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1.
Br J Cancer ; 109(4): 1063-71, 2013 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-23839489

RESUMEN

BACKGROUND: Dual specificity phosphatase 6 (DUSP6) is a member of a family of mitogen-activated protein kinase phosphatases that dephosphorylates and inhibits activated ERK1/2. Dual specificity phosphatase 6 is dynamically regulated in developmental and pathological conditions such as cancer. METHODS: Cancer cell lines were made deficient in DUSP6 by siRNA and shRNA silencing. Sensitivity to anti-EGFR and chemotherapeutic agents was determined in viability and apoptosis assays, and in xenografts established in SCID mice. Cellular effects of DUSP6 inactivation were analysed by proteomic methods, followed by analysis of markers of DNA damage response (DDR) and cell cycle. RESULTS: We determined that depletion of DUSP6 reduced the viability of cancer cell lines and increased the cytotoxicity of EGFR and other targeted inhibitors, and cytotoxic agents, in vitro and in vivo. Subsequent phosphoproteomic analysis indicated DUSP6 depletion significantly activated CHEK2 and p38, which function in the DDR pathway, and elevated levels of phosphorylated H2AX, ATM, and CHEK2, for the first time identifying a role for DUSP6 in regulating DDR. CONCLUSION: Our results provide a novel insight into the DUSP6 function in regulating genomic integrity and sensitivity to chemotherapy in cancer.


Asunto(s)
Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Fosfatasa 6 de Especificidad Dual/fisiología , Receptores ErbB/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Células HEK293 , Humanos , Etiquetado Corte-Fin in Situ , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones SCID , Fosforilación , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
2.
Br J Cancer ; 106(4): 748-55, 2012 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-22240781

RESUMEN

BACKGROUND: Increased Aurora kinase A gene copy number (AURKA-CN) has been reported in metastatic colorectal cancer (mCRC), with unknown relationship to clinical outcome. We correlated increased AURKA-CN in mCRC tumours with KRAS mutation status, overall and progression-free survival (OS, PFS). METHODS: Sixty-one mCRC tumours were analysed for AURKA-CN using q-PCR, and KRAS mutation status by direct sequencing. Expression of AURKA protein was analysed by immunohistochemistry. Cox-proportional hazard method, Kaplan-Meier curves and log-rank statistics were used to estimate and compare the hazard ratios and median survival between the groups. RESULTS: In all, 68% of tumour exhibited high AURKA-CN, and 29% had a KRAS mutation, without correlation between the two. Patients with high AURKA-CN tumours had longer median OS (48.6 vs 18.8 months, P=0.01), with stronger trend among KRAS wild-type tumours (median OS not reached vs 18.8 months, P=0.003). Progression-free survival was longer on first-line or second-line chemotherapy among patients with KRAS wild-type and high vs low AURKA-CN (first: 17.6 vs 5.13 months, P=0.04; second: 10.4 vs 5.1 months, P=0.01). AURKA-CN level did not affect outcomes among patients with KRAS mutant tumours. CONCLUSION: Increased AURKA-CN is common in mCRC tumours and is associated with longer OS and longer PFS during chemotherapy, particularly in KRAS wild-type tumours.


Asunto(s)
Neoplasias Colorrectales/genética , Dosificación de Gen , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Aurora Quinasa A , Aurora Quinasas , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia , Pronóstico , Proteínas Proto-Oncogénicas p21(ras)
3.
Trends Cell Biol ; 10(3): 111-9, 2000 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10675905

RESUMEN

Cellular morphology is determined by the organization of the intracellular actin cytoskeleton, which is influenced by external and internal cues. Focal adhesions are sites at which the actin cytoskeleton is linked to the extracellular matrix by integrin receptor complexes. In addition to providing structural tethering points for cells, integrin receptor complexes transduce signals that influence a broad range of cellular processes, including migration, proliferation, transformation and apoptosis. The Cas proteins (p130Cas, HEF1/Cas-L and Efs/Sin), a family of docking proteins containing multiple interaction domains, are important components of integrin receptor signalling and have been implicated in all of these processes.


