Figure 1.

Plk1 interacts with nuclear SREBP1 during mitosis. (A) HEK293 cells were transfected with FLAG-tagged nuclear SREBP1a (nSREBP1a), either wild-type (WT, amino acids 1–490) or the ΔC mutant (amino acids 1–417), lacking the entire C-terminal domain of nSREBP1a. Cell lysates were inmunoprecipitated with anti-FLAG antibodies. The amounts of immunoprecipitated Plk1 and nSREBP1a, and the levels of nSREBP1a and Plk1 in whole-cell lysates (WCL) were determined by Western blotting, with α-tubulin serving as a loading control. (B) HEK293 cells were transfected with FLAG-nSREBP1a, either WT or the S439A mutant. Cell lysates were immunoprecipitated with anti-FLAG antibodies. The amounts of immunocrecipitated Plk1 and nSREBP1a, and the levels of nSREBP1a, Plk1 and α-tubulin in cell lysates were determined by Western blotting. (C) Cell lysates from HeLa cells were used in peptide pull-down assays, using 2 peptides corresponding to residues 436–442 of human SREBP1a, either non-phosphorylated (Ref) or the same peptide phosphorylated on S439 (P-pept) (upper panel). Myc-PBD, either WT or a binding-deficient mutant (MT), was expressed in HEK293 cells, and the cell lysates were used in peptide pull-down assays, using the same 2 peptides as those described above (lower panel). The bound proteins were subjected to SDS/PAGE and Western blotting using 20% of input as control. (D) Recombinant nSREBP1a, either WT or the S439A mutant, was used in in vitro kinase assays in the absence or presence of recombinant Cdk1/cyclin B. The phosphorylated proteins were mixed with lysates of HEK293 cells expressing GFP-Plk1. The His-tagged nSREBP1a proteins were captured on NiTA-agarose, washed and resolved by SDS/PAGE, followed by Western blotting. The phosphorylation of S439 in nSREBP1a was monitored with a phosphorylation-specific antibody (pS439). (E) HeLa cells were synchronized at the G₁/S transition by a double-thymidine protocol. Cells were collected after the second thymidine block (G₁/S) or 14 h after the release into either nocodazole-containing media (Mit) or normal media (G₁). Cell lysates were incubated with recombinant GST-PBD, either WT or binding-deficient mutant (MT), in the presence of glutathione beads. The amount of nSREBP1 associated with the glutathione beads was determined by Western blotting (left panel). The GST-PBD proteins were detected by coomassie brilliant blue staining (CBB). The levels of nSREBP1 and α-tubulin in cell lysates were determined by Western blotting (right panel) (F) HeLa cells were synchronized at the G₁/S transition by a double-thymidine protocol. Cells were collected after the second thymidine block (G₁/S) or 14 h after the release into either nocodazole-containing media (Mit) or normal media (G₁). Cell lysates were immunoprecipitated with either a control antibody (Gal4, lanes 1–3) or SREBP1 antibody (lanes 4–6). The amounts of immunoprecipitated Plk1 and nSREBP1 were determined by Western blotting (left panel). The levels of nSREBP1, Plk1 and α-tubulin in cell lysates were determined by Western blotting (right panel).