential activation of nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, leading to degranulation in mast cells. A hematopoietic cell–specific adaptor protein, 3BP2, that was originally identified as an Abl SH3-binding protein was rapidly tyrosine phosphorylated by the aggregation of FcεRI on rat basophilic leukemia RBL-2H3 cells. Tyrosine phosphorylation of 3BP2 did not depend on calcium influx from external sources. To examine the role of 3BP2 in mast cells, we overexpressed the SH2 domain of 3BP2 in the RBL-2H3 cells. Overexpression of 3BP2-SH2 domain resulted in a suppression of antigen-induced degranulation as assessed by β-hexosaminidase release. Even though overall tyrosine phosphorylation of cellular protein was not altered, antigen-mediated tyrosine phosphorylation of phospholipase C-γ (PLC-γ) and calcium mobilization were significantly suppressed in the cells overexpressing the 3BP2-SH2 domain. Furthermore, antigen stimulation induced the association of 3BP2-SH2 domain with LAT and other signaling molecule complexes in the RBL-2H3 cells. FcεRI-mediated phosphorylation of JNK and ERK was not affected by the overexpression of 3BP2-SH2 domain. These data indicate that 3BP2 functions to positively regulate the FcεRI-mediated tyrosine phosphorylation of PLC-γ and thereby the signals leading to degranulation.

The Cytokine Signal Inhibitor Lnk Promotes αIIbβ3 Integrin Outside-In Signaling through β3 Tyrosine Phosphorylation in Platelets.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3651-3651
Author(s):  
Koji Eto ◽  
Hitoshi Takizawa ◽  
Satoshi Takaki ◽  
Hidekazu Nishikii ◽  
Atsushi Oda ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Cytokine Signaling ◽  
Adapter Protein ◽  
Clot Retraction ◽  
Hematopoietic Stem ◽  
Kinase Activation ◽  
C Terminus

Abstract Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk−/− mice exhibit a marked increase in numbers of hematopoietic stem cells, megakaryocytes and platelets, presumably due to the lack of negative regulation in thrombopoietin-mediated signals by Lnk. We previously reported that Lnk might play an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk−/− platelets exhibited defects in full spreading on fibrinogen, clot retraction and formation of thrombi on collagen under flow conditions while they showed normal inside-out signaling (Blood, 106 (11):115a, 2005). However the mechanism(s) in which Lnk participates in αIIbβ3 outside-in signaling had not been elucidated. Here we report that in normal platelets Lnk forms a complex with c-Src, Syk, Fyn and adhesion and degranulation promoting adaptor protein (ADAP) but not SLP-76 in a manner dependent on αIIbβ3 ligation and Src kinase activation. c-Src-, but not Syk-, mediated tyrosine phosphorylation of C-terminus in Lnk appeared to be indispensable for the complex formation and Lnk-mediated function. Furthermore we have shown that Lnk is required for the association of Fyn to αIIbβ3 and for β3 subunit tyrosine phosphorylation while activation of non-receptor tyrosine kinases (c-Src and Syk) in proximity to αIIbβ3 is independent of Lnk. Thus, these results provide new insights into Lnk function and the mechanism by which Lnk contributes to integrin signaling in the adhesion responses of platelets.

Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcepsilonRI-activated rat basophilic leukemia (RBL-2H3) cells

1997 ◽  
Vol 110 (18) ◽  
pp. 2215-2225 ◽  
Author(s):  
J.C. Guillemot ◽  
P. Montcourrier ◽  
E. Vivier ◽  
J. Davoust ◽  
P. Chavrier
Keyword(s):  
Mast Cells ◽  
Rho Gtpases ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Membrane Ruffling ◽  
Cytoskeletal Responses ◽  
High Affinity Ige Receptor ◽  
Mutant Forms ◽  
Basophilic Leukemia

Engagement of the high affinity IgE receptor (FcepsilonRI) in mast cells elicits a series of intracellular signalling events including cytoskeletal reorganization and granule exocytosis. To analyze the coupling of receptor activation to specific cytoskeletal responses, we expressed dominant negative mutant forms of the Rho GTPases CDC42 and Rac1 in rat RBL-2H3 tumor mast cells. We show here that dominant inhibition of CDC42 function decreases cell adhesion, interferes with Fc(epsilon)RI-induced actin plaque assembly and reduced the recruitment of vinculin at the cell-substratum interface, while the inhibitory Rac1 mutant abolishes Fc(epsilon)RI-mediated membrane ruffling. The expression of trans-dominant inhibitory forms of either CDC42 or Rac1 significantly inhibited antigen-induced degranulation. Altogether, our results demonstrate that CDC42 and Rac1 control distinct pathways downstream of FcepsilonRI engagement leading either to the induction of actin plaques, or to the production of membrane ruffles. These two pathways are critically involved during the degranulation response induced by Fc(epsilon)RI aggregation.

Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor

1987 ◽  
Vol 7 (2) ◽  
pp. 905-913
Author(s):  
W Yonemoto ◽  
A J Filson ◽  
A E Queral-Lustig ◽  
J Y Wang ◽  
J S Brugge
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tumor Antigen ◽  
Tyrosine Kinases ◽  
Cellular Transformation ◽  
Cellular Protein ◽  
Cellular Proteins ◽  
Transformed Cells ◽  
Major Protein ◽  
Rous Sarcoma ◽  
Phosphotyrosine Phosphatases

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.

Ca2+ channels mediate protein tyrosine kinase activation by endothelin-1

1996 ◽  
Vol 270 (5) ◽  
pp. F790-F797 ◽  
Author(s):  
M. S. Simonson ◽  
Y. Wang ◽  
W. H. Herman
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Mesangial Cells ◽  
Focal Adhesions ◽  
Cellular Protein ◽  
Early Gene ◽  
Glomerular Mesangial Cells ◽  
Protein Tyrosine ◽  
Endothelin 1 ◽  
Ca2 Channels

To investigate the novel interaction between endothelin-1 (ET-1) and cellular protein tyrosine kinases (PTK), we asked whether Ca2+ influx links ET-1 receptors to PTK activation. In glomerular mesangial cells, ET-1 stimulated a biphasic increase in PTK activity in anti-phosphotyrosine immunoprecipitates that temporally correlated with increased tyrosine phosphorylation of cellular proteins. ET-1 increased tyrosine phosphorylation of proteins in the cytosol and in a puncture distribution consistent with focal adhesions. Addition of ionomycin to increase Ca2+ influx stimulated PTK activity, and inhibition of extracellular Ca2+ influx blocked PTK activation by ET-1. ET-1 increased autophosphorylation of pp60c-src, which was mimicked by addition of ionomycin and inhibited by chelation of extracellular Ca2+. In addition, a selective PTK inhibitor blocked induction of c-fos mRNA by ionomycin, suggesting that Ca(2+)-stimulated PTKs contribute to a signaling pathway regulating immediate early gene expression. Taken together, these results demonstrate that ET-1 stimulates nonreceptor PTK activity, including pp60c-src, by activating Ca2+ channels and subsequent influx of extracellular Ca2+.

scholarly journals The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2

2020 ◽  
Vol 295 (12) ◽  
pp. 3932-3944 ◽  
Author(s):  
Melany J. Wagner ◽  
Marilyn S. Hsiung ◽  
Gerald D. Gish ◽  
Rick D. Bagshaw ◽  
Sasha A. Doodnauth ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Cell Movement ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Sh2 Domain ◽  
Tumor Formation ◽  
Eph Receptors ◽  
Eph Receptor ◽  
Varied Composition ◽  
Cell Segregation

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)–mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain–containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and β-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2–ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.

The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 132-138 ◽  
Author(s):  
Laura Velazquez ◽  
Gerald D. Gish ◽  
Peter van der Geer ◽  
Lorne Taylor ◽  
Johanna Shulman ◽  
...  
Keyword(s):  
Mast Cells ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Phospholipid Metabolism ◽  
Sh2 Domain ◽  
Interleukin 3 ◽  
Inositol Phosphatase ◽  
Ptb Domain

The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules—the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain—and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3–dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated β subunit of the IL-3 receptor and with the SH2-containing 5′ inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3–stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3′ kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3–stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.

scholarly journals Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

1994 ◽  
Vol 14 (2) ◽  
pp. 1308-1321 ◽  
Author(s):  
M Autero ◽  
J Saharinen ◽  
T Pessa-Morikawa ◽  
M Soula-Rothhut ◽  
C Oetken ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Lymphocyte Activation ◽  
Sh2 Domain ◽  
Protein Tyrosine Kinases ◽  
Phosphotyrosine Phosphatase ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Family Protein ◽  
Ptpase Activity

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

scholarly journals Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.

1994 ◽  
Vol 14 (8) ◽  
pp. 5108-5113 ◽  
Author(s):  
Y Kawakami ◽  
L Yao ◽  
T Miura ◽  
S Tsukada ◽  
O N Witte ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Immunoglobulin E ◽  
Cross Linking ◽  
Mouse Bone Marrow ◽  
Ionophore A23187 ◽  
Membrane Compartment ◽  
High Affinity Receptor ◽  
Protein Kinase C Activation

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.

scholarly journals Tyrosine phosphorylation of Grb2-associated proteins correlates with phospholipase C gamma 1 activation in T cells.

1996 ◽  
Vol 16 (6) ◽  
pp. 2823-2829 ◽  
Author(s):  
D G Motto ◽  
M A Musci ◽  
S E Ross ◽  
G A Koretzky
Keyword(s):  
T Cell ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Inositol Phosphate ◽  
Critical Role ◽  
Chimeric Protein ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase ◽  
Phospholipase C Gamma

Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.

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