Nek2A mediates G2/M phosphorylation of GAS2L1. (A) GAS2L1 (Bio-FLAG–tagged) was coexpressed with a kinase-dead mutant of GFP-Nek2A (K37R) in HEK293T cells. After pull-down of ectopically expressed GAS2L1, pull-down samples (50%) and cell-extract inputs (5%) were immunoblotted with anti-FLAG and anti-GFP. (B) FLAG-GAS2L1 was transiently expressed with GFP-Nek2A or the K37R mutant in HEK293T cells, and then cell extracts were immunoblotted. An aliquot of the extract from cells coexpressing FLAG-GAS2L1 and GFP-Nek2A was treated with calf intestinal alkaline phosphatase before immunoblotting. (C) Purified GFP-GAS2L1 and S352A mutant proteins were phosphorylated in vitro with GST-Nek2A and then immunoblotted for GAS2L1, pSer352-GAS2L1, and Nek2A. (D) RPE-1 cells were synchronized with aphidicolin and then released into STLC-containing medium. Cells were collected at indicated time points and analyzed by immunoblotting. (E) RPE-1 cells transfected with control siRNA (si-Control) or nek2-targeting siRNA (si-Nek2) were arrested with aphidicolin and then released into STLC-containing medium for 12 h. Cell extracts were immunoblotted with indicated antibodies. Intensity of Ser352-phosphorylated GAS2L1 was measured in GAS2L1 doublets and normalized and is presented as means ± SD from three independent experiments. Unpaired Student’s t test; **, P < 0.001. (F) HEK293T cells were transfected with GAS2L1 constructs and Nek2A WT or kinase-dead mutant K37R. After anti-FLAG immunoprecipitation, immunoprecipitates (50%) and lysate inputs (1%) were immunoblotted with anti-FLAG and anti-GFP antibodies. Anti-pSer352-GAS2L1 immunoblotting was also performed on lysate inputs. IP, immunoprecipitation. The coimmunoprecipitated CH domain was quantified and normalized and is presented as means ± SD from three independent experiments. Unpaired Student’s t test; ***, P < 0.0001. (G) GFP-GAS2L1 or the S352D mutant was overexpressed in asynchronous RPE-1 cells transfected with si-Control or si-Nek2. Centrosomes were labeled through anti-γ-tubulin staining. Boxed areas are enlarged; arrowheads: centrosomes. Centrosome distance was measured in GFP-GAS2L1- and S352D-expressing cells from three independent experiments: n = 157 (si-Control + GFP-GAS2L1 WT), 148 (si-Nek2 + GFP-GAS2L1 WT), 144 (si-Control + GFP-GAS2L1 S352D), and 161 (si-Nek2 + GFP-GAS2L1 S352D). χ2 test; **, P < 0.001; n.s., not significant. Scale bars, 10 µm in cell micrographs and 5 µm in enlarged centrosome images.
Figure 7.

Nek2A mediates G2/M phosphorylation of GAS2L1. (A) GAS2L1 (Bio-FLAG–tagged) was coexpressed with a kinase-dead mutant of GFP-Nek2A (K37R) in HEK293T cells. After pull-down of ectopically expressed GAS2L1, pull-down samples (50%) and cell-extract inputs (5%) were immunoblotted with anti-FLAG and anti-GFP. (B) FLAG-GAS2L1 was transiently expressed with GFP-Nek2A or the K37R mutant in HEK293T cells, and then cell extracts were immunoblotted. An aliquot of the extract from cells coexpressing FLAG-GAS2L1 and GFP-Nek2A was treated with calf intestinal alkaline phosphatase before immunoblotting. (C) Purified GFP-GAS2L1 and S352A mutant proteins were phosphorylated in vitro with GST-Nek2A and then immunoblotted for GAS2L1, pSer352-GAS2L1, and Nek2A. (D) RPE-1 cells were synchronized with aphidicolin and then released into STLC-containing medium. Cells were collected at indicated time points and analyzed by immunoblotting. (E) RPE-1 cells transfected with control siRNA (si-Control) or nek2-targeting siRNA (si-Nek2) were arrested with aphidicolin and then released into STLC-containing medium for 12 h. Cell extracts were immunoblotted with indicated antibodies. Intensity of Ser352-phosphorylated GAS2L1 was measured in GAS2L1 doublets and normalized and is presented as means ± SD from three independent experiments. Unpaired Student’s t test; **, P < 0.001. (F) HEK293T cells were transfected with GAS2L1 constructs and Nek2A WT or kinase-dead mutant K37R. After anti-FLAG immunoprecipitation, immunoprecipitates (50%) and lysate inputs (1%) were immunoblotted with anti-FLAG and anti-GFP antibodies. Anti-pSer352-GAS2L1 immunoblotting was also performed on lysate inputs. IP, immunoprecipitation. The coimmunoprecipitated CH domain was quantified and normalized and is presented as means ± SD from three independent experiments. Unpaired Student’s t test; ***, P < 0.0001. (G) GFP-GAS2L1 or the S352D mutant was overexpressed in asynchronous RPE-1 cells transfected with si-Control or si-Nek2. Centrosomes were labeled through anti-γ-tubulin staining. Boxed areas are enlarged; arrowheads: centrosomes. Centrosome distance was measured in GFP-GAS2L1- and S352D-expressing cells from three independent experiments: n = 157 (si-Control + GFP-GAS2L1 WT), 148 (si-Nek2 + GFP-GAS2L1 WT), 144 (si-Control + GFP-GAS2L1 S352D), and 161 (si-Nek2 + GFP-GAS2L1 S352D). χ2 test; **, P < 0.001; n.s., not significant. Scale bars, 10 µm in cell micrographs and 5 µm in enlarged centrosome images.

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