Polo-Like Kinase 1 Directs Assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF Complex to Initiate Cleavage Furrow Formation (original) (raw)
Figure 2
Plk1 phosphorylates multiple serine residues in HsCyk-4-Nt to stimulate association with Ect2-BRCT.
(A) Schematic of experimental design (upper diagram). Recombinant HsCyk-4-Nt or Ect2-BRCT fused to CBD was incubated in the presence (+) or absence (−) of Plk1-T210D kinase domain and ATP. Soluble HsCyk-4-Nt or Ect2-BRCT was added to the washed beads, mixed, and then separated on SDS-PAGE and Coomassie stained for visualization. Input represents 50% of the total soluble protein incubated with immobilized protein. (B) Immobilized GST and a series of GST–HsCyk-4 truncated proteins as indicated were incubated with full-length recombinant Plk1 T210D (KA) or Plk1 K82M/T210D (KD) and [γ-32P] ATP. Proteins were separated on SDS-PAGE and Coomassie stained for visualization. Incorporation of 32P was visualized by autoradiography. (C) Peptide arrays containing 142 18-mer peptides covering amino acids 1–300 of HsCyk-4 were incubated with recombinant full-length Plk1 T210D or kinase-dead Plk1 K82M/T210D and [γ-32P] ATP. Incorporated 32P was visualized by autoradiography and the intensity at each peptide spot was quantified and plotted relative to the position of the peptide across the array. (D) Schematic representation of HsCyk-4 functional domains (C-C, coiled-coil domain; C1, C1 domain; GAP, GTPase activating protein domain). The red box indicates the predicted Plk1 target peptide sequence identified from the array analysis (amino acids 139–174). Within this region, Ser149, Ser157, Ser164, and Ser170 are shaded to indicate those residues mutated in the HsCyk-4 derivatives analyzed in (E). (E) Recombinant HsCyk-4-Nt and indicated derivatives fused to CBD were assayed for Plk1 phosphorylation and Ect2-BRCT binding as in (A). Fold stimulation (± standard error of the mean) of Ect2-BRCT binding to HsCyk-4-Nt upon Plk1 phosphorylation was determined from at least three independent experiments (bar graph). (F) HeLa cells transfected with H2O (control) or siRNA to deplete endogenous HsCyk-4 were synchronized in anaphase using an MG132 arrest/release protocol. Following treatment with 100 nM BI-2536 or DMSO, cells were fixed and stained with phospho-Ser170 antibodies, and DNA was stained with DAPI.