RESUMEN
RATIONALE: Nkx2.5 is a transcription factor that regulates cardiomyogenesis in vivo and in embryonic stem cells. It is also a common target in congenital heart disease. Although Nkx2.5 has been implicated in the regulation of many cellular processes that ultimately contribute to cardiomyogenesis and morphogenesis of the mature heart, relatively little is known about how it is regulated at a functional level. OBJECTIVE: We have undertaken a proteomic screen to identify novel binding partners of Nkx2.5 during cardiomyogenic differentiation in an effort to better understand the regulation of its transcriptional activity. METHODS AND RESULTS: Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits protein phosphatase 1ß and myosin phosphatase targeting subunit 1 (Mypt1) as novel binding partners. The interaction with protein phosphatase 1 ß/Mypt1 resulted in exclusion of Nkx2.5 from the nucleus and, consequently, inhibition of its transcriptional activity. Exclusion of Nkx2.5 was inhibited by treatment with leptomycin B and was dependent on an Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 colocalized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signaling and Rho-associated protein kinase. Treatment of differentiating mouse embryonic stem cells with Wnt3a resulted in enhanced phosphorylation of endogenous Mypt1, increased nuclear exclusion of endogenous Nkx2.5, and a failure to undergo terminal cardiomyogenesis. Finally, knockdown of Mypt1 resulted in rescue of Wnt3a-mediated inhibition of cardiomyogenesis, indicating that Mypt1 is required for this process. CONCLUSIONS: We have identified a novel interaction between Nkx2.5 and myosin phosphatase. Promoting this interaction represents a novel mechanism whereby Wnt3a regulates Nkx2.5 and inhibits cardiomyogenesis.
Asunto(s)
Inhibidores de Crecimiento/fisiología , Proteínas de Homeodominio/metabolismo , Miocitos Cardíacos/fisiología , Fosfatasa de Miosina de Cadena Ligera/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiología , Proteína Wnt3A/fisiología , Quinasas Asociadas a rho/fisiología , Animales , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Células HEK293 , Proteína Homeótica Nkx-2.5 , Humanos , Ratones , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Proteína Fosfatasa 1/metabolismo , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
Our previous work has demonstrated that the Tudor domain of the 'survival of motor neuron' protein and the Tudor domain-containing protein 3 (TDRD3) are highly similar and that they both have the ability to interact with arginine-methylated polypeptides. TDRD3 has been identified among genes whose overexpression has a strong predictive value for poor prognosis of estrogen receptor-negative breast cancers, although its precise function remains unknown. TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain. We report here that TDRD3 localizes predominantly to the cytoplasm, where it co-sediments with the fragile X mental retardation protein on actively translating polyribosomes. We also demonstrate that TDRD3 accumulates into stress granules (SGs) in response to various cellular stresses. Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process. Pull down experiments identified five novel TDRD3 interacting partners, most of which are potentially methylated RNA-binding proteins. Our findings revealed that two of these proteins, SERPINE1 mRNA-binding protein 1 and DEAD/H box-3 (a gene often deleted in Sertoli-cell-only syndrome), are also novel constituents of cytoplasmic SGs. Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Gránulos Citoplasmáticos/química , Células HeLa , Humanos , Metilación , Datos de Secuencia Molecular , Polirribosomas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
KH-type splicing regulatory protein (KSRP) is closely related to chick zipcode-binding protein 2 and rat MARTA1, which are involved in neuronal transport and localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. KSRP is a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing and mRNA decay. More specifically, KSRP is an essential factor for targeting AU-rich element-containing mRNAs to the exosome. We report here that KSRP is arginine methylated and interacts with the Tudor domain of SMN, the causative gene for spinal muscular atrophy (SMA), in a CARM1 methylation-dependent fashion. These two proteins colocalize in granule-like foci in the neurites of differentiating neuronal cells and the CARM1 methyltransferase is required for normal localization of KSRP in neuronal cells. Strikingly, this interaction is abrogated by naturally-occurring Tudor domain mutations found in human patients affected with severe Type I SMA, a strong indication of its functional significance to the etiology of the disease. We also report for the first time that Q136E and I116F Tudor mutations behave similarly to the previously characterized E134K mutation, and cause loss of Tudor interactions with several cellular methylated proteins. Finally, we show that KSRP is misregulated in the absence of SMN, and this correlated with increased mRNA stability of its mRNA target, p21(cip1/waf1), in spinal cord of mild SMA model mice. Our results suggest SMN can act as a molecular chaperone for methylated proteins involved in RNA metabolism and provide new insights into the pathophysiology of SMA.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/química , Secuencia de Bases , Sitios de Unión , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Cartilla de ADN/genética , Regulación de la Expresión Génica , Humanos , Metilación , Ratones , Ratones Noqueados , Ratones Mutantes , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/etiología , Mutación , Proteínas del Tejido Nervioso/química , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/deficiencia , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas del Complejo SMN , Homología de Secuencia de Aminoácido , Proteína 1 para la Supervivencia de la Neurona Motora , Transactivadores/químicaRESUMEN
PRMT1 is the predominant member of a family of protein arginine methyltransferases (PRMTs) that have been implicated in various cellular processes, including transcription, RNA processing, and signal transduction. It was previously reported that the human PRMT1 pre-mRNA was alternatively spliced to yield three isoforms with distinct N-terminal sequences. Close inspection of the genomic organization in the 5'-end of the PRMT1 gene revealed that it can produce up to seven protein isoforms, all varying in their N-terminal domain. A detailed biochemical characterization of these variants revealed that unique N-terminal sequences can influence catalytic activity as well as substrate specificity. In addition, our results uncovered the presence of a functional nuclear export sequence in PRMT1v2. Finally, we find that the relative balance of PRMT1 isoforms is altered in breast cancer.
Asunto(s)
Empalme Alternativo/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Secuencia Conservada , Exones/genética , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genoma Humano/genética , Salud , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Leucina/genética , Leucina/metabolismo , Datos de Secuencia Molecular , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Class II histone deacetylases (HDAC4, HDAC5, HDAC7 and HDAC9) have been shown to interact with myocyte enhancer factors 2 (MEF2s) and play an important role in the repression of cardiac hypertrophy. We examined the role of HDACs during the differentiation of P19 embryonic carcinoma stem cells into cardiomyocytes. Treatment of aggregated P19 cells with the HDAC inhibitor trichostatin A induced the entry of mesodermal cells into the cardiac muscle lineage, shown by the upregulation of transcripts Nkx2-5, MEF2C, GATA4 and cardiac alpha-actin. Furthermore, the overexpression of HDAC4 inhibited cardiomyogenesis, shown by the downregulation of cardiac muscle gene expression. Class II HDAC activity is inhibited through phosphorylation by Ca2+/calmodulin-dependent kinase (CaMK). Expression of an activated CaMKIV in P19 cells upregulated the expression of Nkx2-5, GATA4 and MEF2C, enhanced cardiac muscle development, and activated a MEF2-responsive promoter. Moreover, inhibition of CaMK signaling downregulated GATA4 expression. Finally, P19 cells constitutively expressing a dominant-negative form of MEF2C, capable of binding class II HDACs, underwent cardiomyogenesis more efficiently than control cells, implying the relief of an inhibitor. Our results suggest that HDAC activity regulates the specification of mesoderm cells into cardiomyoblasts by inhibiting the expression of GATA4 and Nkx2-5 in a stem cell model system.