RESUMEN
Specific subpopulations of neurons in nerve and sensory systems must be developed and maintained, and this is accomplished in significant part by neurotrophins (NTs) and the signaling receptors on which they act, called tyrosine protein kinase receptors (Trks). The neurotrophins-tyrosine protein kinase receptors (NTs/Trks) system is involved in sensory organ regulation, including the visual system. An NTs/Trks system alteration is associated with neurodegeneration related to aging and diseases, including retinal pathologies. An emergent model in the field of translational medicine, for instance, in aging study, is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, and humans share a similar retinal stratigraphy. Nevertheless, according to the authors' knowledge, the occurrence and distribution of the NTs/Trks system in the retina of N. guentheri has never been investigated before. Therefore, the present study aimed to localize neurotrophin BDNF, NGF, and NT-3 and TrkA, TrkB, and TrkC receptors in the N. guentheri retina using the immunofluorescence method. The present investigation demonstrates, for the first time, the occurrence of the NTs/Trks system in N. guentheri retina and, consequently, the potential key role of these proteins in the biology and survival of the retinal cells.
Asunto(s)
Peces Killi , Factores de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso , Humanos , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Retina/metabolismo , Receptor trkA , Neurotrofina 3 , Factor Neurotrófico Derivado del EncéfaloRESUMEN
AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells.
Asunto(s)
Endocitosis , Péptidos y Proteínas de Señalización Intercelular , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/metabolismo , COVID-19/terapia , Clatrina/metabolismo , Clatrina/fisiología , Endocitosis/efectos de los fármacos , Endocitosis/genética , Endocitosis/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
Meningioma (MGM) is the most common type of intracranial tumor in adults. The validation of novel prognostic biomarkers to better inform tumor stratification and clinical prognosis is urgently needed. Many molecular and cellular alterations have been described in MGM tumors over the past few years, providing a rational basis for the identification of biomarkers and therapeutic targets. The role of receptor tyrosine kinases (RTKs) as oncogenes, including those of the ErbB family of receptors, has been well established in several cancer types. Here, we review histological, molecular, and clinical evidence suggesting that RTKs, including the epidermal growth factor receptor (EGFR, ErbB1), as well as other members of the ErbB family, may be useful as biomarkers and therapeutic targets in MGM.
Asunto(s)
Meningioma/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Biomarcadores de Tumor , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Meningioma/fisiopatología , Proteínas Oncogénicas v-erbB/genética , Proteínas Oncogénicas v-erbB/metabolismo , Fosforilación , Pronóstico , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor ErbB-2/metabolismo , Transducción de Señal , Tirosina/metabolismoRESUMEN
Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.
Asunto(s)
COVID-19/etiología , Receptores de Superficie Celular/fisiología , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/fisiología , Animales , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Superficie Celular/antagonistas & inhibidores , Internalización del Virus , Tirosina Quinasa del Receptor Axl , Tratamiento Farmacológico de COVID-19RESUMEN
Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer-/- mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45+ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.
Asunto(s)
Músculo Esquelético/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Regeneración/genética , Tirosina Quinasa c-Mer/fisiología , Animales , Supervivencia Celular/genética , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Noqueados , Desarrollo de Músculos/genética , Mioblastos/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa del Receptor AxlRESUMEN
The control of inflammation is strictly regulated to ensure the adequate intensity and duration of an inflammatory response, enabling the removal of the trigger factors and the restoration of the integrity of the tissues and their functions. This process is coordinated by anti-inflammatory and pro-resolving mediators that regulate the cellular and molecular events necessary to restore homeostasis, and defects in this control are associated with the development of chronic and autoimmune diseases. The TAM family of receptor tyrosine kinases-Tyro3, Axl, and MerTK-plays an essential role in efferocytosis, a key process for the resolution of inflammation. However, new studies have demonstrated that TAM receptor activation not only reduces the synthesis of pro-inflammatory mediators by different cell types in response to some stimuli but also stimulates the production of anti-inflammatory and pro-resolving molecules that control the inflammation. This review provides a comprehensive view of TAM receptor family members as important players in controlling inflammatory responses through anti-inflammatory and pro-resolving actions.