Asunto(s)
Integrinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas , Transducción de Señal/fisiología , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Muerte Celular/fisiología , Movimiento Celular/fisiología , Tamaño de la Célula/fisiología , Transformación Celular Neoplásica/metabolismo , Proteína Sustrato Asociada a CrK , Citoesqueleto/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Proteína p130 Similar a la del Retinoblastoma
4.
Oncogene ; 26(33): 4825-32, 2007 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-17297447

RESUMEN

Human enhancer of invasion, clone 10 (HEI10) (CCNB1IP1) was first described as a RING-finger family ubiquitin ligase that regulates cell cycle by interacting with cyclin B and promoting its degradation. Subsequently, other studies suggested specific upregulation of HEI10 in metastatic melanoma and demonstrated direct interaction between HEI10 and the tumor suppressor Merlin, encoded by the neurofibromatosis 2 gene. These and other results led us to hypothesize that HEI10 also influences the processes of cell migration and metastasis. We here show that cells with depleted HEI10 both migrate more rapidly and invade more effectively than control cells. HEI10 depletion post-transcriptionally increases the expression of a group of promotility regulatory proteins including p130Cas, paxillin, Cdk1 and cyclin B2, but excluding Merlin. Among these, only inhibition of Cdk1/cyclin B activity specifically reversed the motility and invasion of HEI10-depleted cells. Finally, HEI10 is abundantly transcribed in many human tissues, and particularly abundant in some tumor cell lines, suggesting that it may be commonly involved in coordinating cell cycle with cell migration and invasion.


Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/fisiología , Ciclina B/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Cisplatino/farmacología , Proteína Sustrato Asociada a CrK/metabolismo , Citometría de Flujo , Humanos , Microscopía Fluorescente , Paxillin/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Roscovitina , Factores de Tiempo , Ubiquitina-Proteína Ligasas , Proteína de Unión al GTP cdc42/metabolismo
5.
Oncogene ; 25(32): 4389-98, 2006 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-16532029

RESUMEN

Merlin and ezrin are homologous proteins with opposite effects on neoplastic growth. Merlin is a tumor suppressor inactivated in the neurofibromatosis 2 disease, whereas upregulated ezrin expression is associated with increased malignancy. Merlin's tumor suppressor mechanism is not known, although participation in cell cycle regulation has been suggested. To characterize merlin's biological activities, we screened for molecules that would interact with merlin but not ezrin. We identified the cyclin B-binding protein and cell cycle regulator HEI10 as a novel merlin-binding partner. The interaction is mediated by the alpha-helical domain in merlin and the coiled-coil domain in HEI10 and requires conformational opening of merlin. The two proteins show partial subcellular colocalization, which depends on cell cycle stage and cell adhesion. Comparison of Schwann cells and schwannoma cultures demonstrated that the distribution of HEI10 depends on merlin expression. In transfected cells, a constitutively open merlin construct affected HEI10 protein integrity. These results link merlin to the cell cycle control machinery and may help to understand its tumor suppressor function.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Neurofibromina 2/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Humanos , Neurofibromina 2/biosíntesis , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Ratas , Ubiquitina-Proteína Ligasas
6.
Oncogene ; 25(12): 1721-32, 2006 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-16288224

RESUMEN

The highly invasive behavior of glioblastoma cells contributes to the morbidity and mortality associated with these tumors. The integrin-mediated adhesion and migration of glioblastoma cells on brain matrix proteins is enhanced by stimulation with growth factors, including platelet-derived growth factor (PDGF). As focal adhesion kinase (FAK), a nonreceptor cytoplasmic tyrosine kinase, has been shown to promote cell migration in various other cell types, we analysed its role in glioblastoma cell migration. Forced overexpression of FAK in serum-starved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in a twofold enhancement of basal migration and a ninefold enhancement of PDGF-BB-stimulated migration. Both expression of mutant FAK(397F) and the downregulation of FAK with small interfering (si) RNA inhibited basal and PDGF-stimulated migration. FAK overexpression and PDGF stimulation was found to increase the phosphorylation of the Crk-associated substrate (CAS) family member human enhancer of filamentation 1 (HEF1), but not p130CAS or Src-interacting protein (Sin)/Efs, although the levels of expression of these proteins was similar. Moreover downregulation of HEF1 with siRNA, but not p130CAS, inhibited basal and PDGF-stimulated migration. The phosphorylated HEF1 colocalized with vinculin and was associated almost exclusively with 0.1% Triton X-100 insoluble material, consistent with its signaling at focal adhesions. FAK overexpression promoted invasion through normal brain homogenate and siHEF1 inhibited this invasion. Results presented here suggest that HEF1 acts as a necessary and specific downstream effector of FAK in the invasive behavior of glioblastoma cells and may be an effective target for treatment of these tumors.


