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1.
J Biol Chem ; 298(12): 102622, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36272642

RESUMEN

Fibrosis, stiffening and scarring of an organ/tissue due to genetic abnormalities, environmental factors, infection, and/or injury, is responsible for > 40% of all deaths in the industrialized world, and to date, there is no cure for it despite extensive research and numerous clinical trials. Several biomarkers have been identified, but no effective therapeutic targets are available. Human galectin-3 is a chimeric gene product formed by the fusion of the internal domain of the collagen alpha gene [N-terminal domain (ND)] at the 5'-end of galectin-1 [C-terminal domain (CRD)] that appeared during evolution together with vertebrates. Due to the overlapping structural similarities between collagen and galectin-3 and their shared susceptibility to cleavage by matrix metalloproteases to generate circulating collagen-like peptides, this review will discuss present knowledge on the role of collagen and galectin-3 as biomarkers of fibrosis. We will also highlight the need for transformative approaches targeting both the ND and CRD domains of galectin-3, since glycoconjugate binding by the CRD is triggered by ND-mediated oligomerization and the therapies targeted only at the CRD have so far achieved limited success.


Asunto(s)
Colágeno , Fibrosis , Galectina 3 , Animales , Humanos , Biomarcadores , Colágeno/genética , Colágeno/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Vertebrados , Glicoconjugados , Metaloproteinasas de la Matriz
2.
Cancer Metastasis Rev ; 40(1): 297-302, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-32929561

RESUMEN

In the past decade, the development of immune checkpoint inhibitors in oncological clinical settings was in the forefront. However, the interest in musculoskeletal tumor patients as candidates for checkpoint inhibition remains underserved. Here, we are forwarding evidence proposing that galectin-3 (Gal-3) is an additional immune factor in the checkpoint processes. This review is the result of a large-scale cohort study depicting that overexpression of Gal-3 was widely prevalent in patients with musculoskeletal tumors, whereas T cell infiltrations were generally suppressed in the tumor microenvironment. Targeting Gal-3 would serve as a novel immune checkpoint inhibitor candidate in patients afflicted with aggressive musculoskeletal tumors.


Asunto(s)
Galectina 3 , Neoplasias , Estudios de Cohortes , Humanos , Inhibidores de Puntos de Control Inmunológico , Neoplasias/tratamiento farmacológico , Linfocitos T , Microambiente Tumoral
3.
J Transl Med ; 19(1): 459, 2021 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-34743703

RESUMEN

BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. METHODS: To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. RESULTS: Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. CONCLUSIONS: As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.


Asunto(s)
Neoplasias , Anticuerpos de Cadena Única , Antígenos CD28/genética , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , ARN Guía de Kinetoplastida , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T
4.
Biochim Biophys Acta ; 1833(5): 976-86, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23360980

RESUMEN

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using (32)P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating the C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the down-regulation of RKIP and MST2.


Asunto(s)
MAP Quinasa Quinasa 1/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Células COS , División Celular , Chlorocebus aethiops , Regulación hacia Abajo , Humanos , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo
5.
J Biol Chem ; 287(8): 5192-8, 2012 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-22232548

RESUMEN

Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer.


Asunto(s)
Galectina 3/química , Galectina 3/metabolismo , Antígeno Prostático Específico/metabolismo , Proteolisis , Tirosina/metabolismo , Línea Celular Tumoral , Quimiotaxis , Espacio Extracelular/metabolismo , Humanos , Modelos Moleculares , Neovascularización Patológica/metabolismo , Fosfohidrolasa PTEN/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-abl/metabolismo
6.
Biochem Biophys Res Commun ; 434(1): 155-61, 2013 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-23541576

RESUMEN

Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus-cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3-nucleus translocation rate. Overall, the results show Nup98's involvement in nuclear-cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.


