Fig. 2. Nek6 phosphorylates Eg5 in vitro at a conserved site that is phosphorylated during mitosis. (A) Recombinant GST-HsEg5 or His₆-XlEg5 were phosphorylated by FLAG-Nek6. The insets show ³²P incorporation with time (upper gel, left to right with no kinase in rightmost lane) and a Coomassie-blue stain of the substrate (lower gel). (B) Myelin basic protein (MBP, positive control), maltose-binding protein (MalBP), or MalBP fusion proteins containing the Eg5 segments indicated were incubated with [-³²P]ATP/Mg²⁺ and either FLAG-Nek6 (odd-numbered lanes) or pre-activated FLAG-Nercc1 (even-numbered lanes) for 30 minutes followed by SDS-PAGE. The upper panels show Coomassie-blue stains of the gels and the lower panels the corresponding autoradiographs. White arrowheads indicate Nercc1 and black arrowheads indicate Nek6. Asterisks indicate the location of MBP (lanes 3 and 4) or MalBP fusion proteins (7-12). The left gel is 12% acrylamide, the right gel 7.5%; Nek6 is run off the latter. (C) HsEg5, human Eg5; MmEg5, mouse Eg5; XlEg5, X. laevis Eg5; KLP61F, D. melanogaster KLP61F; bmk-1, C. elegans BMK-1; BimC, A. nidulans BimC; KIP1/Cin8p, S. cerevisiae KIP1/Cin8p; Cut7, S. pombe Cut7. Identical and conserved residues are shaded; regions conserved around the C-terminal CDK1 site (BimC box) and Nek6 site (LXXS*) are boxed. (D) Using normal IgG (NIgG) and anti-HsEg5 antibodies, immunoprecipitates were prepared from extracts of ³²P-labelled U2OS cells growing exponentially (Exp) or incubated overnight in 0.25 mM nocodazole (M), and subjected to SDS-PAGE; a PhosphoImage of the gel is shown. (E) Left, two-dimensional tryptic phosphopeptide map of endogenous ³²P-Eg5 immunoprecipitated from ³²P-labelled mitotic U2OS cells, visualized by PhosphorImager. Right, phosphopeptide map of purified recombinant ³²P-GST-Eg5 phosphorylated in vitro by immunoprecipitated mitotic CDK1. The gray arrows mark phosphopeptides attributed to CDK1; the black arrow indicates a ³²P-peptide evident in digests of mitotic Eg5 that is not present in digests of CDK1-phosphorylated ³²P-Eg5. (F) Top left, two-dimensional phosphopeptide map of endogenous Eg5 immunoprecipitated from ³²P-labelled mitotic U2OS cells; right (top and bottom), two-dimensional maps of recombinant GST-Eg5 wild type (wt; top right) and GST-Eg5[Ser1033Ala] (bottom right) phosphorylated in vitro by FLAG-Nek6; note in the lower right map the absence of the most anodally migrating ³²P-peptide seen in the upper right map, which encompasses Ser1033-P. Bottom left, a mixture of comparable amounts of the digests of mitotic ³²P-Eg5 and FLAG-Nek6-phosphorylated ³²P-Eg5. The arrow marks the phosphopeptide common to the two digests, which contains Ser1033. In the digests of mitotic ³²P-Eg5 (E, left; F, upper left), this spot contains 3% of total ³²P, estimated by PhosphorImager.