Asunto(s)
Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Quinasa 1 de Adhesión Focal/metabolismo , Glioblastoma/patología , Invasividad Neoplásica/patología , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Glioblastoma/metabolismo , Humanos , Immunoblotting , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Interferente Pequeño
7.
Mol Cell Biol ; 15(10): 5820-9, 1995 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-7565735

RESUMEN

Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Genes Reporteros , Saccharomyces cerevisiae/genética , Serina Endopeptidasas , Proteínas Bacterianas/genética , Secuencia de Bases , Genes Reporteros/genética , Secuencias Hélice-Asa-Hélice , Cinética , Leucina , Datos de Secuencia Molecular , Mutación , Regiones Operadoras Genéticas , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Activación Transcripcional , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales , beta-Galactosidasa/genética
8.
Mol Cell Biol ; 12(7): 3006-14, 1992 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-1620111

RESUMEN

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Regiones Operadoras Genéticas/fisiología , Proteínas Represoras/genética , Serina Endopeptidasas , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Transcripción Genética
9.
Mol Cell Biol ; 21(15): 5094-108, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11438665

RESUMEN

The dynamic regulation of focal adhesions is implicated in cellular processes of proliferation, differentiation, migration, and apoptosis. The focal adhesion-associated docking protein HEF1 is cleaved by caspases during both mitosis and apoptosis. Common to both of these cellular processes is the loss of focal adhesions, transiently during mitosis and permanently during apoptosis. The proteolytic processing of HEF1 during both mitosis and apoptosis therefore posits a general role for HEF1 as a sensor of altered adhesion states. In this study, we find that HEF1 undergoes proteolytic processing specifically in response to cellular detachment, while HEF1 proteolysis is prevented by specific integrin receptor ligation and focal adhesion formation. We show that overexpression of a C-terminal caspase-derived 28-kDa HEF1 peptide causes cellular rounding that is demonstrably separable from apoptosis. Mutation of the divergent helix-loop-helix motif found in 28-kDa HEF1 significantly reduces the induction of apoptosis by this peptide, while deletion of the amino-terminal 28 amino acids of 28-kDa HEF1 completely abrogates the induction of apoptosis. Conversely, these mutations have no effect on the rounding induced by 28-kDa HEF1. Finally, we detect a novel focal adhesion targeting domain located in the C terminus of HEF1 and show that this activity is necessary for HEF1-induced cell spreading. Together, these data suggest that proteolytic and other posttranslational modifications of HEF1 in response to loss of adhesion serve to modulate the disassembly of focal adhesions.


Asunto(s)
Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Clorometilcetonas de Aminoácidos/metabolismo , Secuencia de Aminoácidos , Apoptosis , Western Blotting , Inhibidores de Caspasas , Caspasas/metabolismo , Adhesión Celular , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular , Supervivencia Celular , Adhesiones Focales/metabolismo , Eliminación de Gen , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mitosis , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/química , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/genética , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transfección , Células Tumorales Cultivadas
10.
Mol Cell Biol ; 18(6): 3540-51, 1998 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9584194

RESUMEN

HEF1, p130(Cas), and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115(HEF1), p105(HEF1), p65(HEF1), and p55(HEF1). We show that p115(HEF1) and p105(HEF1) are different phosphorylation states of the full-length HEF1. p55(HEF1), however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105(HEF1) and p115(HEF1) being rapidly upregulated upon induction of cell growth, whereas p55(HEF1) is produced specifically at mitosis. While p105(HEF1) and p115(HEF1) are predominantly cytoplasmic and localize to focal adhesions, p55(HEF1) unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55(HEF1). In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.


Asunto(s)
Compartimento Celular , Ciclo Celular , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adhesión Celular , Proteínas de Ciclo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , ADN Complementario/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células HeLa , Humanos , Metiltransferasas/metabolismo , Mitosis , Fosfoproteínas/genética , Conformación Proteica , Huso Acromático/metabolismo , Transfección , Células Tumorales Cultivadas
11.
Mol Cell Biol ; 18(4): 1935-45, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9528765

RESUMEN

Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4- phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.


Asunto(s)
ARN Polimerasa II/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , Células HeLa , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Unión Proteica , Conformación Proteica , ARN Polimerasa II/genética
12.
Mol Cell Biol ; 20(14): 5184-95, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10866674

RESUMEN

HEF1 (human enhancer of filamentation 1) is a member of a docking protein family that includes p130(Cas) and Efs. Through assembly of multiple protein interactions at focal adhesion sites, these proteins activate signaling cascades in response to integrin receptor binding of the extracellular matrix. The HEF1 protein is cell cycle regulated, with full-length forms cleaved in mitosis at a caspase consensus site to generate an amino-terminal 55-kDa form that localizes to the mitotic spindle. The identification of a caspase cleavage site in HEF1 led us to investigate whether HEF1 belongs to a select group of caspase substrates cleaved in apoptosis to promote the morphological changes characteristic of programmed cell death. Significantly, inducing expression of HEF1 in MCF-7 or HeLa cells causes extensive apoptosis, as assessed by multiple criteria. Endogenous HEF1 is cleaved into 65- and 55-kDa fragments and a newly detected 28-kDa form in response to the induction of apoptosis, paralleling cleavage of poly(ADP-ribose) polymerase and focal adhesion kinase (FAK); the death-promoting activity of over-expressed HEF1 is associated with production of the 28-kDa form. While the generation of the cleaved HEF1 forms is caspase dependent, the accumulation of HEF1 forms is further regulated by the proteasome, as the proteasome inhibitors N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl and lactacystin enhance their stability. Finally, the induction of HEF1 expression also increases Jun N-terminal protein kinase (JNK) activation, and activated JNK colocalizes with HEF1, implicating this pathway in HEF1 action. Based on these results, we propose that dysregulation of HEF1 and its family members along with FAK may signal the destruction of focal adhesion sites and regulate the onset of apoptosis.


Asunto(s)
Apoptosis/fisiología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasa 7 , Inhibidores de Caspasas , Caspasas/metabolismo , Muerte Celular/fisiología , Proteína Sustrato Asociada a CrK , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Leupeptinas/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis , Complejos Multienzimáticos/metabolismo , Mutación , Oligopéptidos/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Isoformas de Proteínas/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Transducción de Señal , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología
13.
Mol Cell Biol ; 16(7): 3327-37, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8668148

RESUMEN

Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domain-containing protein that is similar in structure to pl30cas, a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130cas, the expression of HEF1 appears to be tissue specific. Further, whereas p130cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130cas, as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130cas, transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Clonación Molecular , Proteína Sustrato Asociada a CrK , Cartilla de ADN , ADN Complementario , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Biblioteca de Genes , Células HeLa , Humanos , Mamíferos , Metionina , Datos de Secuencia Molecular , Oncogenes , Fosfoproteínas/biosíntesis , Fosfoproteínas/química , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Homología de Secuencia de Aminoácido , Tirosina
14.
Mol Cell Biol ; 15(4): 2304-10, 1995 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-7891724

RESUMEN

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Activación Enzimática , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad
15.
Mol Biol Cell ; 6(7): 759-75, 1995 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-7579693

RESUMEN

Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS.


Asunto(s)
ARN Polimerasa II/fisiología , Saccharomyces cerevisiae/citología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Respuesta al Choque Térmico , Humanos , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos , ARN Polimerasa II/química , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Temperatura
16.
Oncogene ; 35(13): 1643-56, 2016 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-26165839

RESUMEN

Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, a systems biology approach was used. A library of small interfering RNAs targeting an estrogen receptor (ER)- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent ER-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mammalian target of rapmycin (mTOR) inhibitors in estrogen-independent cells but not in estrogen-dependent cells. Phosphoproteomic profiles from reverse-phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with ER-positive breast cancers.


Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular/genética , Resistencia a Antineoplásicos/genética , Estrógenos/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Supresoras de Tumor/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Supresoras de Tumor/metabolismo
17.
Oncogene ; 15(9): 1043-9, 1997 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-9285558

RESUMEN

Krev-1/rap1A is an evolutionarily conserved Ras-family GTPase whose cellular function remains unclear, but which has been proposed to function as a tumor suppressor gene, and may act as a Ras antagonist. To elucidate Krev-1 activity, we have used LexA-Krev-1 in a two-hybrid screen of a HeLa cell cDNA library. Of the two cDNA classes isolated, one contained a single isolate encoding the known Krev-1 interactor Raf, while the second contained multiple isolates coding for a previously undescribed protein which we have designated Kritl (for Krev Interaction Trapped 1). The full length Krit1 cDNA encodes a protein of 529 amino acids, with an amino-terminal ankyrin repeat domain and a novel carboxy-terminal domain required for association with Krit1. Krit1 interacted strongly with Krev-1 but only weakly with Ras, suggesting it might specifically regulate Krev-1 activities. Krit1 mRNA and protein are expressed endogenously at low levels, with tissue specific variation. Intriguingly, the Krit cDNA has been mapped by FISH to chromosome 7q21-22, a region known to be frequently deleted or amplified in multiple forms of cancer.


