RESUMO
OBJECTIVES: This study aims to investigate the influence of glucose regulated protein (GRP) 78 on osteoblast differentiation in periodontal ligament fibroblasts (PDLFs) under cyclic mechanical stretch and determine the underlying mechanism. METHODS: FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth. GRP78High and GRP78Low subpopulation were obtained by flow sorting. Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src. Western blot analysis was used to detect the protein expression of Runt-related gene 2 (RUNX2), Osterix, osteocalcin (OCN), and osteopontin (OPN). Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src. The formation of cellular mineralized nodules was determined by alizarin red staining. RESULTS: GRP78 was heterogeneously expressed in PDLFs, and GRP78High and GRP78Low subpopulations were obtained by flow sorting. The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch (P<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (P<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (P<0.05) after cyclic mechanical stretch. CONCLUSIONS: GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.
Assuntos
Diferenciação Celular , Proteínas de Choque Térmico , Osteoblastos , Ligamento Periodontal , Proteínas Proto-Oncogênicas pp60(c-src) , Transdução de Sinais , Estresse Mecânico , Humanos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese , Osteopontina/metabolismo , Ligamento Periodontal/metabolismo , Ligamento Periodontal/citologia , Fosforilação , Quinases da Família src/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismoRESUMO
Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.
Assuntos
Matriz Extracelular , Adesões Focais , Quinases da Família src , Adesões Focais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Quinases da Família src/metabolismo , Quinases da Família src/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Metaloproteinases da Matriz/metabolismoRESUMO
C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.
Assuntos
Técnicas Biossensoriais , Proteína Tirosina Quinase CSK , Transferência Ressonante de Energia de Fluorescência , Humanos , Animais , Proteína Tirosina Quinase CSK/metabolismo , Ratos , Quinases da Família src/metabolismo , Fosforilação , Microdomínios da Membrana/metabolismo , Domínios de Homologia de srcRESUMO
One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 µM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.
Assuntos
Artrite Experimental , Reabsorção Óssea , NF-kappa B , Fatores de Transcrição NFATC , Osteoclastos , Osteogênese , Animais , Camundongos , Fatores de Transcrição NFATC/metabolismo , Células RAW 264.7 , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Reabsorção Óssea/prevenção & controle , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Artrite Experimental/metabolismo , Artrite Experimental/induzido quimicamente , Osteogênese/efeitos dos fármacos , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Proteína Tirosina Quinase CSK/metabolismo , Simulação de Acoplamento Molecular , Quinases da Família src/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain (R)-LW-Srci-8 with nearly 75-fold improved potency (IC50 = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C-F···CâO interactions that may contribute to tight binding. The kinact and Ki values validated the improved binding affinity and decreased warhead reactivity of (R)-LW-Srci-8 for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by (R)-LW-Srci-8. Intriguingly, (R)-LW-Srci-8 preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.
Assuntos
Proteína Tirosina Quinase CSK , Inibidores de Proteínas Quinases , Humanos , Proteína Tirosina Quinase CSK/antagonistas & inibidores , Proteína Tirosina Quinase CSK/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Quinases da Família srcRESUMO
In this work, readily achievable synthetic pathways were utilized for construction of a library of N/S analogues based on the pyrazolopyrimidine scaffold with terminal alkyl or aryl fragments. Subsequently, we evaluated the anticancer effects of these novel analogs against the proliferation of various cancer cell lines, including breast, colon, and liver lines. The results were striking, most of the tested molecules exhibited strong and selective cytotoxic activity against the MDA-MB-231 cancer cell line; IC50 1.13 µM. Structure-activity relationship (SAR) analysis revealed that N-substituted derivatives generally enhanced the cytotoxic effect, particularly with aliphatic side chains that facilitated favorable target interactions. We also investigated apoptosis, DNA fragmentation, invasion assay, and anti-migration effects, and discussed their underlying molecular mechanisms for the most active compound 7c. We demonstrated that 7c N-propyl analogue could inhibit MDA-MB-231 TNBC cell proliferation by inducing apoptosis through the regulation of vital proteins, namely c-Src, p53, and Bax. In addition, our results also revealed the potential of these compounds against tumor metastasis by downregulating the invasion and migration modes. Moreover, the in vitro inhibitory effect of active analogs against c-Src kinase was studied and proved that might be the main cause of their antiproliferative effect. Overall, these compelling results point towards the therapeutic potential of these derivatives, particularly those with N-substitution as promising candidates for the treatment of TNBC type of breast cancer.
Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Proteína Tirosina Quinase CSK/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Quinases da Família src , Relação Estrutura-Atividade , Pirimidinas/química , Pirimidinas/farmacologia , Pirazóis/química , Pirazóis/farmacologiaRESUMO
Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.
Assuntos
Reabsorção Óssea , Podossomos , Humanos , Osteoclastos/metabolismo , Podossomos/metabolismo , Actinas/metabolismo , Diferenciação Celular/fisiologia , Reabsorção Óssea/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Colágeno/metabolismo , Oligopeptídeos/metabolismoRESUMO
BACKGROUND: Cancer cells exhibit selective metabolic reprogramming to promote proliferation, invasiveness, and metastasis. Sphingolipids such as sphingosine and sphinganine have been reported to modulate cell death processes in cancer cells. However, the potential of extracellular sphinganine and its mimetic compounds as inducers of cancer cell death has not been thoroughly investigated. METHODS: We obtained extracellular conditioned medium from HCT-116 cells treated with the previously reported anticancer composition, goat urine DMSO fraction (GUDF). The extracellular metabolites were purified using a novel and in-house developed vertical tube gel electrophoresis (VTGE) technique and identified through LC-HRMS. Extracellular metabolites such as sphinganine, sphingosine, C16 sphinganine, and phytosphingosine were screened for their inhibitory role against intracellular kinases using molecular docking. Molecular dynamics (MD) simulations were performed to study the inhibitory potential of a novel designed modified mimetic sphinganine (MMS) (Pubchem CID: 162625115) upon c-Src kinase. Furthermore, inhibitory potential and ADME profile of MMS was compared with luteolin, a known c-Src kinase inhibitor. RESULTS: Data showed accumulation of sphinganine and other sphingolipids such as C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0) in the extracellular compartment of GUDF-treated HCT-116 cells. Molecular docking projected c-Src kinase as an inhibitory target of sphinganine. MD simulations projected MMS with strong (-7.1 kcal/mol) and specific (MET341, ASP404) binding to the inhibitory pocket of c-Src kinase. The projected MMS showed comparable inhibitory role and acceptable ADME profile over known inhibitors. CONCLUSION: In summary, our findings highlight the significance of extracellular sphinganine and other sphingolipids, including C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0), in the context of drug-induced cell death in HCT-116 cancer cells. Furthermore, we demonstrated the importance of extracellular sphinganine and its modified mimetic sphinganine (MMS) as a potential inhibitor of c-Src kinase. These findings suggest that MMS holds promise for future applications in targeted and combinatorial anticancer therapy.
Assuntos
Neoplasias , Esfingosina , Esfingosina/análogos & derivados , Humanos , Esfingosina/farmacologia , Esfingosina/metabolismo , Proteína Tirosina Quinase CSK , Simulação de Acoplamento Molecular , Esfingolipídeos/metabolismo , Ceramidas/farmacologia , Neoplasias/patologiaRESUMO
OBJECTIVE: C-terminal Src kinase (CSK), a sarcoma (Src) homologous family kinase, is one of the most important negative regulators. It acts as a tumor suppressor by inhibiting the activity of Src family tyrosine kinases. Paradoxically, CSK is highly expressed in a variety of common tumors. Therefore, we report the expression profile of CSK in pan-cancer patients, focusing on the prognostic value, immune infiltration pattern, and biological function of CSK in gastric cancer. MATERIALS AND METHODS: We used the TCGA database to analyze CSK expression, clinical relevance, prognostic significance, assessment of the tumor immune microenvironment, and GO and Kegg enrichment analysis based on co-expressed genes using a bioinformatics approach. RESULTS: CSK is a protective factor in gastric cancer, and its expression correlates with the level of immune cell infiltration and immune checkpoint molecules. CONCLUSIONS: Our findings suggest that CSK is an independent prognostic factor in gastric cancer and may predict molecular targeting and immunotherapy and provide ideas for its therapeutic strategy.