Asunto(s)
Inflamación , Proteínas Tirosina Quinasas Receptoras , Humanos , Inflamación/prevención & control , Proteínas Tirosina Quinasas Receptoras/fisiologíaRESUMEN
In Caenorhabditis elegans, axon regeneration is activated by a signaling cascade through the receptor tyrosine kinase (RTK) SVH-2. Axonal injury induces svh-2 gene expression by degradation of the Mad-like transcription factor MDL-1. In this study, we identify the svh-24/sdz-33 gene encoding a protein containing F-box and F-box-associated domains as a regulator of axon regeneration in motor neurons. We find that sdz-33 is required for axon injury-induced svh-2 expression. SDZ-33 targets MDL-1 for poly-ubiquitylation and degradation. Furthermore, we demonstrate that SDZ-33 promotes axotomy-induced nuclear degradation of MDL-1, resulting in the activation of svh-2 expression in animals. These results suggest that the F-box protein is required for RTK signaling in the control of axon regeneration.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury via the Ets-like transcription factor ETS-4, whose transcriptional activity is inhibited by the Mad-like transcription factor MDL-1. Axon injury leads to the degradation of MDL-1, and this is linked to the activation of ETS-4 transcriptional activity. In this study, we identify the sdz-33 gene encoding a protein containing an F-box domain as a regulator of axon regeneration. We demonstrate that MDL-1 is poly-ubiquitylated and degraded through the SDZ-33-mediated 26S proteasome pathway. These results reveal that an F-box protein promotes axon regeneration by degrading the Mad transcription factor.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas F-Box/fisiología , Regeneración Nerviosa/fisiología , Aminoácidos/metabolismo , Animales , Animales Modificados Genéticamente , Axones/fisiología , Axotomía , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al ADN/genética , Proteínas F-Box/genética , Neuronas Motoras/fisiología , Regeneración Nerviosa/genética , Plásmidos , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , UbiquitinaRESUMEN
The recent extensive spread of Zika virus has led to increased interest in the development of early diagnostic tests. To the best of our knowledge, this is the first study to demonstrate the successful use of phage display to identify affinity peptides for quantitative analysis of AXL, a tyrosine kinase receptor involved in Zika virus entry. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for AXL using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors.
Asunto(s)
Péptidos/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Virus Zika/fisiología , Secuencia de Aminoácidos , Línea Celular , Dicroismo Circular , Espectroscopía Dieléctrica , Ensayo de Inmunoadsorción Enzimática , Humanos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
Breast cancer was one of the first malignancies to benefit from targeted therapy, i.e., treatments directed against specific markers. Inhibitors against HER2 are a significant example and they improved the life expectancy of a large cohort of patients. Research on new biomarkers, therefore, is always current and important. AXL, a member of the TYRO-3, AXL and MER (TAM) subfamily, is, today, considered a predictive and prognostic biomarker in many tumor contexts, primarily breast cancer. Its oncogenic implications make it an ideal target for the development of new pharmacological agents; moreover, its recent role as immune-modulator makes AXL particularly attractive to researchers involved in the study of interactions between cancer and the tumor microenvironment (TME). All these peculiarities characterize AXL as compared to other members of the TAM family. In this review, we will illustrate the biological role played by AXL in breast tumor cells, highlighting its molecular and biological features, its involvement in tumor progression and its implication as a target in ongoing clinical trials.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Movimiento Celular/genética , Movimiento Celular/fisiología , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Terapia Molecular Dirigida/métodos , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Inhibidores de Proteínas Quinasas/uso terapéutico , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Microambiente Tumoral/genética , Microambiente Tumoral/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαiâ¢ßγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell's decision to "go" vs. "grow." These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Células COS , Chlorocebus aethiops , Receptores ErbB/metabolismo , Células HEK293 , Células HeLa , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Fosforilación , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal , Tirosina/metabolismoAsunto(s)
Moléculas de Adhesión Celular/antagonistas & inhibidores , Oncología Médica , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Moléculas de Adhesión Celular/fisiología , Desarrollo de Medicamentos/métodos , Desarrollo de Medicamentos/tendencias , Humanos , Oncología Médica/métodos , Oncología Médica/tendencias , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Proteínas Tirosina Quinasas Receptoras/fisiologíaRESUMEN
Osteosarcoma is the most common malignant bone tumor in adolescence and childhood. Metastatic osteosarcoma has a poor prognosis with an overall 5-year survival rate of approximately 20%. TAS-115 is a novel multiple receptor tyrosine kinase inhibitor that is currently undergoing clinical trials. Using the mouse highly lung-metastatic osteosarcoma cell line, LM8, we showed that TAS-115 suppressed the growth of subcutaneous grafted tumor and lung metastasis of osteosarcoma at least partially through the inhibition of platelet-derived growth factor receptor alpha, AXL, and Fms-like tyrosine kinase 3 phosphorylation. We also show that these signaling pathways are activated in various human osteosarcoma cell lines and are involved in proliferation. Our results suggest that TAS-115 may have potential for development into a novel treatment for metastatic osteosarcoma.
