Figure 2.

Plk1 phosphorylates T424, S467 and S486 in nuclear SREBP1 during mitosis. (A) In vitro kinase assay with recombinant nSREBP1a and Plk1. The levels and phosphorylation (pT424, pS467 and pS486) of nSREBP1a were monitored by Western blotting. (B) Recombinant nSREBP1a was used in in vitro kinase assays with mitotic HeLa extracts (Mit) in the absence or presence of the Plk1 inhibitor BTO-1 (5, 10 and 25 μM). The levels and phosphorylation (pT424, pS467 and pS486) of nSREBP1a were monitored by Western blotting. (C) Recombinant nSREBP1a was used in in vitro kinase assays with extracts from HeLa cells transfected with either control or Plk1 siRNA. The levels and phosphorylation (pT424, pS467 and pS486) of nSREBP1a were monitored by Western blotting (left panel). The efficiency of the Plk1 knockdown was monitored by Western blotting, with α-tubulin serving as a loading control (right panel). (D) Recombinant nSREBP1a, either WT or the S439A mutant, was used in in vitro kinase assays with mitotic HeLa extracts. The levels and phosphorylation (pT424, pS467 and pS486) of nSREBP1a were monitored by Western blotting. (E) HEK293 cells were transfected with nSREBP1a or nSREBP1c and left untreated or treated with nocodazole. After immunoprecipitation, the levels and phosphorylation (pT424, pS467 and pS486) of the respective nSREBP1 protein were determined by Western blotting. (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). The levels and phosphorylation (pT424, pS467 and pS486) of nSREBP1 were determined by Western blotting following immunoprecipitation of SREBP1.