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 phosphosylation 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 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.

Genetic perspective on the role of the autophagy-lysosome pathway in Parkinson disease

“…In another model, it was demonstrated that knockout of GAK results in destabilization of the lysosomal membrane and leakage of iron, causing DNA damage. 28 Genetic markers around GPNMB were identified and replicated in several GWASs, with approximately 10% risk reduction. 7,9 Interestingly, GPNMB is an important gene in melanoma, 187 a skin malignancy that is associated with PD.…”

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase

“…5 Thus, GAK is essential for cell growth because GAK knockout (GAK ¡/¡ ) mice display embryonic lethality 6 and mouse embryonic fibroblasts (MEFs) derived from GAK ¡/¡ mice fail to divide and ultimately become senescent. 7 This phenotype is independent of the Ser/Thr kinase activity of GAK because the cell cycle of MEFs derived from knockout mice expressing a kinase-dead form of GAK is normal. 8 GAK is overexpressed in nuclei of various cancer cells and the GAK expression level is positively correlated with malignancy in surgical specimens from prostate cancer patients.…”

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain

“…For adenoviral infection, cells plated 1 day before infection were incubated in fresh DMEM culture medium with adenovirus expressing Cre recombinase (Ad-Cre; Vector Biolabs) at a multiplicity of infection (MOI) of 200 in the presence of 5 µg/ml polybrene (Sigma-Aldrich), as described previously (Olszewski et al, 2014). The stable cell lines were treated with adenovirus as described above to knockout the endogenous GAK.…”