scholarly journals Role of the protein tyrosine phosphatase SHP-1 (Src homology phosphatase-1) in the regulation of interleukin-3-induced survival, proliferation and signalling

2002 ◽  
Vol 368 (3) ◽  
pp. 885-894 ◽  
Author(s):  
Nicholas R.D. PALING ◽  
Melanie J. WELHAM
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Interleukin 3 ◽  
Extracellular Signal ◽  
Dependent Cell ◽  
Src Homology ◽  
Β Chain

The tyrosine phosphatase SHP-1 (Src homology phosphatase-1) has been widely implicated as a negative regulator of signalling in immune cells. We have investigated in detail the role of SHP-1 in interleukin-3 (IL-3) signal transduction by inducibly expressing wild-type (WT), C453S (substrate-trapping) and R459M (catalytically inactive) forms of SHP-1 in the IL-3-dependent cell line BaF/3. Expression of WT SHP-1 had little impact on IL-3-induced proliferation, but enhanced apoptosis following IL-3 withdrawal. Expression of R459M SHP-1 increased the proliferative response of BaF/3 cells to IL-3 and increased cell survival at low doses of IL-3 and following IL-3 withdrawal. Investigation into the biochemical consequences resulting from expression of these SHP-1 variants demonstrated that the β chain of the IL-3 receptor (Aic2A) was hypo-phosphorylated in cells expressing WT SHP-1 and hyper-phosphorylated in those expressing R459M SHP-1. Further, ectopic expression of the trapping mutant, C453S SHP-1, protected Aic2A from dephosphorylation, suggesting that Aic2A is a SHP-1 substrate in BaF/3 cells. Examination of overall levels of tyrosine phosphorylation demonstrated that they were not perturbed in these transfectants. Activation-specific phosphorylation of STAT (signal transducer and activator of transcription) 5a/b, protein kinase B and ERK (extracellular-signal-regulated kinase)-1 and −2 was also unaffected by expression of WT or R459M SHP-1. However, overall levels of IL-3-induced tyrosine phosphorylation of STAT5 were reduced upon expression of WT SHP-1 and increased when R459M SHP-1 was expressed, consistent with STAT5 being a potential SHP-1 substrate. These results demonstrate that SHP-1 acts to negatively regulate IL-3-driven survival and proliferation, potentially via regulation of tyrosine phosphorylation of Aic2A and STAT5.

scholarly journals Erythropoietin and Interleukin-3 Activate Tyrosine Phosphorylation of CBL and Association With CRK Adaptor Proteins

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3166-3174 ◽  
Author(s):  
Dwayne L. Barber ◽  
Jacqueline M. Mason ◽  
Toru Fukazawa ◽  
Kris A. Reedquist ◽  
Brian J. Druker ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Hematopoietic Cells ◽  
Adaptor Protein ◽  
Normal Growth ◽  
Adaptor Proteins ◽  
Erythroid Cell ◽  
Interleukin 3 ◽  
Dependent Cell ◽  
Src Homology

Abstract Transformation of hematopoietic cells by the Bcr-abl oncoprotein leads to constitutive tyrosine phosphorylation of a number of cellular polypeptides that function in normal growth factor-dependent cell proliferation. Recent studies have shown that the CrkL adaptor protein and the Cbl protooncoprotein are constitutively tyrosine phosphorylated and form a preformed complex in cells expressing Bcr-abl. In the current study, we have examined cytokine-dependent tyrosine phosphorylation of Cbl and its association with Crk proteins. Erythropoietin (EPO) and interleukin-3 induced a dose and time-dependent tyrosine phosphorylation of Cbl in both EPO-dependent Ba/F3 and DA-3 transfectants, and the erythroid cell line HCD-57. Furthermore, once phosphorylated, Cbl associated with Crk adaptor proteins. Of the three Crk isoforms expressed in hematopoietic cells (CrkL, CrkII, and CrkI), tyrosine phosphorylated Cbl binds preferentially to CrkL and CrkII. The amount of Cbl associated with CrkL and CrkII exceeded the fraction of Cbl associated with Grb2 indicating that unlike other receptor systems, the Cbl-Crk association represents the dominant complex of Cbl in growth factor-stimulated hematopoietic cells. In factor-dependent hematopoietic cell lines, CrkL constitutively associated with the guanine nucleotide release factor, C3G, which is known to interact via Crk src-homology 3 (SH3) domains. Our data suggest that the inducible Cbl-Crk association is a proximal component of a signaling pathway downstream of multiple cytokine receptors.

scholarly journals Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin

2001 ◽  
Vol 360 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Michael D. SCHALLER ◽  
Erik M. SCHAEFER
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Phosphorylation ◽  
Binding Sites ◽  
Signalling Molecules ◽  
Src Homology 2 ◽  
Dependent Cell ◽  
Src Homology ◽  
Tyrosine Phosphorylated Protein ◽  
Src Homology 2 Domain

