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Src Kinase Activity (src + kinase_activity)
Selected AbstractsInhibition of Src Kinase Activity by 4-Anilino-5,10-dihydropyrimido[4,5-b]quinolines.CHEMINFORM, Issue 51 2003Diane H. Boschelli Abstract For Abstract see ChemInform Abstract in Full Text. [source] A FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assemblyCYTOSKELETON, Issue 1 2008Priscila M. F. Siesser Abstract Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK ,/, mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK ,/, cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Sustained MAPK activation is dependent on continual NGF receptor regenerationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2004Dongru Qiu It still remains intriguing how signal specificity is achieved when different signals are relayed by the common intracellular signal transduction pathways. A well documented example for signal specificity determination is found in rat phaeochromocytoma PC12 cells where epidermal growth factor (EGF) stimulation produces a transient mitogen-activated protein kinase (MAPK) activation and leads to cell proliferation while nerve growth factor (NGF) initiates a sustained MAPK activation and induces cell differentiation. In this simulation, we demonstrated that NGF-induced sustained MAPK activation may mainly depend on continual regeneration of NGF receptors and that the presence of a small pool of surface receptors is enough to maintain a sustained MAPK activation. On the other hand, MAPK activation is not significantly sensitive to the half-life of internalized receptors and the levels of NGF-specific MAPK phosphatase MAP kinase phosphatase-3 (MKP-3), though cytoplasmic persistence of internalized NGF-bound receptors and the MKP-3 dependent feedback control also contribute to the sustaining of MAPK activation. These results are consistent with the recent experimental evidence that persistent tyrosine receptor kinase A (TrkA) activity is necessary to maintain transcription in the differentiating PC12 cells (Chang et al. 2003) and a sustained Src kinase activity is detected in response to NGF stimulation (Gatti 2003). It is suggested that sustained or transient MAPK activation induced by different growth factor and neurotrophins, which is crucial to their signaling specificity, could be satisfactorily accounted for by their specific receptor turnover kinetics rather than by the activation of specific downstream signaling cascades. [source] Identification and functional characterization of an Src homology domain 3 domain-binding site on CblFEBS JOURNAL, Issue 23 2006Archana Sanjay§ Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540,551), which corresponds to residues 540,551 of Cbl, inhibited the binding of a GST,Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540,551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543,548 reduced Src binding to the Cbl 479,636 fragment significantly more than mutating the prolines in the PPVPPR(494,499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543,548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540,546) sequence is a functionally important binding site for Src. [source] Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Vijayalakshmi Thamilselvan Abstract Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0,±,3.0% (n,=,3; P,<,0.001) and primary cancer cells by 41.0,±,3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5,±,0.13 fold) and Western analysis (1.34,±,0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 µM), PP2 (20 µM), and actin-cytoskeleton stabilizer phalloidin (10 µM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0,±,2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection. Published 2004 Wiley-Liss, Inc. [source] Continuous requirement for pp60-Src and phospho-paxillin during fibronectin matrix assembly by transformed cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Iwona Wierzbicka-Patynowski Fibronectin (FN) matrix assembly is an integrin-mediated process that is regulated by both the extracellular environment and intracellular signaling pathways. The activity of Src-family kinases is important for initiation of FN assembly by normal fibroblasts. Here we report that in HT1080 fibrosarcoma cells, Src kinase activity is required not only for the assembly of FN matrix but also for the maintenance of FN matrix fibrils at the cell surface. Dexamethasone-induced FN fibril formation by these cells was completely blocked for at least 24 h when Src-family kinase activity was inhibited by either PP1 or SU6656. Inhibition of Src after significant matrix had already been assembled, resulted in an increased rate of loss of detergent-insoluble FN. Binding of activation-dependent integrin antibodies reveals a role for Src in maintaining integrin activity. The requirement for Src kinase activity appears to depend, in part, on phosphorylation of paxillin at tyrosine 118 (Y118). Phospho-paxillin co-localized with FN fibrils, and overexpression of GFP-paxillin but not of GFP-paxillinY118F enhanced cell-mediated assembly of FN. Our results indicate that Src maintains FN matrix at the cell surface through its effect on integrin activity and paxillin phosphorylation. J. Cell. Physiol. 210: 750,756, 2007. © 2006 Wiley-Liss, Inc. [source] c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aortaTHE JOURNAL OF PHYSIOLOGY, Issue 16 2008Rong Lu Serotonin (5-hydroxytryptamine, 5-HT) receptors (5-HTRs) play critical roles in brain and cardiovascular functions. In the vasculature, 5-HT induces potent vasoconstrictions, which in aorta are mainly mediated by activation of the 5-HT2AR subtype. We previously proposed that one signalling mechanism of 5-HT-induced vasoconstriction could be c-Src, a member of the Src tyrosine kinase family. We now provide evidence for a central role of c-Src in 5-HT2AR-mediated contraction. Inhibition of Src kinase activity with 10 ,m 4-amino-5-(4-chlorophenyl)-7-(t -butyl)pyrazolo[3,4-d]pyrimidine (PP2) prior to contraction resulted in ,90,99% inhibition of contractions induced by 5-HT or by ,-methyl-5-HT (5-HT2R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A2 mimetic, U46619, and had no significant action on phenylephrine-induced contractions. 5-Hydroxytryptamine increased Src kinase activity and PP2-sensitive tyrosine-phosphorylated proteins. As expected for c-Src identity, PP2 pretreatment inhibited 5-HT-induced contraction with an IC50 of ,1 ,m. Ketanserin (10 nm), a 5-HT2A antagonist, but not antagonists of 5-HT2BR (100 nm SB204741) or 5-HT2CR (20 nm RS102221), prevented 5-HT-induced contractions, mimicking PP2 and implicating 5-HT2AR as the major receptor subtype coupled to c-Src. In HEK 293T cells, c-Src and 5-HT2AR were reciprocally co-immunoprecipitated and co-localized at the cell periphery. Finally, 5-HT-induced Src activity was unaffected by inhibition of Rho kinase, supporting a role of c-Src upstream of Rho kinase. Together, the results highlight c-Src activation as one of the early and pivotal mechanisms in 5-HT2AR contractile signalling in aorta. [source] Quinolinic acid modulates the activity of src family kinases in rat striatum: in vivo and in vitro studiesJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Alessio Metere Abstract Quinolinic acid (QA) has been shown to evoke neurotoxic events via NMDA receptor (NMDAR) overactivation and oxidative stress. NMDARs are particularly vulnerable to free radicals, which can modulate protein tyrosine kinase (PTK) and phosphotyrosine phosphatase (PTP) activities. The src family of tyrosine kinases are associated with the NMDAR complex and regulate NMDA channel function. Because QA is an NMDAR agonist as well as a pro-oxidant agent, we investigated whether it may affect the activity of PTKs and PTPs in vivo and in vitro. In synaptosomes prepared from striata dissected 15 min, 30 min or 15 days after bilateral injection of QA we observed modulation of the phosphotyrosine pattern; a significant decrease in PTP activity; and a sustained increase in c-src and lyn activity at 15 and 30 min after treatment with QA, followed by a decrease 2 weeks later. Striatal synaptosomes treated in vitro with QA showed time- and dose-dependent modulation of c-src and lyn kinase activities. Moreover, the nitric oxide synthase inhibitor NG -nitro- l -arginine-methyl ester, the NMDAR antagonist d -2-amino-5-phosphonovaleric acid and pyruvate suppressed the QA-induced modulation of c-src activity. These findings suggest a novel feature of QA in regulating src kinase activity through the formation of reactive radical species and/or NMDAR overactivation. [source] | |||||||||||||