NEK2

Cilia effects upon perturbation of NEK2

Cilia number / % ciliated:

Incrased cilia number

Loss-of-function effect:

Longer cilia

Overexpression effect:

Shorter cilia

Ciliogenesis screen results (4 screens)

• Kim2016: Not Reported
• Wheway et al. 2015 (siRNA) [siRNA]: No effect PMID:26167766
• Breslow et al. 2018 (CRISPR) [CRISPR]: No Significant Effect PMID:29459680
• Roosing et al. 2015 (siRNA) [siRNA]: No effect PMID:26595381

Phenotypes

Mouse phenotype:

abnormal locomotor behavior, increased fasting circulating glucose level, enlarged lymph nodes

Mouse ciliopathy phenotype:

abnormal auditory brainstem response, increased heart weight, abnormal kidney morphology

Human ciliopathy phenotype:

retinitis pigmentosa

Subcellular localization

basal body, centrosome, cilia, cytosol, microtubules, mother centriole, nucleus

Functional category

• Ciliary assembly/disassembly
• Trafficking (BBSome, small GTPases, vesicular transport, ATPases)
• Actin & cytoskeleton regulation
• Protein processing & maturation

Function

Phosphorylates the centrosomal linker proteins C- p1, Cep68 and Rootletin to promote centriole separation, and is involved in the development of left-right asymmetry by regulating balancing ciliogenesis and resorption (26493400). Phosphorylates Kif24, a depolymerising kinesin (26290419). Influences centrosome-anchored organisation and promotes cilium disassembly at the onset of mitosis (22613497). NEK2 removes distal appendage components from the mother centriole, preventing cilia mainte nce during mitosis (32211891)., Furthermore, siRNA-mediated depletion of Nek2 in hTERT-RPE1 cells has been reported to reduce cilium formation (Graser et al., 2007, Mikule et al., 2007). These findings raise the possibility that Nek2 has a ciliary function. In addition, we report that siRNA-mediated depletion of Nek2 compromises the ability of cells to resorb primary cilia before the onset of mitosis, while overexpression of catalytically active Nek2A reduces ciliation and cilium length in serum-starved cells. Based on these findings, we propose that Nek2 has a role in promoting cilium disassembly at the onset of mitosis. We also present evidence that recruitment of Nek2 to the proximal ends of centrioles is dependent on one of its substrates, the centrosome cohesion protein C-Nap1. Consistent with our previous studies, we observed a dramatic increase in ciliation in cycling RPE1 cells depleted of Kif24, as compared with controls (Fig. 3a and Supplementary Fig. 4A). In addition, we found that depletion of Nek2 mimicked silencing of Kif24 (Fig. 3a and Supplementary Fig. 4A). However, co-silencing of Nek2 and Kif24 did not further increase the frequency of ciliation as compared with individual siRNA treatments, suggesting that they could work in a coordinated manner. Further, we stably expressed each protein (alone or together) in RPE1 cells and provoked quiescence through serum starvation. Since endogenous levels of Nek2 and Kif24 are negligible after serum starvation (Supplementary Fig. 3C), this allowed us to test the impact of altering the levels of each protein while limiting the expression of its partner (Fig. 3b). We found that ciliation was significantly diminished on expression of Nek2 or Kif24, and this effect was considerably more pronounced in cells co-expressing both proteins (Fig. 3b). The effect of Nek2 was dependent on its kinase activity, since expression of an inactive form of Nek2 (KD Nek2) overrode the effect of Kif24 expression (compare Fig. 4e,f and Supplementary Fig. 5D,E). Of note, cellular quiescence was not impaired in cells expressing Kif24 and/or Nek2, suggesting that reduced ciliation after ectopic expression did not result from cell cycle perturbations (Supplementary Fig. 4B). We next tested the impact of depleting Nek2 or Kif24 in cells ectopically expressing its partner. (26290419)