Abstract
Many proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown. Here we identify a mechanism that favours cilium disassembly and maintains the disassembled state. We show that co-localization of the S/G2 phase kinase, Nek2 and Kif24 triggers Kif24 phosphorylation, inhibiting cilia formation. We show that Kif24, a microtubule depolymerizing kinesin, is phosphorylated by Nek2, which stimulates its activity and prevents the outgrowth of cilia in proliferating cells, independent of Aurora A and HDAC6. Our data also suggest that cilium assembly and disassembly are in dynamic equilibrium, but Nek2 and Kif24 can shift the balance toward disassembly. Further, Nek2 and Kif24 are overexpressed in breast cancer cells, and ablation of these proteins restores ciliation in these cells, thereby reducing proliferation. Thus, Kif24 is a physiological substrate of Nek2, which regulates cilia disassembly through a concerted mechanism involving Kif24-mediated microtubule depolymerization.
Highlights
Many proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown
We performed in vitro kinase assays using glutathione S-transferase (GST)-tagged Kif[24] fragments produced in bacteria and flag-tagged WT or Nek2-KD purified from HEK293 cells, which confirmed that Kif[24] was directly phosphorylated by Nek[2] (Fig. 1c)
We previously reported that Kif[24] suppresses the aberrant growth of centriolar microtubules and primary cilia formation through its kinesin motif by showing that (1) abnormally long centrioles generated by Cep[97] depletion in U2OS cells were abolished by expression of active Kif[24], but not by a catalytically inactive (KEC) form, and (2) a purified fragment of Kif[24] containing the kinesin motif alone was able to depolymerize microtubules in vitro[21]
Summary
Many proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown. We show that Kif[24], a microtubule depolymerizing kinesin, is phosphorylated by Nek[2], which stimulates its activity and prevents the outgrowth of cilia in proliferating cells, independent of Aurora A and HDAC6. Nek[2] and Kif[24] are overexpressed in breast cancer cells, and ablation of these proteins restores ciliation in these cells, thereby reducing proliferation. The basal subtypes, which include triple-negative breast cancers, have been shown to ciliate, albeit with very low frequency[8,10] These cells are thought to originate from the myoepithelial layer of the mammary gland, which is highly ciliated in both mouse and human tissue[8,10,12]. Our results suggest the potential to target these enzymes in tumour cells
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