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Fig. 4. Identification of Ser-904 and Ser-905 of CHO1/MKLP-1 as two major Plk1 phosphorylation sites. (A) GST-fused CHO1/MKLP-1 domains were purified (left) and subjected to in vitro kinase reactions with purified Plk1 (right). Only the tail domain (residues 643-953) was strongly phosphorylated by Plk1, indicated by the arrowhead on the right. (B,C) Different regions of the tail domain fused with GST were purified and subjected to in vitro kinase reactions with Plk1. The stars indicate the positions of the proteins based on Coomassie Blue staining. The size markers are indicated on the left. The region 901-953 contains Plk1 phosphorylation sites. (D) Wild-type or mutant GST fusion proteins of the 901-953 region were subjected to kinase assays with recombinant Plk1. (E) Wild-type or mutant GST fusion proteins of the 801-900 region (left) or the 643-953 region (right) were subjected to kinase assays with recombinant Plk1. The 2A mutant contains the mutations Ser805Ala and Ser807Ala; the 4A mutant contains the mutations Ser805Ala, Ser807Ala, Ser904Ala and Ser905Ala. The arrow on the left indicates the position of GST-Plk1, and the stars indicate the positions of the proteins based on Coomassie Blue staining. (F) Purified GST-fused CHO1 C-terminus (residues 406953) was subjected to kinase assay with either kinase defective (KM) or wild-type (WT) GST-Plk1. (G) CHO1 is highly phosphorylated during mitosis. Asynchronous or mitotic (treated with 200 ng ml–1 nocodazole for 10 hours) HeLa cells were labeled with [32P]orthophosphate for 4 hours. Cell lysates were either directly analysed on an anti-Plk1 western blot or immunoprecipitated with anti-CHO1 antibody first, then detected with anti-CHO1 western blot. Phosphorylated CHO1 was visualized by autoradiography and quantified using a phosphorimager. (H) HeLa cells were transfected with pBS/U6-Plk1 to deplete Plk1 first, then treated with nocodazole for 10 hours and labeled in vivo as in (G). (I) Cells were transfected with GFP-CHO1 wild-type or 4A mutant as indicated. 30 hours after transfection, cells were incubated with nocodazole for 10 hours and labeled with [32P]-orthophosphate for additional 4 hours. Cell lysates were immunoprecipitated with anti-GFP antibody and phosphorylated GFP-CHO1 was quantified using a phosphorimager.
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