Asunto(s)
Repetición de Anquirina , Cromosomas Humanos Par 7 , Proteínas de Unión al GTP/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteína KRIT1 , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas de Unión al GTP rap
18.
Oncogene ; 18(35): 4891-8, 1999 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-10490823

RESUMEN

AKT2 is a serine/threonine kinase implicated in human ovarian and pancreatic cancers. AKT2 is activated by a variety of growth factors and insulin via phosphatidylinositol 3-kinase (PI3K). However, its normal cellular role is not well understood. To gain insight into the function of AKT2, we performed yeast two-hybrid system to screen for interacting proteins. Using this technique, we identified a novel interactor, designated APPL, which contains a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a leucine zipper, classes of motifs defined in signaling molecules as functional interaction domains with specific targets. The PH domain of APPL shows similarity to those found in GTPase-activating proteins such as oligophrenin-1 and Graf, whereas its PTB domain exhibits homology with CED-6, an adaptor protein that promotes engulfment of apoptotic cells, and IB1, a transactivator of the GLUT2 gene. APPL is highly expressed in skeletal muscle, heart, ovary and pancreas, tissues in which AKT2 mRNA is abundant. APPL interacts with the inactive form of AKT2; moreover, APPL binds to the PI3K catalytic subunit, p110alpha. These data suggest that APPL is an adaptor that may tether inactive AKT2 to p110alpha in the cytoplasm and thereby may expedite recruitment of AKT2 and p110alpha to the cell membrane upon mitogenic stimulation. Furthermore, the APPL gene was mapped to human chromosome 3p14.3-p21.1, where deletions and other rearrangements have often been reported in a variety of tumor types. The identification of APPL may facilitate further analysis of the physiological and oncogenic activities of AKT2.


Asunto(s)
Proteínas Portadoras/metabolismo , Cromosomas Humanos Par 3/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Activación Enzimática , Femenino , Humanos , Leucina Zippers , Datos de Secuencia Molecular , Neoplasias Ováricas , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotirosina/metabolismo , Mapeo Físico de Cromosoma , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Células Tumorales Cultivadas , Levaduras/genética , Levaduras/metabolismo
19.
Oncogene ; 17(5): 603-10, 1998 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-9704926

RESUMEN

As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast two-hybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that specifically interacts with the amino terminus of EWS. This association was confirmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 specifically increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may influence promoter selectivity.


Asunto(s)
Gelsolina , Proteínas de Fusión Oncogénica/metabolismo , ARN Polimerasa II/metabolismo , Receptores Citoplasmáticos y Nucleares , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Fusión Artificial Génica , Sitios de Unión , Clonación Molecular , Expresión Génica , Humanos , Ratones , Proteínas de Microfilamentos , Hibridación de Ácido Nucleico , Proteínas de Fusión Oncogénica/genética , Proteínas/genética , Proteína Proto-Oncogénica c-fli-1 , ARN Polimerasa II/química , ARN Polimerasa II/genética , Proteína EWS de Unión a ARN , Saccharomyces cerevisiae , Sarcoma de Ewing , Transactivadores , Factores de Transcripción/genética , Activación Transcripcional , Células Tumorales Cultivadas
20.
Oncogene ; 12(9): 1921-9, 1996 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-8649853

RESUMEN

Arg and c-Abl represent the mammalian members of the Abelson family of nonreceptor protein-tyrosine kinases. To gain insight into the biological role of Arg we used the two-hybrid approach to identify interacting proteins. Using a C-terminal segment of Arg we identified a novel protein, ArgBP1 (Arg binding protein 1). ArgBP1 contains a C-terminal SH3 domain, several PEST sequences, a serine rich domain and an SH3 binding site. ArgBP1 is ubiquitously expressed as two transcripts of approximately 2.2 kb and approximately 8 kb with highest levels in brain, heart and testis. The association of ArgBP1 with Arg in living cells was confirmed by coimmunoprecipitation in cotransfected COS cells. Analysis of the mechanism of association indicated that the ArgBP1 SH3 domain binds to a C-terminal Arg SH3-binding site, and that an N-terminal ArgBP1 proline-rich sequence binds to the Arg SH3 domain. Immunostaining indicated that the subcellular localization of ArgBP1 is cytoplasmic. The similarity of the ArgBP1 expression pattern and subcellular localization to those of Arg and the potential for a highly specific and potentially strong association mediated by two pairs of SH3 domain/proline-rich motif interactions, suggest that ArgBP1 is likely to be a regulator and/or effector of Arg function.


Asunto(s)
Proteínas Portadoras/genética , Sistema Nervioso Central/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Secuencia de Bases , Proteínas Portadoras/metabolismo , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Unión Proteica , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Xenopus/genética
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