Figure 4.

Nek2A Binding, but Not Its Kinase Activity, Is Pre-required for Cep85 Phosphorylation by Plk1

(A) HEK293T cells were infected with indicated lentiviral shRNAs for 30 hr before co-transfection with FLAG-Cep85 and HA-Plk1. Cell lysates were prepared and subjected to immunoblotting 20 hr post-transfection. HA, hemagglutinin.

(B) HeLa cells were infected with indicated lentiviral shRNAs 20 hr before synchronization with a double thymidine block. After releasing, cells were harvested at indicated time points followed by immunoblotting (IB) with indicated antibodies.

(C–E) HeLa cells were infected with indicated lentiviral shRNAs for 36 hr before fixation for co-immunostaining with antibodies against γ-tubulin (red) and phosphorylated Cep85-Thr392 (green) (C). The boxed areas in (C) are shown at a higher magnification directly below the corresponding image. The relative fluorescent intensities of γ-tubulin and Cep85pT392 were quantitated and plotted in (D) and (E), respectively. In each group 40 cells were quantified. Data are mean ± SEM; ns, not significant; *p < 0.05; ****p < 0.0001; scale bar, 5 μm.

(F) The recombinant GST-Cep85 and immunoprecipitated kinases were subjected to in vitro kinase assays with [γ-³²P]-ATP. CBB, Coomassie brilliant blue stain; IB, immunoblotting.

(G) The recombinant GST-Cep85 was incubated with HEK293T cell lysates transfected with indicated constructs before in vitro kinase assays with [γ-³²P]-ATP. The mobility shift of Cep85 was visualized by CBB staining after prolonged separation (p.s.) of samples on an SDS-PAGE.

(H) GST-Cep85 was pre-bound with either active Nek2A-WT or kinase-dead Nek2A-K37R before in vitro kinase assays in the presence of either active Plk1-T210D (Plk1-TD) or inactive Plk1-K83M (Plk1-KM). After reaction, the samples were washed to remove unbound proteins before immunoblotting. Prolonged separation followed by CBB staining or western blot with anti-pT392 antibody was used to reveal phosphorylated Cep85. Western blot with anti-HA antibody was applied to detected Nek2A proteins remaining on Cep85 after the kinase reaction. The amount of retained Nek2A was quantitated and plotted in the lower panel. Data are mean ± SEM from three independent experiments, ****p < 0.0001.

(I) Schematic diagram of Nek2A deletion mutants are shown in the upper panel. GST-Cep85 was preincubated with cell lysates containing WT, N terminus (N), or C terminus (C) of Nek2A before in vitro kinase assays, followed by similar treatments as mentioned in (H). The intensity of Cep85pT392 and the amount of Nek2A proteins that remained on GST-Cep85 were quantitated and are shown below the representative western blots, respectively.

See also Figures S3 and S4.