Asunto(s)
Galectina 3/metabolismo , Proteínas de Complejo Poro Nuclear/fisiología , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte Activo de Núcleo Celular/genética , Proliferación Celular , Progresión de la Enfermedad , Células HeLa , Humanos , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patología , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/fisiología , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/genética , Transducción de Señal , beta Catenina/metabolismo
7.
Biochim Biophys Acta ; 1813(8): 1453-64, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21621563

RESUMEN

FoxO3 is a member of FoxO family transcription factors that mediate cellular functions downstream of AKT. FoxO3 phosphorylation by AKT generates binding sites for 14-3-3, which in-turn regulates FoxO3 transcriptional activity and localization. We examine here the functional significance of AKT-FoxO3 interaction and further detail the mechanistic aspects of FoxO3 regulation by AKT and 14-3-3. Our data show that AKT overexpression increases the steady-state levels of FoxO3 protein in a manner dependent on AKT activity and its ability to bind FoxO3. Characterization of the AKT-FoxO3 interaction shows that the three AKT phosphorylation-site-recognition motifs (RxRxxS/T) present on FoxO3, which are required for FoxO3 phosphorylation, are dispensable for AKT binding, suggesting that AKT has a docking point on FoxO3 distinct from the phosphorylation-recognition motifs. Development of a FoxO3 mutant deficient in 14-3-3 binding (P34A), which can be phosphorylated by AKT, established that 14-3-3 binding and not AKT phosphorylation per se controls FoxO3 transcriptional activity. Intriguingly, 14-3-3 binding was found to stabilize FoxO3 by inhibiting its dephosphorylation and degradation rates. Collectively, our data support a model where both AKT and 14-3-3 positively regulate FoxO3 in addition to their established negative roles and that 14-3-3 availability could dictate the fate of phosphorylated FoxO3 toward degradation or recycling.


Asunto(s)
Proteínas 14-3-3/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Sitios de Unión , Línea Celular , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Células Hep G2 , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Oncotarget ; 13: 534-550, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35309869

RESUMEN

The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.


Asunto(s)
Cadenas Pesadas de Miosina , Miosina Tipo IIA no Muscular , Proteínas del Citoesqueleto , ADN/genética , Desoxirribosa , Humanos , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Pentosas , Fosfatos , ARN Interferente Pequeño , Azúcares
9.
Biochim Biophys Acta ; 1803(10): 1198-205, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20600357

RESUMEN

Galectin-3, a beta-galactoside-binding lectin, is found in cellular and extracellular location of the cell and has pleiotropic biological functions such as cell growth, cell adhesion and cell-cell interaction. It may exhibit anti- or pro-apoptotic activity depending on its localization and post-translational modifications. Two important post-translational modifications of galectin-3 have been reported: its cleavage and phosphorylation. Cleavage of galectin-3 was reported to be involved with angiogenic potential and apoptotic resistance. Phosphorylation of galectin-3 regulates its sugar-binding ability. In this report we have identified novel tyrosine phosphorylation sites in galectin-3 as well as the kinase responsible for its phosphorylation. Our results demonstrate that tyrosines at positions 79, 107 and 118 can be phosphorylated in vitro and in vivo by c-Abl kinase. Tyrosine 107 is the main target of c-Abl. Expression of galectin-3 Y107F mutant in galectin-3 null SK-Br-3 cells leads to morphological changes and increased motility compared to wild type galectin-3. Further investigation is needed to better understand the functional significance of the novel tyrosine phosphorylated sites of galectin-3.


Asunto(s)
Galectina 3/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-abl/metabolismo , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Galectina 3/genética , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-abl/genética , Especificidad por Sustrato , Tirosina/genética , Tirosina/metabolismo
10.
Int J Cancer ; 127(11): 2530-41, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-20162566

RESUMEN

Galectin-3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin-3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)-2/-9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin-3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin-3 in induction of angiogenesis and (iii) determination of the galectin-3 domain responsible for induction of angiogenic response. Galectin-3 null breast cancer cells BT-459 were transfected with either cleavable full-length galectin-3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial-endothelial cell interactions and angiogenesis were compared to noncleavable galectin-3. BT-549-H(64) cells harboring cleavable galectin-3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT-549-P(64) cells harboring noncleavable galectin-3. BT-549-H(64) cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT-549 cells transfected with galectin-3 peptides indicate that amino acids 1-62 and 33-250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin-3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin-3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression.