Asunto(s)
Osteosarcoma/metabolismo , Quinolinas/farmacología , Tiourea/análogos & derivados , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares , Ratones , Ratones Endogámicos C3H , Osteosarcoma/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinolinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Transducción de Señal/efectos de los fármacos , Tiourea/metabolismo , Tiourea/farmacología , Tirosina Quinasa 3 Similar a fms/metabolismo , Tirosina Quinasa 3 Similar a fms/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers due to its aggressive and metastatic nature. PDA is characterized by a rich tumor stroma with abundant macrophages, fibroblasts, and collagen deposition that can represent up to 90% of the tumor mass. Activation of the tyrosine kinase receptor AXL and expression of its ligand growth arrest-specific protein 6 (Gas6) correlate with a poor prognosis and increased metastasis in pancreatic cancer patients. Gas6 is a multifunctional protein that can be secreted by several cell types and regulates multiple processes, including cancer cell plasticity, angiogenesis, and immune cell functions. However, the role of Gas6 in pancreatic cancer metastasis has not been fully investigated. In these studies we find that, in pancreatic tumors, Gas6 is mainly produced by tumor associated macrophages (TAMs) and cancer associated fibroblasts (CAFs) and that pharmacological blockade of Gas6 signaling partially reverses epithelial-to-mesenchymal transition (EMT) of tumor cells and supports NK cell activation, thereby inhibiting pancreatic cancer metastasis. Our data suggest that Gas6 simultaneously acts on both the tumor cells and the NK cells to support pancreatic cancer metastasis. This study supports the rationale for targeting Gas6 in pancreatic cancer and use of NK cells as a potential biomarker for response to anti-Gas6 therapy.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Neoplasias Pancreáticas/patología , Animales , Fibroblastos Asociados al Cáncer/fisiología , Línea Celular Tumoral , Plasticidad de la Célula , Colágeno/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neovascularización Patológica/etiología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Macrófagos Asociados a Tumores/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Insulina/fisiología , Proteínas de la Membrana/fisiología , Proteínas ras/fisiología , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Receptores ErbB , Femenino , Edición Génica , Estudios de Asociación Genética , Longevidad/genética , Masculino , Proteínas de la Membrana/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Péptidos de Invertebrados , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Alas de Animales/crecimiento & desarrollo , Alas de Animales/ultraestructura , Proteínas ras/genéticaRESUMEN
This study was performed to investigate the role of PTK7 in esophageal squamous cell carcinoma (ESCC) stem-like cells (CSCs). PTK7 expression in ESCCs identified by RT-qPCR, and CSC-like cells were isolated from populations of NEC and TE-1 cells. The CSC-like cells were verified by flow cytometric analyses performed using CD34 and CD133 antibodies, and RT-qPCR and western blot assays were used to examine the self-renewal capability of CSC-like cells. CSC-like cells treated with PTK7 siRNA or a P53-specific inhibitor (PFTα) were analyzed for their sphere formation capacity and their apoptosis and migration/invasion capabilities by sphere formation, flow cytometry, and transwell assay, respectively. Their levels of P53, MKK3, and cleaved caspase 3 expression were examined by western blot analysis. Our results revealed that a majority of the isolated CSC-like cells were CD34+/CD133+ double positive cells. Nango, Sox2, and OCT4 were dramatically increased in the separated CSC-like cells, which had the pluripotency and self-renewal properties of stem cells. Additional, PTK7 was dramatically upregulated in the ESCC tissues and CSC-like cells. An investigation of the function of CSC-like cells revealed that knockdown of PTK7 reduced their sphere formation, promoted apoptosis, and suppressed their migration and invasion abilities, all of which could be significantly reversed by PFTα. Mechanistic studies showed that PFTα could attenuate the upregulation of P53, MKK3, and cleaved caspase 3 expression that was induced by PTK7 knockdown in CSC-like cells. PTK7 increased the malignant behaviors of CSC-like cells derived from ESCC cells by regulating p53. Therefore, this study suggests PTK7 as an underlying target for therapy against ESCC.
