title: Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions.
abstract: Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division, signaling, differentiation, and metabolism. It is expressed in mammalian cells as three closely related isoforms: alpha, beta/delta, and gamma1. These isoforms differ in their relative affinities for proteins, termed targeting subunits, that mediate their intracellular localization and substrate specificity. Because of the dynamic nature of these interactions, it is important to find experimental approaches that permit direct analyses of PP1 localization and PP1-targeting subunit interactions in live cells. When transiently or stably expressed as fluorescent protein (FP) fusions, the three isoforms are active phosphatases with distinct localization patterns and can interact with both endogenous and exogenous targeting subunits. Their changing spatio-temporal distributions can be monitored both throughout the cell cycle and following cellular perturbations by time-lapse fluorescence microscopy, and turnover rates of intracellular pools of the protein calculated by fluorescence recovery after photobleaching (FRAP). Interactions with targeting subunits can be visualized in vivo by fluorescence resonance energy transfer (FRET), using techniques such as sensitized emission, acceptor photobleaching, or fluorescence lifetime imaging.
authors.affiliation: Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB Complex, University of Dundee, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Trinkle-Mulcahy
authors.forename: Janet
authors.initials: J
authors.lastname: Chusainow
authors.forename: Yun Wah
authors.initials: YW
authors.lastname: Lam
authors.forename: Sam
authors.initials: S
authors.lastname: Swift
authors.forename: Angus
authors.initials: A
authors.lastname: Lamond
mesh_terms: D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1