Asunto(s)
Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Galectina 3/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Sustitución de Aminoácidos , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Quimiotaxis/fisiología , Técnicas de Cocultivo , Células Endoteliales/patología , Femenino , Galectina 3/genética , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Polimorfismo de Nucleótido Simple , Transfección
11.
Am J Pathol ; 174(4): 1515-23, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19286570

RESUMEN

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.


Asunto(s)
Biomarcadores de Tumor/análisis , Galectina 3/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Galectina 3/genética , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Methods Mol Biol ; 2055: 301-322, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31502158

RESUMEN

Recent advances in cancer immunotherapy have shed new light on the possibility to cure most, if not all, cancer patients with further development of various treatment options. The emergency of a new genome editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR) technology, revolutionized the biomedical research field. We envision application of the CRISPR technology in cancer research, diagnosis, and therapy will markedly speed up the development of new treatment options for cancer patients. The CRISPR system and its applications in biomedical research will be discussed with an emphasis on cancer immunotherapy and biomarker development.


Asunto(s)
Biomarcadores de Tumor/genética , Edición Génica/métodos , Neoplasias/genética , Sistemas CRISPR-Cas , Humanos , Inmunoterapia , Neoplasias/tratamiento farmacológico , Resultado del Tratamiento
13.
Methods Enzymol ; 635: 251-266, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32122549

RESUMEN

Emergency of the CRISPR technology, a new genome editing tool, revolutionized the biomedical research field in the past 6 years. At the same time, recent advances in cancer immunotherapy reinvigorated our hope to cure most if not all cancer patients with further development of various treatment options. A combination of the CRISPR technology with immuno-oncology research will undoubtedly accelerate the development of new cancer therapies. This review will focus on the CRISPR system and its applications in immune-oncology including identification of immune-oncology gene targets, generation of cancer animal models, and enabling better cell design and manufacture for adoptive cellular therapies.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias , Animales , Sistemas CRISPR-Cas/genética , Edición Génica , Humanos , Neoplasias/genética , Neoplasias/terapia , Tecnología
14.
Mol Biol Cell ; 17(3): 1141-53, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16407412

RESUMEN

The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Fosfo-Específicos/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Factor de Crecimiento Epidérmico/farmacología , Expresión Génica , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/genética , Serina/metabolismo
15.
Biochim Biophys Acta ; 1773(8): 1196-212, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17555829

RESUMEN

The Ras-Raf-MAPK pathway regulates diverse physiological processes by transmitting signals from membrane based receptors to various nuclear, cytoplasmic and membrane-bound targets, coordinating a large variety of cellular responses. Function of Raf family kinases has been shown to play a role during organism development, cell cycle regulation, cell proliferation and differentiation, cell survival and apoptosis and many other cellular and physiological processes. Aberrations along the Ras-Raf-MAPK pathway play an integral role in various biological processes concerning human health and disease. Overexpression or activation of the pathway components is a common indicator in proliferative diseases such as cancer and contributes to tumor initiation, progression and metastasis. In this review, we focus on the physiological roles of Raf kinases in normal and disease conditions, specifically cancer, and the current thoughts on Raf regulation.


Asunto(s)
Neoplasias/enzimología , Quinasas raf/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Humanos , Invertebrados , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias/etiología , Neoplasias/genética , Neoplasias/terapia , Oncogenes , Homología de Secuencia de Aminoácido , Transducción de Señal , Quinasas raf/antagonistas & inhibidores , Quinasas raf/química , Quinasas raf/genética
16.
Mol Biol Cell ; 16(10): 4733-44, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16093354

RESUMEN

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.


Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Factor de Crecimiento Epidérmico/fisiología , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Serina/metabolismo
17.
Cell Cycle ; 16(16): 1526-1533, 2017 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-28650766

RESUMEN

Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16(Ink4a)-positive cells from the tissues of mice was shown to extend a healthy lifespan. Here, we describe a population of mesenchymal cells isolated from mice that are highly p16(INK4a)-positive are proficient in proliferation but lack other properties of cellular senescence. These data, along with earlier reports on p16(Ink4a)-positive macrophages, indicate that p16(Ink4a)-positive and senescent cell populations only partially intersect, therefore, extending the list of potential cellular targets for anti- aging therapies.


Asunto(s)
Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Proliferación Celular , Humanos , Antígenos Comunes de Leucocito/metabolismo , Ratones Endogámicos C57BL
18.
Aging (Albany NY) ; 9(8): 1867-1884, 2017 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-28768895

RESUMEN

Constitutive p16Ink4a expression, along with senescence-associated ß-galactosidase (SAßG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAßG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAßG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAßG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAßG-positive macrophages.


Asunto(s)
Proliferación Celular , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Macrófagos Peritoneales/enzimología , beta-Galactosidasa/metabolismo , Tejido Adiposo/citología , Envejecimiento/metabolismo , Animales , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Genotipo , Factores Inmunológicos/farmacología , Activación de Macrófagos , Macrófagos Peritoneales/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Transducción de Señal , Factores de Tiempo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
19.
Aging (Albany NY) ; 8(7): 1294-315, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27391570

RESUMEN

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and ß-galactosidase activity at pH6.0 (ß-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/ß-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/ß-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/ß-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/ß-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.


Asunto(s)
Envejecimiento/metabolismo , Senescencia Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Macrófagos/metabolismo , beta-Galactosidasa/metabolismo , Animales , Liposomas/metabolismo , Ratones
20.
Oncotarget ; 6(23): 19592-604, 2015 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-26158764

RESUMEN

Galectin-3 (Gal-3, LGALS3) is a pleotropic versatile, 29-35 kDa chimeric gene product, and involved in diverse physiological and pathological processes, including cell growth, homeostasis, apoptosis, pre-mRNA splicing, cell-cell and cell-matrix adhesion, cellular polarity, motility, adhesion, activation, differentiation, transformation, signaling, regulation of innate/adaptive immunity, and angiogenesis. In multiple diseases, it was found that the level of circulating Gal-3 is markedly elevated, suggesting that Gal-3-dependent function is mediated by specific interaction with yet an unknown ubiquitous cell-surface protein. Recently, we showed that Gal-3 attenuated drug-induced apoptosis, which is one of the mechanisms underlying multidrug resistance (MDR). Here, we document that MDR could be mediated by Gal-3 interaction with the house-keeping gene product e.g., Na+/K+-ATPase, and P-glycoprotein (P-gp). Gal-3 interacts with Na+/K+-ATPase and induces the phosphorylation of P-gp. We also find that Gal-3 binds P-gp and enhances its ATPase activity. Furthermore Gal-3 antagonist suppresses this interaction and results in a decrease of the phosphorylation and the ATPase activity of P-gp, leading to an increased sensitivity to doxorubicin-mediated cell death. Taken together, these findings may explain the reported roles of Gal-3 in diverse diseases and suggest that a combined therapy of inhibitors of Na+/K+-ATPase and Gal-3, and a disease specific drug(s) might be superior to a single therapeutic modality.


Asunto(s)
Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Galectina 3/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Proteínas Sanguíneas , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Activación Enzimática , Femenino , Galectina 3/farmacología , Galectinas , Células HeLa , Humanos , Masculino , Neoplasias/genética , Neoplasias/patología , Fenotipo , Fosforilación , Unión Proteica , Proteómica/métodos , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/genética , Transfección , Familia-src Quinasas/metabolismo
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