Paxillin is a focal-adhesion-associated, tyrosine-phosphorylated protein. In cells transformed by the src, crk or BCR-Abl oncogenes, the phosphotyrosine content of paxillin is elevated. In normal cells paxillin functions in signalling following integrin-dependent cell adhesion or exposure to a number of stimuli, including growth factors and neuropeptides. These stimuli induce tyrosine phosphorylation of paxillin, regulating the association of Src homology 2 domain-containing signalling molecules with paxillin. There are multiple sites of tyrosine phosphorylation on paxillin. To elucidate the role of paxillin in transducing signals in response to various stimuli, it is essential to identify all of the sites of phosphorylation on paxillin and to define which residues are phosphorylated in response to distinct stimuli. We describe two new sites of tyrosine phosphorylation on paxillin, residues at positions 40 and 88. Using paxillin variants with phenylalanine substitutions at phosphorylation sites and phospho-specific paxillin antibodies, tyrosine phosphorylation of paxillin in response to distinct stimuli was examined. The results demonstrate that Tyr31 and Tyr118, which are binding sites for Crk, are major sites of tyrosine phosphorylation following cell adhesion or stimulation with platelet-derived growth factor or angiotensin II. Thus multiple stimuli may elicit similar signalling events downstream of paxillin.

scholarly journals TRB3 Blocks Adipocyte Differentiation through the Inhibition of C/EBPβ Transcriptional Activity

2007 ◽  
Vol 27 (19) ◽  
pp. 6818-6831 ◽  
Author(s):  
Olivier Bezy ◽  
Cecile Vernochet ◽  
Stephane Gesta ◽  
Stephen R. Farmer ◽  
C. Ronald Kahn
Keyword(s):  
Mammalian Cells ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Biological Processes ◽  
Preadipocyte Differentiation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Important Negative Regulator ◽  
Regulatory Sites

ABSTRACT TRB3 has been implicated in the regulation of several biological processes in mammalian cells through its ability to influence Akt and other signaling pathways. In this study, we investigated the role of TRB3 in regulating adipogenesis and the activity of adipogenic transcription factors. We find that TRB3 is expressed in 3T3-L1 preadipocytes, and this expression is transiently suppressed during the initial days of differentiation concomitant with induction of C/EBPβ. This event appears to be a prerequisite for adipogenesis. Overexpression of TRB3 blocks differentiation of 3T3-L1 cells at a step downstream of C/EBPβ. Ectopic expression of TRB3 in mouse fibroblasts also inhibits the C/EBPβ-dependent induction of PPARγ2 and blocks their differentiation into adipocytes. This inhibition of preadipocyte differentiation by TRB3 appears to be the result of two complementary effects. First, TRB3 inhibits extracellular signal-regulated kinase activity, which prevents the phosphorylation of regulatory sites on C/EBPβ. Second, TRB3 directly interacts with the DR1 domain of C/EBPβ in the nucleus, further inhibiting both its ability to bind its response element and its ability to transactivate the C/EBPα and a-FABP promoters. Thus, TRB3 is an important negative regulator of adipogenesis that acts at an early step in the differentiation cascade to block the C/EBPβ proadipogenic function.

scholarly journals Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2)

2003 ◽  
Vol 376 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Helen WHEADON ◽  
Christine EDMEAD ◽  
Melanie J. WELHAM
Keyword(s):  
Signal Transduction ◽  
Cell Survival ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Adaptor Protein ◽  
Functional Responses ◽  
Protein Tyrosine ◽  
Interleukin 3 ◽  
Src Homology

The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 –14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281–29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764–23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911–914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the β-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in suboptimal IL-3 and upon IL-3 withdrawal. These findings indicate that SHP-2 plays an important role in mediating the anti-apoptotic effect of IL-3 and raises the possibility that PECAM-1 participates in the modulation of cytokine-induced signals.

Erythropoietin Induces Tyrosine Phosphorylation of the Interleukin-3 Receptor β Subunit (βIL3 ) and Recruitment of Stat5 to Possible Stat5-Docking Sites in βIL3

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4327-4336 ◽  
Author(s):  
Hiroshi Chin ◽  
Hiroshi Wakao ◽  
Atsushi Miyajima ◽  
Ryuichi Kamiyama ◽  
Nobuyuki Miyasaka ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Β Subunit ◽  
Interleukin 3 ◽  
Amino Acid Region ◽  
Dependent Cell ◽  
Src Homology ◽  
Docking Sites ◽  
Carboxy Terminal ◽  
Dose Dependent

