title: Disruption of clathrin-mediated trafficking causes centrosome overduplication and senescence.
abstract: The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.
authors.affiliation: Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Maciej B
authors.initials: MB
authors.lastname: Olszewski
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Chandris
authors.forename: Bum-Chan
authors.initials: BC
authors.lastname: Park
authors.forename: Evan
authors.initials: E
authors.lastname: Eisenberg
authors.forename: Lois E
authors.initials: LE
authors.lastname: Greene
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016922:Cellular Senescence; D018385:Centrosome / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004705:Endocytosis; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D010641:Phenotype; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism
chemical_list: D033962:Adaptor Protein Complex 2; D002966:Clathrin; C534962:GAK protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt
keywords: DNA damage; centrosome; clathrin; endocytosis; overduplication; senescence; trafficking