title: Phosphorylation of filamin A by Cdk1 regulates filamin A localization and daughter cell separation.
abstract: In cell culture, many adherent mammalian cells undergo substantial actin cytoskeleton rearrangement prior to mitosis as they detach from the extracellular matrix and become spherical. At the end of mitosis, the actin cytoskeleton is required for cytokinesis and the reassembly of interphase structures as cells spread and reattach to substrate. To understand the processes regulating mitotic cytoskeletal remodeling, we studied how mitotic phosphorylation regulates filamin A (FLNa). FLNa is an actin-crosslinking protein that was previously identified as a cyclin-dependent kinase 1 (Cdk1) binding partner and substrate in vitro. Using quantitative label-based mass spectrometry, we find that FLNa serines 1084, 1459 and 1533 are phosphorylated in mitotic HeLa cells and all three sites match the phosphorylation consensus sequence of Cdk1. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine residues and expressed GFP-tagged FLNa(S1084A,S1459A,S1533A) (FLNa-AAA GFP) in a FLNa-deficient human melanoma cell line called M2. M2 cells expressing FLNa-AAA GFP have enhanced FLNa-AAA GFP and actin localization at sites of contact between daughter cells, impaired post-mitotic daughter cell separation and defects in cell migration. Therefore, mitotic phosphorylation of FLNa is important for successful cell division and interphase cell behavior.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, Canada ON K1H 8M5. Electronic address: sandy.gy.szeto@gmail.com.
authors.forename: Sandy G Y
authors.initials: SGY
authors.lastname: Szeto
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, Canada ON K1H 8M5; Ottawa Institute of Systems Biology, Faculty of Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, Canada ON K1H 8M5. Electronic address: williams.liz.c@gmail.com.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Williams
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, Canada ON K1H 8M5; Ottawa Institute of Systems Biology, Faculty of Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, Canada ON K1H 8M5. Electronic address: arudner@uottawa.ca.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Rudner
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, Canada ON K1H 8M5. Electronic address: jlee@uottawa.ca.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Lee
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000378:metabolism; D016203:CDC2 Protein Kinase; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D056744:Cyclin B1 / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D048749:Cytokinesis / Q000502:physiology; D064448:Filamins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008545:Melanoma; D008938:Mitosis / Q000502:physiology*; D009154:Mutation; D010766:Phosphorylation; D011554:Pseudopodia / Q000502:physiology
chemical_list: D000199:Actins; D056744:Cyclin B1; D064448:Filamins; D049452:Green Fluorescent Proteins; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases
keywords: Actin cytoskeleton remodeling; Cyclin-dependent kinase 1; Filamin A; Mitosis; Mitotic cell rounding