PLK1-mediated phosphorylation of NFBD1 is essential for the M phase entry.

<p>(A) Schematic representation of PST domains of wild-type NFBD1 and mutant form of NFBD1 termed GST-PST(T847A). (B) <i>In </i><i>vitro</i> kinase reaction. GST, GST-PST, or GST-PST(T847A) were purified using glutathione Sepharose beads (right panel) and incubated with purified PLK1 in the presence of [γ-³²P]ATP. The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography (left panel). (C) Enforced expression of PLK1 but not the kinase-deficient mutant form of PLK1 [PLK1(K28M)] induces phospho-histone H3. HeLa cells were transiently transfected with the indicated expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and immunoblotted with the indicated antibodies. (D) Expression of GFP-PST and GFP-PST(T847A). HeLa cells were transiently transfected with the expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and immunoblotted with an anti-GFP antibody. (E and F) GFP-PST but not GFP-PST(T847A) inhibits the phosphorylation of histone H3. HeLa cells were transiently transfected with the expression plasmid for FLAG-PLK1 alone or FLAG-PLK1 and increasing amounts of GFP-PST (E) or GFP-PST(T847A) (F). Forty-eight hours after transfection, whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.