Assuntos
Neoplasias Gástricas , Quinases da Família src , Humanos , Quinases da Família src/metabolismo , Fosforilação , Proteína Tirosina Quinase CSK/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Prognóstico , Biomarcadores/metabolismo , Microambiente TumoralRESUMO
New chromene derivatives were synthesized based on 4-(3,4-dimethoxy)-4H-chromene scaffold. All target compounds exhibited cytotoxic activity against HepG2 cells (IC50 = 2.40-141.22 µM). Chromens 5 and 9 showed superior cytotoxicity over staurosporine (IC50 = 18.27 µM) and vinblastine (IC50 = 5.20 µM). c-Src kinase inhibition assay of compounds 5 and 9 displayed the dominant c-Src inhibitory activity of 5 (IC50 = 0.184 µM) over 9 (IC50 = 0.288 µM). The safety of the most potent compound 5 against normal WI-38 cells was confirmed via its IC50 of 115.75 µM comparable with 5-FU (IC50 = 16.28 µM). Moreover, the promising chromene 5 displayed potent cytotoxicity against resistant HepG2 cells with IC50 of 26.03 µM comparable with 5-FU (IC50 = 42.68 µM). The most active chromene 5 arrested the HepG2 cell cycle at the S phase and induced a 29-fold increase in the total number of apoptotic cells indicating pre-G1 apoptosis. The ability of compound 5 to induce apoptosis was supported via elevation of caspase-3, caspase-7, caspase-9 and proapoptotic Bax protein levels in addition to downregulation of the antiapoptotic Bcl2 protein. Molecular docking studies of compound 5 showed good binding interaction pattern inside c-Src kinase enzyme active site.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Estrutura Molecular , Relação Estrutura-Atividade , Benzopiranos/química , Simulação de Acoplamento Molecular , Proteína Tirosina Quinase CSK/metabolismo , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Hepáticas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Antineoplásicos/química , Apoptose , Fluoruracila/farmacologia , Desenho de FármacosRESUMO
The pyrimidine and fused pyrimidine ring systems play vital roles to inhibit the c-Src kinase. The Src kinase is made of different domains but the kinase domain is responsible for inhibition of Src kinase. In which the kinase domain is the main domain that is made of several amino acids. The Src kinase is inhibited by its inhibitors when it is activated by phosphorylation. Although dysregulation of Src kinase caused cancer in the late nineteenth century, medicinal chemists have not explored it extensively; therefore it is still regarded as a cult pathway. There are numerous FDA-approved drugs on the market, yet novel anticancer drugs are still in demand. Existing medications have adverse effects and drug resistance owing to rapid protein mutation. In this review, we discussed the activation process of Src kinase, chemistry of pyrimidine ring and its different synthetic routes, as well as the recent development in c-Src kinase inhibitors containing pyrimidine and their biological activity, SAR, and selectivity. The c-Src binding pocket has been predicted in detail to discover the vital amino acids which will interact with inhibitors. The potent derivatives were docked to discover the binding pattern. The derivative 2 established three hydrogen bonds with the amino acid residues Thr341 and Gln278 and had the greatest binding energy of -13.0 kcal/mol. The top docked molecules were further studied for ADMET studies. The derivative 1, 2, and 43 did not show any violation of Lipinski's rule. All derivatives used for the prediction of toxicity showed toxicity.
