RESUMEN
Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.
Asunto(s)
Citoesqueleto de Actina/química , Actinas/química , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/química , Estrógenos/química , Invasividad Neoplásica , Animales , Células CACO-2 , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Perros , Estradiol/química , Femenino , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Ganglios Linfáticos/patología , Células MCF-7 , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos NOD , Metástasis de la Neoplasia , Transcripción GenéticaRESUMEN
Protein serine/threonine phosphatase 2A (PP2A) is a major cellular enzyme implicated in the control of a wide variety of biological processes. The predominant form of PP2A in cells is a heterotrimeric holoenzyme (ABC) consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although numerous signal transduction pathways are known to be regulated by PP2A, the identity of the PP2A holoenzymes controlling each pathway remains unclear. Studies aimed at elucidating substrates for individual PP2A holoenzymes have been hindered by the limited availability of purified endogenous holoenzymes and the inability to differentiate cellular roles of closely related PP2A holoenzymes. In this chapter, we describe a strategy for the functional expression of select FLAG-tagged regulatory B subunits in human embryonic kidney-293 cells and subsequent purification of PP2A holoenzymes containing the FLAG-tagged B subunit and endogenous A and C subunits (ABFLAGC). Biochemical analyses of the purified ABFLAGC holoenzymes reveal that they exhibit virtually indistinguishable specific activities and sensitivities to inhibitors as compared to the corresponding endogenous PP2A holoenzymes. The strategy described herein provides a straightforward method to purify individual PP2A holoenzymes from target mammalian cells for subsequent in vitro studies, as well as a powerful approach to identify cellular substrates and roles for each holoenzyme.
Asunto(s)
Holoenzimas/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Línea Celular , Expresión Génica , Holoenzimas/genética , Humanos , Immunoblotting , Inmunoprecipitación , Fosfoproteínas Fosfatasas/aislamiento & purificación , Proteína Fosfatasa 2 , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismoRESUMEN
High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2delta assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC(50)-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.
RESUMEN
MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.
Asunto(s)
Proteínas 14-3-3/metabolismo , MAP Quinasa Quinasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 3/metabolismo , Serina/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Catálisis , Línea Celular , Activación Enzimática/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Isoenzimas/metabolismo , MAP Quinasa Quinasa 6/metabolismo , MAP Quinasa Quinasa Quinasa 3/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , FN-kappa B/fisiología , Presión Osmótica , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Serina/genética , Transducción de Señal/genética , Treonina/genéticaRESUMEN
Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.
Asunto(s)
Alquilación , Inhibidores Enzimáticos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Biotina/análogos & derivados , Biotina/farmacología , Western Blotting , Catálisis , Línea Celular/efectos de los fármacos , Cisteína/química , Cisteína/metabolismo , Activación Enzimática/efectos de los fármacos , Holoenzimas/aislamiento & purificación , Holoenzimas/metabolismo , Humanos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/aislamiento & purificación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Conformación Proteica , Proteína Fosfatasa 2 , Transducción de Señal/genética , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Balpha/delta regulatory subunits by approximately 3-fold or knock-down of Balpha by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Balpha/delta subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Balpha knock-down cells but elevated in Balpha- and Bdelta-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Balpha or Bdelta. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABalphaC and ABdeltaC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABalphaC and ABdeltaC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , MAP Quinasa Quinasa 1/biosíntesis , MAP Quinasa Quinasa 2/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Proteína Quinasa 3 Activada por Mitógenos/biosíntesis , Fosfoproteínas Fosfatasas/química , Proteínas Proto-Oncogénicas c-raf/biosíntesis , Western Blotting , Dominio Catalítico , Línea Celular , Dimerización , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Epítopos/química , Humanos , Immunoblotting , Inmunoprecipitación , Luciferasas/metabolismo , Sistema de Señalización de MAP Quinasas , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Proteína Fosfatasa 2 , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/química , Transducción de Señal , Tetraciclina/farmacología , TransfecciónRESUMEN
A key regulator of many kinase cascades, heterotrimeric protein serine/threonine phosphatase 2A (PP2A), is composed of catalytic (C), scaffold (A), and variable regulatory subunits (B, B', B'' gene families). In neuronal PC12 cells, PP2A acts predominantly as a gatekeeper of extracellular signal-regulated kinase (ERK) activity, as shown by inducible RNA interference of the Aalpha scaffolding subunit and PP2A inhibition by okadaic acid. Although okadaic acid potentiates Akt/protein kinase B and ERK phosphorylation in response to epidermal, basic fibroblast, or nerve growth factor, silencing of Aalpha paradoxically has the opposite effect. Epidermal growth factor receptor Tyr phosphorylation was unchanged following Aalpha knockdown, suggesting that chronic Akt and ERK hyperphosphorylation leads to compensatory down-regulation of signaling molecules upstream of Ras and blunted growth factor responses. Inducible exchange of wild-type Aalpha with a mutant with selective B' subunit binding deficiency implicated PP2A/B' heterotrimers as Akt modulators. Conversely, silencing of the B-family regulatory subunits Balpha and Bdelta led to hyperactivation of ERK stimulated by constitutively active MEK1. In vitro dephosphorylation assays further support a role for Balpha and Bdelta in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs. Thus, receptor tyrosine kinase signaling cascades leading to Akt and ERK activation are modulated by PP2A holoenzymes with distinct regulatory properties.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sustancias de Crecimiento/metabolismo , Fosfoproteínas Fosfatasas/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Células Cultivadas , Dimerización , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Silenciador del Gen , Genes Reporteros , Immunoblotting , Sistema de Señalización de MAP Quinasas , Mutación , Factor de Crecimiento Nervioso/metabolismo , Ácido Ocadaico/farmacología , Células PC12 , Fosforilación , Proteína Fosfatasa 2 , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Ratas , Transducción de SeñalRESUMEN
Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Serina/metabolismo , Proteínas 14-3-3 , Células 3T3 , Animales , Células COS , Células Cultivadas , Humanos , Insectos/citología , MAP Quinasa Quinasa Quinasa 3 , Quinasas Quinasa Quinasa PAM/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.