Skip to main content
. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Nat Cell Biol. 2014 Dec 15;17(1):31–43. doi: 10.1038/ncb3076

Figure 2. PLK1 phosphorylates Cep68 on Ser332 to promote βTrCP-mediated Cep68 degradation.

Figure 2

a) Ser332 is necessary for Cep68 degradation in prometaphase. HeLa cells expressing inducible FLAG-tagged Cep68, Cep68(Δ331-337), or Cep68(S332A) were synchronized by double-thymidine arrest. Cells were released and harvested at the G1/S transition, in G2 phase (8 hours after release), or in prometaphase (PM). Cell lysates were immunoblotted as indicated.

b) PLK1 inhibition prevents the degradation of endogenous Cep68. HeLa cells were synchronized by double-thymidine arrest, treated with nocodazole seven hours after release, and incubated for a further five hours to allow cells to enter prometaphase (PM). Where indicated, cells were treated with PLK1 inhibitor (BI2536). Cep68 was immunoprecipitated from cell lysates and immunoblotted as indicated. Cep68 interacts with Cep215 both in asynchronous cells and when Cep68 is stabilized by PLK1 inhibition.

c) PLK1 depletion, but not Nek2 depletion, prevents the degradation of Cep68. HeLa cells expressing inducible FLAG-Cep68 were transfected with siRNAs to βTrCP, Nek2, or PLK1. Cells in prometaphase were harvested and imunoblotted as indicated.

d) PLK1 inhibition prevents phosphorylation of Cep68 on Ser332 in vivo. HeLa cells expressing either inducible FLAG-Cep68 or FLAG-Cep68(S332A) were released from a double-thymidine arrest. Seven hours after release, cells were treated with nocodazole and, where indicated, either BI2536 (a PLK1 inhibitor) or MLN4924 (a CRL inhibitor). Cells were then harvested at the indicated time points. Cep68 or Cep68(S332A) were immunoprecipitated from cell lysates using an anti-FLAG resin. Whole cell lysates (WCL) and immunoprecipitates were immunoblotted as indicated.