Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro

2003 ◽  
Vol 285 (5) ◽  
pp. G861-G869 ◽  
Author(s):  
Douglas A. Potoka ◽  
Jeffrey S. Upperman ◽  
Xiao-Ru Zhang ◽  
Joshua R. Kaplan ◽  
Seth J. Corey ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Src Kinase ◽  
Dependent Manner ◽  
Gut Barrier ◽  
Enterocyte Proliferation ◽  
Barrier Failure ◽  
Enterocyte Apoptosis ◽  
Gut Barrier Failure

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO-may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO-on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO-or decomposed ONOO-in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2′-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO-treatment in vitro inhibited enterocyte proliferation. ONOO-treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO-blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO-may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.

scholarly journals Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils

2003 ◽  
Vol 370 (2) ◽  
pp. 687-694 ◽  
Author(s):  
Fredrik MELANDER ◽  
Tommy ANDERSSON ◽  
Karim DIB
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
E3 Ligase ◽  
In Vitro Assay ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Β2 Integrin

An early and critical event in β2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent β2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) β2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected β2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in β2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.

scholarly journals Src acts as the target of matrine to inhibit the proliferation of cancer cells by regulating phosphorylation signaling pathways

2020 ◽  
Author(s):  
Xi Zhang ◽  
Hui Xu ◽  
Xiaoyang Bi ◽  
Guoqing Hou ◽  
Andong Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Src Kinase ◽  
Kinase Domain ◽  
Akt Phosphorylation ◽  
In Vivo Experiments

Background and Purpose: Identification of accurate targets is essential for a successful development of targeted therapy in cancer. Studies have shown that matrine has antitumor activity against many types of cancers. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Experimental Approach: The effect of matrine on the proliferation of cancer cells were examined by MTT assay. Pull-down assay and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were performed to explore the target of matrine. A series of in vitro and in vivo experiments were conducted to reveal the mechanisms by which matrine targeted Src to regulate the downstream signaling pathways of Src in cancer cells. Key Results: Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with matrine-amino coupling resins (MA beads) and LC-MS/MS identified Src as the target of matrine. Src kinase domain is required for its interaction with matrine and Ala392 in the kinase domain participated in matrine-Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3 and PI3K/Akt signaling pathways. Conclusions and Implications: Collectively, matrine targeted Src, inhibited kinase activity and down-regulated its downstream MAPK/ERK, JAK2/STAT3 and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells.

scholarly journals Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259

2002 ◽  
Vol 22 (10) ◽  
pp. 3237-3246 ◽  
Author(s):  
Amardeep S. Dhillon ◽  
Claire Pollock ◽  
Helge Steen ◽  
Peter E. Shaw ◽  
Harald Mischak ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Dependent Protein ◽  
Kinase Pathway

ABSTRACT The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5290-5300
Author(s):  
S M Murphy ◽  
M Bergman ◽  
D O Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Kinase Activity ◽  
Src Kinase ◽  
Intact Cells ◽  
Sh3 Domains ◽  
Mutant Proteins ◽  
Dependent Growth

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

scholarly journals Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (9) ◽  
pp. 5290-5300 ◽  
Author(s):  
S M Murphy ◽  
M Bergman ◽  
D O Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Kinase Activity ◽  
Src Kinase ◽  
Intact Cells ◽  
Sh3 Domains ◽  
Mutant Proteins ◽  
Dependent Growth

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

scholarly journals Src Acts as the Target of Matrine to Inhibit the Proliferation of Cancer Cells by Regulating Phosphorylation Signaling Pathways

2020 ◽  
Author(s):  
Xi Zhang ◽  
Hui Xu ◽  
Xiaoyang Bi ◽  
Guoqing Hou ◽  
Andong Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Src Kinase ◽  
Kinase Domain ◽  
Competitive Binding Assay ◽  
In Vivo Experiments

Abstract Background: Identification of accurate targets is essential for a successful development of targeted therapy in cancer. Studies have shown that matrine has antitumor activity against many types of cancers, including lung cancer, breast cancer, liver cancer, pancreatic cancer, ovarian cancer and leukemia, etc. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Methods: The effect of matrine on the proliferation of cancer cells were examined by MTT assay. Pull-down assay with matrine-amino coupling resins (MA beads) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were performed to explore the target of matrine. The target of matrine was further validated by competitive binding assay, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS). A series of in vitro and in vivo experiments were conducted to reveal the mechanisms by which matrine targeted Src to regulate the downstream signaling pathways of Src in cancer cells. Results: Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with MA beads and LC-MS/MS identified Src as the target of matrine. The findings provided solid evidences that matrine directly bound to Src and Src kinase domain is required for its interaction with matrine and Ala392 in the kinase domain participated in matrine-Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419 in Src kinase domain. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3 and PI3K/Akt signaling pathways via targeting Src.Conclusions: Collectively, matrine targeted Src, inhibited its kinase activity and down-regulated its downstream MAPK/ERK, JAK2/STAT3 and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells.

scholarly journals Constitutive Association of BRCA1 and c-Abl and Its ATM-Dependent Disruption after Irradiation

2002 ◽  
Vol 22 (12) ◽  
pp. 4020-4032 ◽  
Author(s):  
Nicolas Foray ◽  
Didier Marot ◽  
Voahangy Randrianarison ◽  
Nicole Dalla Venezia ◽  
Didier Picard ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Direct Interaction ◽  
Cell Cycle Checkpoint ◽  
Dependent Manner ◽  
Tyrosine Kinase Activity ◽  
Abl Tyrosine Kinase ◽  
C Terminus

ABSTRACT BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

scholarly journals Src acts as the target of matrine to inhibit the proliferation of cancer cells by regulating phosphorylation signaling pathways

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Xi Zhang ◽  
Hui Xu ◽  
Xiaoyang Bi ◽  
Guoqing Hou ◽  
Andong Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Src Kinase ◽  
Kinase Domain ◽  
Akt Phosphorylation ◽  
Cellular Thermal Shift Assay

AbstractStudies have shown that matrine has antitumor activity against many types of cancers. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with matrine-amino coupling resins and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) identified Src as the target of matrine. Cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) provided solid evidences that matrine directly bound to Src. Bioinformatics prediction and pull-down experiment demonstrated that Src kinase domain was required for its interaction with matrine and Ala392 in the kinase domain participated in matrine–Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419 in Src kinase domain. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3, and PI3K/Akt signaling pathways via targeting Src. Collectively, matrine targeted Src, inhibited its kinase activity, and down-regulated its downstream MAPK/ERK, JAK2/STAT3, and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Sign in / Sign up

Export Citation Format

Share Document