Abstract
ABSTRACTDuring mitotic entry, centrosomes separate to establish the bipolar spindle. Delays in centrosome separation can perturb chromosome segregation and promote genetic instability. However, interphase centrosomes are physically tethered by a proteinaceous linker composed of C-Nap1 (also known as CEP250) and the filamentous protein rootletin. Linker disassembly occurs at the onset of mitosis in a process known as centrosome disjunction and is triggered by the Nek2-dependent phosphorylation of C-Nap1. However, the mechanistic consequences of C-Nap1 phosphorylation are unknown. Here, we demonstrate that Nek2 phosphorylates multiple residues within the C-terminal domain of C-Nap1 and, collectively, these phosphorylation events lead to loss of oligomerization and centrosome association. Mutations in non-phosphorylatable residues that make the domain more acidic are sufficient to release C-Nap1 from the centrosome, suggesting that it is an increase in overall negative charge that is required for this process. Importantly, phosphorylation of C-Nap1 also perturbs interaction with the core centriolar protein, Cep135, and interaction of endogenous C-Nap1 and Cep135 proteins is specifically lost in mitosis. We therefore propose that multisite phosphorylation of C-Nap1 by Nek2 perturbs both oligomerization and Cep135 interaction, and this precipitates centrosome disjunction at the onset of mitosis.
Highlights
Centrosomes are the primary microtubule-organizing centers in animal cells
Using a fluorescence recovery after photobleaching (FRAP) assay, we found that the bulk of C-Nap1 and rootletin remained stably associated with the centrosomes, with C-Nap1 exhibiting a small fraction (,10%) of turnover with a half-life (t1/2) of,30 seconds, but no recovery of rootletin (Fig. 1B,C)
We examined the localization of C-Nap1 and Cep135 in cells in which centrosomes were prematurely split in response to Nek2 overexpression or addition of epidermal growth factor (EGF), both of which induce centrosome splitting in a Nek2-dependent manner (Faragher and Fry, 2003; Mardin et al, 2013)
Summary
Centrosomes are the primary microtubule-organizing centers in animal cells. They are composed of centrioles, short cylinders of nine triplets of highly stabilized microtubules arranged into a pinwheel configuration and surrounded by pericentriolar material (PCM) from which microtubules are nucleated (Bornens, 2012; Doxsey et al, 2005). The two centrioles that are present in G1 are not identical; the older mother centriole has distal and sub-distal appendages, which the younger daughter centriole lacks. As cells progress into S and G2, each centriole is duplicated, with a new centriole, known as a procentriole, growing in close proximity to the proximal end of both mother and daughter centrioles. Many cancer cells possess more than two centrosomes, and considerable effort is being put into understanding the mechanisms that lead to the generation of these supernumerary or amplified centrosomes (Bettencourt-Dias and Glover, 2007; Nigg and Stearns, 2011)
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