RESUMEN
Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. Renal cysts also arise in mice and frogs lacking Bicaudal C (BicC), a conserved RNA-binding protein containing K-homology (KH) domains and a sterile alpha motif (SAM). However, a role for BicC in cilia function has not been demonstrated. Here, we report that targeted inactivation of BicC randomizes left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore, depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain concentrates BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. These results suggest a model whereby BicC links the orientation of cilia with PCP, possibly by regulating RNA silencing in P-bodies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Tipificación del Cuerpo/fisiología , Proteínas Portadoras/metabolismo , Polaridad Celular , Cilios , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Portadoras/genética , Línea Celular , Cilios/metabolismo , Cilios/ultraestructura , Proteínas Dishevelled , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Nodal/genética , Proteína Nodal/metabolismo , Fosfoproteínas/genética , Interferencia de ARN , Proteínas de Unión al ARN , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus , Xenopus laevis/anatomía & histología , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1(-)(/-) knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.
Asunto(s)
Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Silenciador del Gen/fisiología , MicroARNs/genética , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/fisiopatología , Transducción de Señal/fisiología , Regiones no Traducidas 3'/genética , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas Portadoras/genética , Línea Celular , AMP Cíclico/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/fisiopatología , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genéticaRESUMEN
PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.