FIGURE 6.

FOXK2 is phosphorylated by CDK·cyclin complexes at serine 368 in vivo. A, gel mobility shifts in the migration of WT and phosphodeficient mutant S368A/S423A GFP-FOXK2 proteins. FOXK2 expression was induced by doxycycline treatment of stably transfected HeLa cells. Cells were grown in the presence or absence of nocodazole (Noc) for 16 h and treated with or without roscovitin (Rosc) for 2 h before harvesting. Cell lysates were subjected to immunoprecipitation (IP) using anti-FOXK2 antibody followed by immunoblot (IB) with FOXK2 antibody. B, in vitro kinase assays using GST-FOXK2(189–660) and recombinant CDK1·cyclin B or CDK2·/cyclin A, Western blots (top) using an anti-phospho-Ser³⁶⁸ FOXK2 antibody and a Coomassie-stained gel of the samples (bottom) are shown. The asterisks represent degradation products. C, HeLa cells containing stably integrated doxycycline (dox)-inducible WT and S368A/S423A mutant forms of EGFP-tagged FOXK2 were either left uninduced (−) or induced (dox) for 24 h. FOXK2 was isolated by immunoprecipitation with anti-FOXK2 antibody and detected by immunoblot using an anti-phospho-Ser³⁶⁸ FOXK2 antibody or anti-FOXK2 antibody. Input levels of FOXK2 and ERK2 were detected by immunoblot. D, endogenous FOXK2 was isolated by immunoprecipitation from asynchronous (Asy) or U2OS cells synchronized in G₁, S, G₂, or M phases. Precipitated proteins detected by immunoblot using anti-phospho-Ser³⁶⁸ FOXK2 or FOXK2 antibody and input proteins by direct immunoblot with the indicated antibodies. E, endogenous FOXK2 phosphorylation was analyzed as in D from asynchronous U2OS cells or cells treated with nocodazole for 16 h, with or without purvalanol A (PuA) treatment. Mitotic cells were collected by manual shake off (lanes 3 and 4). F, U2OS cells were transfected with siRNA duplexes against either CDK1 (left) or cyclin B (right) where indicated. 30 h after transfection, cells were grown in the absence (Asy) or presence (Noc) of nocodazole for 16 h before harvesting. Cell lysates were analyzed as in D.