Assuntos
Antineoplásicos , Quinases da Família src , Quinases da Família src/química , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Pirimidinas/farmacologia , Pirimidinas/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Aminoácidos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
PURPOSE: To observe the effects of oxidative stress on vascular endothelial growth factor (VEGF) and connections of lens epithelial cells. METHODS: Human lens epithelium of patients with age-related cataract (ARC), both SRA01/04 cells and whole mice lens stimulated by H2O2 were employed. VEGF in human aqueous humor of ARC-patients and the supernatant of SRA01/04 cells was determined by ELISA. The expressions of VEFG in human lens epithelium were detected by immunofluorescence staining. Multiple linear regression analysis and spearman rank-order correlation were used to determine the associations between VEGF and parameters of ARC individuals. In H2O2-induced SRA01/04 cells, Catalase (CAT), PP1 (inhibitor of c-Src kinase) and Avastin (VEGF antibody) were used to inhibit the effects of H2O2, activation of c-Src kinase and VEGF, which were detected by Western blot. The alterations of ZO-1 and N-cadherin were tested by immunofluorescence staining and Western blot. In H2O2-induced whole lens, the changes of opacification area in different treatment of inhibitors were observed. RESULTS: The secretion of VEGF in aqueous humor and expression of VEGF in the lens epithelium of ARC patients increased significantly with age. In H2O2-induced SRA01/04 cells, the VEGF in the supernatant was increased with the culture duration and the dose of H2O2. The expressions of p-Src418 and VEGF were also up-regulated, whereas the expressions of ZO-1 and N-cadherin were down-regulated. CAT effectively prevented these changes induced by H2O2, while PP1 inhibited not only p-Src418 but also up-regulation of VEGF, Avastin partially inhibited VEGF up-regulation. Both PP1 and Avastin prevented down-regulation of ZO-1 and N-cadherin, respectively, but Avastin combined with PP1 had no significant synergistic effects. In H2O2-induced cataract, CAT prevented development of opacification area effectively, and PP1 and Avastin did partially. CONCLUSIONS: Oxidative stress disrupts connections of lens epithelial cells by activating c-Src/VEGF, inhibiting which may prevent cataract.
Assuntos
Catarata , Cristalino , Humanos , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Bevacizumab/farmacologia , Peróxido de Hidrogênio/farmacologia , Catarata/metabolismo , Cristalino/metabolismo , Células Epiteliais/metabolismo , Estresse Oxidativo , Caderinas , ApoptoseRESUMO
Using dasatinib linked to E3 ligase ligands, we identified a potent and selective dual Csk/c-Src PROTAC degrader. We then replaced dasatinib, the c-Src-directed ligand, with a conformation-selective analogue that stabilizes the αC-helix-out conformation of c-Src. Using the αC-helix-out ligand, we identified a PROTAC that is potent and selective for c-Src. We demonstrated a high degree of catalysis with our c-Src PROTACs. Using our c-Src PROTACs, we identified pharmacological advantages of c-Src degradation compared to inhibition with respect to cancer cell proliferation.
Assuntos
Ubiquitina-Proteína Ligases , Dasatinibe/farmacologia , Proteína Tirosina Quinase CSK/metabolismo , Ligantes , Proliferação de Células , Ubiquitina-Proteína Ligases/metabolismo , ProteóliseRESUMO
Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human cancers (extracellular, e-Src). Phosphoproteomics has demonstrated the existence of 114 potential extracellular e-Src substrates in addition to Tissue Inhibitor of Metalloproteinases 2. Here, we present a protocol to characterize secreted tyrosine-phosphorylated substrates as a result of c-Src expression and secretion. We describe steps for collecting cell secretomes and extracts, performing antibody treatment and Ni-NTA pull-down, and detecting protein-protein interaction and substrate Y-phosphorylation. This protocol is adaptable for studies examining the function of other extracellular kinases. For complete details on the use and execution of this protocol, please refer to Backe et al. (2023)1 and Sánchez-Pozo et al. (2018).2.
