FIG. 9.

In vitro phosphorylation of Nlp by Nek2 and Plk1. (A) Purified MBP-X-Nlp_262-552, MBP alone, and casein were phosphorylated in vitro by wild-type (WT) or kinase-inactive (KR) human Nek2A expressed in insect cells. Samples were separated by SDS-PAGE, stained with Coomassie blue (bottom panel), and exposed to autoradiography (top panel). (B) Western blots indicating equal expression (anti-Myc, top panel) and immunoprecipitation (anti-Nek2, bottom panel) of wild-type and kinase-inactive Myc-Nek2A proteins from transfected HeLa cells for use in kinase assays. (C) GST-Nlp-N-term (Nlp) or GST-Nlp-N-term-Δ8 (Δ8) was phosphorylated in vitro with Nek2A kinases immunoprecipitated from transfected HeLa cells or purified Plk1 expressed in insect cells, as indicated. (D) ³²P incorporation into the Nlp proteins as shown in panel C was determined by scintillation counting. The level of phosphorylation of the GST-Nlp-N-term protein by wild-type Nek2 (1-4) and Plk1 (5, 6) was set as 100%. The histogram presents the average of three independent experiments. (E). GST-Nlp-N-term was incubated with either wild-type (left panels) or kinase-inactive (right panels) immunoprecipitated Nek2A for 40 min in the presence of unlabeled ATP. Purified Plk1 was then added together with [γ-³²P]ATP and samples were taken at the times indicated before separation by SDS-PAGE. Gels were stained with Coomassie blue (CB) and exposed to autoradiography (³²P). In each of five independent experiments, the phosphorylation of Nlp by Plk1 was always greater after incubation with wild-type compared to kinase-inactive Nek2A. (F) Kinase assays were performed as in panel E except that phosphorylation of Nlp by Plk1 was assayed after incubation with either immunoprecipitated Nek2A (left panels) or protein G-Sepharose beads alone (right panels).