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. 2013 May 21;4:1882. doi: 10.1038/ncomms2879

Figure 3. Ser99 is a novel phosphorylation site required for Plk1 binding to 14-3-3γ.

Figure 3

(a,b) The indicated Myc–Plk1 fragments were transiently expressed in HeLa cells. In b, Myc–Plk1 fragments (residues 1–371) were mutated at the indicated Ser/Thr to Ala. Each mitotic extract was subjected to GST pull-down assays using GST-14-3-3γ (c) Endogenous Plk1 was immunoprecipitated from interphase (I) or mitotic (M) extract. Before the immunoblotting, the immunoprecipitates were treated with or without λPPase. (d) Specificity of anti-Plk1-pS99 was analysed by the peptide competition assays. The position of heavy (h.c.) or light (l.c.) chain of anti-Plk1 is also indicated. (e) The effect of 14-3-3γ or Akt1/2 silencing on cell-cycle progression after DTB synchronization. (f) The effect of Bora silencing on Plk1 phosphorylation. (g–j) Myc–Plk1 WT or its variant was induced by the addition of doxycycline (Dox) in Tet-On HeLa cells (g, h). In j, Myc-tagged human Plk1 (WT), fly Polo or nematode PLK1 was transiently introduced in HeLa cells. Each anti-Myc immunoprecipitate from mitotic cells was subjected to immunoblotting or IP kinase assays. Fold activation relative to Plk1 WT is shown in the graph. The alignment of Plk1 protein sequences around Ser99 is shown in i.