RESUMEN
The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformación Proteica , Proteína Fosfatasa 1/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Calorimetría , Ensamble y Desensamble de Cromatina/fisiología , Clonación Molecular , Biología Computacional , Cristalización , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Dominios y Motivos de Interacción de Proteínas/genética , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (t(Thresh) and d(Thresh)) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Luz , Microscopía/métodos , Algoritmos , Análisis por Conglomerados , Simulación por Computador , Escherichia coli/citología , Colorantes Fluorescentes/metabolismo , Imagenología Tridimensional , CinéticaRESUMEN
Neurabin and spinophilin are neuronal scaffolding proteins that play important roles in the regulation of synaptic transmission through their ability to target protein phosphatase 1 (PP1) to dendritic spines where PP1 dephosphorylates and inactivates glutamate receptors. However, thus far, it is still unknown how neurabin and spinophilin themselves are targeted to these membrane receptors. Spinophilin and neurabin contain a single PDZ domain, a common protein-protein interaction recognition motif, which are 86% identical in sequence. We report the structures of both the neurabin and spinophilin PDZ domains determined using biomolecular NMR spectroscopy. These proteins form the canonical PDZ domain fold. However, despite their high degree of sequence identity, there are distinct and significant structural differences between them, especially between the peptide binding pockets. Using two-dimensional 1H-15N HSQC NMR analysis, we demonstrate that C-terminal peptide ligands derived from glutamatergic AMPA and NMDA receptors and cytosolic proteins directly and differentially bind spinophilin and neurabin PDZ domains. This peptide binding data also allowed us to classify the neurabin and spinophilin PDZ domains as the first identified neuronal hybrid class V PDZ domains, which are capable of binding both class I and II peptides. Finally, the ability to bind to glutamate receptor subunits suggests that the PDZ domains of neurabin and spinophilin are important for targeting PP1 to C-terminal phosphorylation sites in AMPA and NMDA receptor subunits.