scholarly journals Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

1996 ◽  
Vol 16 (3) ◽  
pp. 1027-1034 ◽  
Author(s):  
T Jelinek ◽  
P Dent ◽  
T W Sturgill ◽  
M J Weber
Keyword(s):  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Tyrosine Phosphatase ◽  
3T3 Cells ◽  
Level Of Activity ◽  
Active Pool ◽  
Ptp 1B ◽  
Nih 3T3

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.

scholarly journals Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin

1996 ◽  
Vol 314 (2) ◽  
pp. 401-404 ◽  
Author(s):  
Dean B. REARDON ◽  
Steven L. WOOD ◽  
David L. BRAUTIGAN ◽  
Graeme I. BELL ◽  
Paul DENT ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Somatostatin Receptor ◽  
3T3 Cells ◽  
Serine Kinase ◽  
Protein Tyrosine ◽  
Active Membrane ◽  
Nih 3T3

Human somatostatin receptor 3 (‘hsstr3’) was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase, and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro.

Abstract 12231: PECAM1 is Essential for Flow-mediated Gab1 Tyrosine Phosphorylation and Signaling in vitro and in vivo

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Suowen Xu ◽  
Marina Koroleva ◽  
Keigi Fujiwara ◽  
Zheng Gen Jin
Keyword(s):  
Tyrosine Phosphorylation ◽  
Wheel Running ◽  
Tyrosine Phosphatase ◽  
Common Mechanism ◽  
Membrane Localization ◽  
No Production ◽  
Voluntary Wheel Running ◽  
Voluntary Wheel

Introduction: Impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued NO production is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Specific signaling cascades, generated by vascular endothelial cells (ECs) in response to laminar flow, modulate EC structure and functions, NO production in particular. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation. However, the upstream mechanism that regulates Gab1 tyrosine phosphorylation remains unclear. Hypothesis: We hypothesized that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Methods: Western blot, en face staining and voluntary wheel running. Results: Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in a flow signaling pathway as well as HGF-induced signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K by LY294002 decreased flow, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. Conclusions: These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs

scholarly journals Characteristics of Food Protein-Derived Antidiabetic Bioactive Peptides: A Literature Update

2021 ◽  
Vol 22 (17) ◽  
pp. 9508
Author(s):  
Nhung Thi Phuong Nong ◽  
Jue-Liang Hsu
Keyword(s):  
Bioactive Peptides ◽  
Protein Hydrolysate ◽  
Tyrosine Phosphatase ◽  
Food Protein ◽  
Dpp Iv ◽  
Glucosidase Inhibitors ◽  
Commercial Applications ◽  
Ptp 1B

Diabetes, a glucose metabolic disorder, is considered one of the biggest challenges associated with a complex complication of health crises in the modern lifestyle. Inhibition or reduction of the dipeptidyl peptidase IV (DPP-IV), alpha-glucosidase, and protein-tyrosine phosphatase 1B (PTP-1B) enzyme activities or expressions are notably considered as the promising therapeutic strategies for the management of type 2 diabetes (T2D). Various food protein-derived antidiabetic bioactive peptides have been isolated and verified. This review provides an overview of the DPP-IV, PTP-1B, and α-glucosidase inhibitors, and updates on the methods for the discovery of DPP-IV inhibitory peptides released from food-protein hydrolysate. The finding of novel bioactive peptides involves studies about the strategy of separation fractionation, the identification of peptide sequences, and the evaluation of peptide characteristics in vitro, in silico, in situ, and in vivo. The potential of bioactive peptides suggests useful applications in the prevention and management of diabetes. Furthermore, evidence of clinical studies is necessary for the validation of these peptides’ efficiencies before commercial applications.

scholarly journals Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4495-4501 ◽  
Author(s):  
T Tauchi ◽  
JE Damen ◽  
K Toyama ◽  
GS Feng ◽  
HE Broxmeyer ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Tyrosine Phosphatase ◽  
Erythropoietin Receptor ◽  
Sh2 Domains ◽  
Proliferation And Differentiation ◽  
Stimulation Of

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.

scholarly journals Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
D Shalloway ◽  
P J Johnson ◽  
E O Freed ◽  
D Coulter ◽  
W A Flood
Keyword(s):  
3T3 Cells ◽  
Focus Formation ◽  
Adenovirus E1a ◽  
Rous Sarcoma ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

FoxF1 is Required for Ciliogenesis and Distribution of Sonic Hedgehog Signaling Components in Cilium

2019 ◽  
Vol 19 (5) ◽  
pp. 326-334
Author(s):  
Lu Huang ◽  
Marco Tjakra ◽  
Desha Luo ◽  
Lin Wen ◽  
Daoxi Lei ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
3T3 Cells ◽  
Shh Signaling ◽  
Nih 3T3 ◽  
Signaling Components ◽  
Nih 3T3 Cells

Background: In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh) signaling in many cases. However, the role of FoxF1 in cilium remains unknown. Here, we showed an essential role of FoxF1 in the regulation of ciliogenesis and in the distribution of Shh signaling components in cilium. Methods: NIH/3T3 cells were serum starved for 24h to induce cilium. Meanwhile, shRNA was used to knockdown the FoxF1 expression in the cells and CRISPR/Cas9 was used to generate the FoxF1 zebrafish mutant. The mRNA and protein expression of indicated genes were detected by the qRT-PCR and western blot, respectively. Immunofluorescence staining was performed to detect the cilium and Shh components distribution. Results: FoxF1 knockdown decreased the cilium length in NIH/3T3 cells. Meanwhile, the disruption of FoxF1 function inhibited the expression of cilium-related genes and caused an abnormal distribution of Shh components in the cilium. Furthermore, homozygous FoxF1 mutants exhibited defective development of pronephric cilium in early zebrafish embryos. Conclusion: Together, our data illustrated that FoxF1 is required for ciliogenesis in vitro and in vivo and for the proper localization of Shh signaling components in cilium.

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.

scholarly journals Dynamic Interaction of PTPμ with Multiple Cadherins In Vivo

1998 ◽  
Vol 141 (1) ◽  
pp. 287-296 ◽  
Author(s):  
Susann M. Brady-Kalnay ◽  
Tracy Mourton ◽  
Joseph P. Nixon ◽  
Gregory E. Pietz ◽  
Michael Kinch ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Cross Reactivity ◽  
Receptor Protein ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Cell Biol ◽  
E Cadherin

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.

scholarly journals 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.

scholarly journals Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation.

1995 ◽  
Vol 15 (7) ◽  
pp. 3805-3812 ◽  
Author(s):  
B Matoskova ◽  
W T Wong ◽  
A E Salcini ◽  
P G Pelicci ◽  
P P Di Fiore
Keyword(s):  
Tumor Cell ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Egf Receptor ◽  
Human Tumor ◽  
Tumor Cell Lines ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy), corresponding to the 50% effective dose of EGF for mitogenesis, approximately 3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (approximately 5%) similar to that associated with potent mitogenic response in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and shc might be part of a pathway which is preferentially selected in some tumors. Cooperation between these two transducers was further indicated by the finding of their in vivo association. This association was, at least in part, dependent on recognition of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alterations can contribute to the malignant phenotype.

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