Asunto(s)
Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/fisiología , Neoplasias Esofágicas/patología , Células Madre Neoplásicas/patología , Proteínas Tirosina Quinasas Receptoras/fisiología , Línea Celular Tumoral , HumanosRESUMEN
A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.
Asunto(s)
Dominio AAA/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Dominio AAA/genética , Dominio Catalítico , Dimerización , Activación Enzimática , Humanos , Ligandos , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Tirosina Quinasas/fisiología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Tirosina/químicaRESUMEN
BACKGROUND AND AIMS: The survival benefit of sorafenib for patients with hepatocellular carcinoma (HCC) is unsatisfactory due to the development of adaptive resistance. Increasing evidence has demonstrated that drug resistance can be acquired by cancer cells by activating a number of signaling pathways through receptor tyrosine kinases (RTKs); nevertheless, the detailed mechanism for the activation of these alternative pathways is not fully understood. APPROACH AND RESULTS: Given the physiological role of Src homology 2 domain-containing phosphatase 2 (SHP2) as a downstream effector of many RTKs for activation of various signaling cascades, we first found that SHP2 was markedly up-regulated in our established sorafenib-resistant cell lines as well as patient-derived xenografts. Upon sorafenib treatment, adaptive resistance was acquired in HCC cells through activation of RTKs including AXL, epidermal growth factor receptor, EPH receptor A2, and insulin-like growth factor 1 receptor, leading to RAS/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), and AKT reactivation. We found that the SHP2 inhibitor SHP099 abrogated sorafenib resistance in HCC cell lines and organoid culture in vitro by blocking this negative feedback mechanism. Interestingly, this sensitization effect was also mediated by induction of cellular senescence. SHP099 in combination with sorafenib was highly efficacious in the treatment of xenografts and genetically engineered models of HCC. CONCLUSIONS: SHP2 blockade by SHP099 in combination with sorafenib attenuated the adaptive resistance to sorafenib by impeding RTK-induced reactivation of the MEK/ERK and AKT signaling pathways. SHP099 in combination with sorafenib may be a safe therapeutic strategy against HCC.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Piperidinas/administración & dosificación , Pirimidinas/administración & dosificación , Proteínas Tirosina Fosfatasas con Dominio SH2/antagonistas & inhibidores , Sorafenib/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Combinación de Medicamentos , Humanos , Piperidinas/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/fisiología , Sorafenib/farmacologíaRESUMEN
BACKGROUND Worldwide, hepatocellular carcinoma (HCC) accounts for 80-90% of all cases of primary liver cancer, and is one of the ten most common malignancies. This study used bioinformatics analysis to identify genes associated with patient outcome in stages I-IV HCC and the gene pathways that distinguished between normal liver and liver cells and HCC and human HCC cell lines. MATERIAL AND METHODS Target genes were defined as those that had marketed drugs or drugs under development targeting a specific gene and acquired from the Clarivate Analytics Integrity Database. Differential expression gene analysis, co-expression network analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, survival analysis and receiver operating characteristic (ROC) curve analysis were used to explore the similarities and differences in gene expression profiles, functional associations, and survival in stage I-IV HCC. Normal liver cells (HL-7702) and HCC cell lines (HepaRG, HepG2, SK-Hep1, and Huh7) were studied using Western blot and quantitative reverse transcription PCR (RT-qPCR). RESULTS Hierarchical gene clustering identified target genes that distinguished between HCC and normal liver tissue. For stages I-IV HCC, there were seven commonly upregulated target genes