Fig. 4.

Kif4 specifically binds to unphosphorylated PRC1. (A) HeLa cells were synchronized at the G₂/M boundary by a thymidine/nocodazole treatment. Mitotic cells were collected by shake-off, released into fresh medium, and harvested at 10, 30, 50, 70, or 90 min after release. Percentages of cells at different stages of mitosis were determined by counting under a microscope. Whole-cell lysates (WCL) were made, subjected to SDS/PAGE, and immunoblotted with corresponding antibodies as indicated. (B) Lysates as in A were immunoprecipitated by anti-PRC1 or anti-Kif4 antibodies. The immunoprecipitates (IP) were subjected to SDS/PAGE and immunoblotted with anti-Kif4, anti-phospho-PRC1, or anti-PRC1 antibodies. (C) One microgram of bacterially expressed glutathione agarose-bound GST or GST-PRC1 was incubated with or without 0.5 μg of purified baculovirus-expressed Cdc2/cyclin B1 (K1/B1) in the presence of 100 μM ATP for 2 h at 30°C. (Middle) After washing, 1/10th of beads were subjected to SDS/PAGE and immunoblotted with anti-phospho-PRC1 antibodies. The rest of the beads were then incubated with 200 μg of early mitotic HeLa cell lysates (10 min release from a thymidine/nocodazole block) for 2 h at 4°C. (Top) After washing, bead-bound proteins were subjected to SDS/PAGE and immunoblotted with anti-Kif4 antibodies. (Bottom) One microgram of GST, GST-PRC1, and Cdk-phosphorylated GST-PRC1 was used in the pull-down assay on SDS/PAGE by Coommasie blue staining. (D) The proposed model for cell cycle-dependent translocation of PRC1 to the plus ends of interdigitating MTs by Kif4 during the metaphase-to-anaphase transition (for details, see text). APC, anaphase promoting complex.