Abstract The receptors for erythropoietin (Epo) and interleukin-3 (IL-3) both induce the ligand-dependent activation of the Jak2 tyrosine kinase. Activated Jak2 then phosphorylates these receptors and thereby recruits various signaling molecules containing the Src homology (SH)-2 domain, including Stat5, to the tyrosine phosphorylated receptors. In the present study, we demonstrate that Epo stimulation induces unidirectional cross-phosphorylation of the IL-3 receptor β subunit (βIL3) on tyrosines and its rapid and transient association with Stat5 in murine IL-3–dependent cell lines engineered to express the Epo receptor (EpoR). Using cell lines expressing various EpoR mutants, it was demonstrated that the Epo-induced tyrosine phosphorylation of βIL3 is dependent on the membrane-proximal EpoR cytoplasmic region involved in the activation of Jak2, but not on the extracellular and transmembrane regions or on the carboxy-terminal 145 amino acid region containing all the intracellular tyrosine residues. It was also shown that IL-3 induces rapid and dose-dependent association of Jak2 with βIL3. However, Epo failed to induce any detectable association of βIL3 with Jak2 or the EpoR. The present study also demonstrates that in IL-3–stimulated cells, an ovine Stat5 mutant harboring a substitution of Tyr694 to Phe, which abolishes the tyrosine phosphorylation required for activation, fails to dimerize with endogenous Stat5, shows sustained binding with tyrosine-phosphorylated βIL3, and inhibits the tyrosine phosphorylation of endogenous Stat5. These results suggest that βIL3 may have Stat5 docking sites, similar to those found in the EpoR, that facilitate the activation of Stat5 by Jak2 and raise the possibility that Epo may cross-activate or transmodulate the IL-3 receptor signaling pathways.

scholarly journals Sprouty3 and Sprouty4, Two Members of a Family Known to Inhibit FGF-Mediated Signaling, Exert Opposing Roles on Proliferation and Migration of Glioblastoma-Derived Cells

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 808 ◽  
Author(s):  
Burcu Emine Celik-Selvi ◽  
Astrid Stütz ◽  
Christoph-Erik Mayer ◽  
Jihen Salhi ◽  
Gerald Siegwart ◽  
...  
Keyword(s):  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Brain Cancer ◽  
Mapk Pathway ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Oncogenic Potential ◽  
And Migration ◽  
Proliferation And Migration

Dysregulation of receptor tyrosine kinase-induced pathways is a critical step driving the oncogenic potential of brain cancer. In this study, we investigated the role of two members of the Sprouty (Spry) family in brain cancer-derived cell lines. Using immunoblot analyses we found essential differences in the pattern of endogenous Spry3 and Spry4 expression. While Spry4 expression was mitogen-dependent and repressed in a number of cells from higher malignant brain cancers, Spry3 levels neither fluctuated in response to serum withdrawal nor were repressed in glioblastoma (GBM)-derived cell lines. In accordance to the well-known inhibitory role of Spry proteins in fibroblast growth factor (FGF)-mediated signaling, both Spry proteins were able to interfere with FGF-induced activation of the MAPK pathway although to a different extent. In response to serum solely, Spry4 exerts its role as a negative regulator of MAPK activation. Ectopic expression of Spry4 inhibited proliferation and migration of GBM-originated cells, positioning it as a tumor suppressor in brain cancer. In contrast, elevated Spry3 levels accelerated both proliferation and migration of these cell lines, while repression of Spry3 levels using shRNA caused a significant diminished growth and migration velocity rate of a GBM-derived cell line. This argues for a tumor-promoting function of Spry3 in GBMs. Based on these data we conclude that Spry3 and Spry4 fulfill different if not opposing roles within the cancerogenesis of brain malignancies.

scholarly journals Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

2001 ◽  
Vol 12 (7) ◽  
pp. 2171-2183 ◽  
Author(s):  
Juan Ángel Fresno Vara ◽  
Ma Aurora Domı́nguez Cáceres ◽  
Augusto Silva ◽  
Jorge Martı́n-Pérez
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Herbimycin A ◽  
Serine Kinases ◽  
Dependent Cell

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [3H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, andodc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.

scholarly journals Loss offlflTriggers JNK-Dependent Cell Death inDrosophila

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Jiuhong Huang ◽  
Lei Xue
Keyword(s):  
Cell Death ◽  
Function Analysis ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Jnk Pathway ◽  
Dependent Cell ◽  
Jnk Activation ◽  
First Time

falafel(flfl) encodes aDrosophilahomolog of human SMEK whosein vivofunctions remain elusive. In this study, we performed gain-of-function and loss-of-function analysis inDrosophilaand identified flfl as a negative regulator of JNK pathway-mediated cell death. While ectopic expression offlflsuppresses TNF-triggered JNK-dependent cell death, loss offlflpromotes JNK activation and cell death in the developing eye and wing. These data report for the first time an essential physiological function offlflin maintaining tissue homeostasis and organ development. As the JNK signaling pathway has been evolutionary conserved from fly to human, a similar role of PP4R3 in JNK-mediated physiological process is speculated.

Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts

1993 ◽  
Vol 13 (8) ◽  
pp. 4648-4656
Author(s):  
R B Birge ◽  
J E Fajardo ◽  
C Reichman ◽  
S E Shoelson ◽  
Z Songyang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Tyrosine Phosphorylation ◽  
Cell Transformation ◽  
Tyrosine Phosphatase ◽  
State Level ◽  
Sh2 Domain ◽  
Sequence Specificity ◽  
Src Homology

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

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