EPHB1, LTK, NTRK2, PTK7, TBK1, TIE1, and TLR3, which were mainly involved in immune and signaling transduction pathways. PTK7 was highly expressed in stage I-IV HCC and was an independent prognostic marker for reduced overall survival (OS). CONCLUSIONS Bioinformatics analysis, combined with patient survival analysis, identified PTK7 gene expression as a potential therapeutic target and prognostic biomarker for all stages of HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Moléculas de Adhesión Celular/genética , Biología Computacional/métodos , Proteínas Tirosina Quinasas Receptoras/genética , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/metabolismo , Moléculas de Adhesión Celular/fisiología , Línea Celular Tumoral , China , Análisis por Conglomerados , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Estadificación de Neoplasias , Pronóstico , Mapas de Interacción de Proteínas/genética , Curva ROC , Proteínas Tirosina Quinasas Receptoras/fisiología , Transcriptoma/genéticaRESUMEN
Gut-homing γδ T cells are induced by chemokines and cell adhesion molecules and play a critical role in homeostasis and mucosal immunity; however, little is known regarding their upstream regulators. We investigated the role of Axl as a specific regulator of chemokines and cell adhesion molecule in the distribution of intestinal γδ T cells. The population of γδ T-cell receptor-positive cells including Vγ1 and Vγ7 subsets was remarkably increased in the intraepithelial lymphocytes of Axl-/- mice compared with those of wild-type (WT) mice. An increased number of migrated γδ T cells were observed in the coculture with intraepithelial cells from Axl-/- mice. The mRNA expression level of chemokine (C-C motif) ligand (CCL) 25 was specifically higher in the small intestine of Axl-/- mice than in WT mice. In adoptive transfer, the migration of both thymic and extrathymic γδ T cells was increased in Axl-/- mice. The activation of Axl signaling down-regulated CCL25 expression via ERK signaling pathway and reduced the population of γδ T cells. Systemic dissemination was suppressed in Axl-/- mice infected with Salmonella typhimurium. Thus, our findings suggest that Axl plays a critical role in regulating the migration of γδ T cells for the maintenance of homeostasis and bacterial resistance.-Kim, S.-M., Park, M., Yee, S.-M., Ji, K.-Y., Lee, E.-H., Nguyen, T.-V., Nguyen, T. H.-L., Jang, J., Kim, E.-M., Choi, H.-R., Yun, C.-H., Kang, H.-S. Axl is a key regulator of intestinal γδ T-cell homeostasis.
Asunto(s)
Células Epiteliales/inmunología , Homeostasis , Intestino Delgado/inmunología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Fiebre Tifoidea/inmunología , Animales , Movimiento Celular , Células Cultivadas , Quimiocinas CC/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestino Delgado/metabolismo , Intestino Delgado/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Salmonella typhimurium/fisiología , Fiebre Tifoidea/metabolismo , Fiebre Tifoidea/microbiología , Tirosina Quinasa del Receptor AxlRESUMEN
Receptor tyrosine kinases (RTK) have been the most prevalent therapeutic targets in anti-cancer drug development. However, the emergence of drug resistance toward single target RTK inhibitors remains a major challenge to achieve long-term remissions. Development of alternative RTK inhibitory strategies that bypass drug resistance is much wanted. In the present study, we found that selected cell surface RTKs were inhibited and crosslinked into detergent resistant complexes by oligomeric but not monomeric concanavalin A (ConA). The inhibition of RTKs by ConA led to suppression of pro-survival pathways and induction of apoptosis in multiple cancer cell lines, while overexpression of constitutively activated protein kinase B (AKT) reversed the apoptotic effect. However, major cell stress sensing checkpoints were not influenced by ConA. To our knowledge, selective crosslinking and inhibition of cell surface receptors by ConA-like molecules might represent a previously unidentified mechanism that could be potentially exploited for therapeutic development.