Assuntos
Proteínas Tirosina Quinases , Quinases da Família src , Humanos , Quinases da Família src/metabolismo , Fosforilação , Proteína Tirosina Quinase CSK/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismoRESUMO
In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição AP-1 , Humanos , Proteína Tirosina Quinase CSK/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Inflamação/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol/farmacologia , Resveratrol/metabolismo , Quinases da Família src/metabolismo , Trombina/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phospho-motif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrate- and enzyme-acting kinases during autophosphorylation.
Assuntos
Quinases da Família src , Fosforilação , Proteína Tirosina Quinase CSK/metabolismo , Domínio Catalítico , Quinases da Família src/metabolismoRESUMO
Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.
Assuntos
Transdução de Sinais , Quinases da Família src , Camundongos , Animais , Proteína Tirosina Quinase CSK/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Fosforilação , Linfócitos B/metabolismo , Camundongos KnockoutRESUMO
Endothelial phospholipase Cγ (PLCγ) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.
Assuntos
Permeabilidade Capilar , Carcinoma de Células Renais , Neoplasias Renais , Animais , Humanos , Camundongos , Permeabilidade Capilar/genética , Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Tirosina Quinase CSK/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is recognized for its poor prognosis and limited options for treatment. Circular RNA KIF4A (circKIF4A) was documented to be abnormally overexpressed in TNBC and was correlated with a poor survival rate. The objective of this study is to further examine the functional role of circKIF4A and its underlying mechanism. CircKIF4A was significantly upregulated in TNBC and the knockdown of circKIF4A suppressed TNBC cell proliferation, migration, and invasion. CircKIF4A was directly bound to EIF4A3, which interacted with SDC1. Knockdown of circKIF4A reduced interaction between EIF4A3 and SDC1 as well as SDC1 mRNA stability. SDC1 activated the c-src/FAK signaling pathways and finally promoted TNBC progression. circKIF4A induced TNBC progress in the in vivo mouse model via SDC1. CircKIF4A interacts with EIF4A3 to stabilize SDC1 mRNA, which activates the c-src/FAK signaling pathways and promotes TNBC progression. This may provide a potential therapy for TNBC treatment.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Proteína Tirosina Quinase CSK/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Cinesinas/genética , RNA Circular , Transdução de Sinais , Quinases da Família src , Sindecana-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Morphine diminishes pain, but its long-term use is compromised by tolerance and hyperalgesia. Studies implicate δ receptors, ß-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. We examined mechanical sensitivity using automated von Frey in wild-type (WT) and transgenic male and female C57Bl/6 mice before and after hind paw inflammation by complete Freund's adjuvant (CFA). CFA-evoked hypersensitivity ceased on day 7 in WT but persisted for the 15-day testing period in µ-/- . Recovery was delayed until day 13 in δ-/- . We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. Restoration to basal sensitivity in WT occurred with increased δ expression. By contrast, κ expression was reduced, while µ remained unchanged. Daily morphine reduced hypersensitivity in WT on day 3 compared to controls; however, hypersensitivity recurred on day 9 and beyond. By contrast, WT had no recurrence of hypersensitivity in the absence of daily morphine. We used ß-arrestin2-/- , δ-/- and Src inhibition by dasatinib in WT to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. Like morphine tolerance, MIH in this model requires δ receptors, ß-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling. KEY POINTS: Morphine is effective for treating severe acute pain, but tolerance and hypersensitivity often develop during its use in treating chronic pain. It is unclear whether these detrimental effects share similar mechanisms; if so, it might be possible to develop a single approach to minimise both phenomena. Mice deficient in µ receptors, δ receptors or ß-arrestin2 and wild type mice treated with the Src inhibitor dasatinib exhibit negligible morphine tolerance. We show that these same approaches also prevent the development of morphine-induced hypersensitivity during persistent inflammation. This knowledge identifies strategies, such as the use of Src inhibitors, which may mitigate tolerance and morphine induced hyperalgesia.