title: Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells.
abstract: When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.
authors: Institute of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland.
authors: Michael H A
authors: MH
authors: Schmitz
authors: Michael
authors: M
authors: Held
authors: Veerle
authors: V
authors: Janssens
authors: James R A
authors: JR
authors: Hutchins
authors: Otto
authors: O
authors: Hudecz
authors: Elitsa
authors: E
authors: Ivanova
authors: Jozef
authors: J
authors: Goris
authors: Laura
authors: L
authors: Trinkle-Mulcahy
authors: Angus I
authors: AI
authors: Lamond
authors: Ina
authors: I
authors: Poser
authors: Anthony A
authors: AA
authors: Hyman
authors: Karl
authors: K
authors: Mechtler
authors: Jan-Michael
authors: JM
authors: Peters
authors: Daniel W
authors: DW
authors: Gerlich
mesh_terms: D048750:Cell Nucleus Division / Q000187:drug effects / Q000502:physiology; D002875:Chromosomes / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007399:Interphase / Q000502:physiology; D007976:Leupeptins / Q000494:pharmacology; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D010766:Phosphorylation / Q000502:physiology; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D008941:Spindle Apparatus / Q000378:metabolism; D014162:Transfection; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D005419:Flavonoids; D006657:Histones; C424166:KPNB1 protein, human; D007976:Leupeptins; C517191:PPP2R1A protein, human; C512974:PPP2R2A protein, human; D010880:Piperidines; D034741:RNA, Small Interfering; D028961:beta Karyopherins; C077990:alvocidib; D018844:Cyclin-Dependent Kinases; C517189:PPP2CA protein, human; D054648:Protein Phosphatase 2; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde