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. 2015 Dec 9;6:10076. doi: 10.1038/ncomms10076

Figure 2. PLK1 regulation of the separase-dependent cleavage of PCNT during mitotic exit.

Figure 2

(a) HeLa cells were treated with BI2536 during mitotic exit and forced to mitotic exit with ZM447439 (ZM). Lysates were subjected to immunoblot analyses with antibodies specific to PCNT, cyclin B1 and GAPDH. (b) Immunoblot analysis was carried out to determine the PCNT cleavage in PLK1-depleted HeLa cells during the forced mitotic exit. (c) Specific cleavage of ectopic FLAG-PCNT-Myc (WT) and FLAG-PCNTR2231A-Myc (RA) was determined in the presence of BI2536. (d) Summary of the PLK1 phosphorylation of PCNT. PLK1 phosphorylates 22 specific sites of PCNT (Supplementary Fig. 1) and these sites are marked with circles. Among them, nine phosphorylation sites (T2154, T2160, S2183, S2189, S2222, S2259, S2267, S2318 and T2324; green line) near the separase cleavage site (R2231; red) are critical for the PCNT cleavage. Two sites at the N-terminal end (S1235 and S1241; yellow circles) are essential for centrosome maturation. (e) Immunoblot analyses were carried out to determine specific cleavage of ectopic FLAG-PCNT7A-Myc (7A) and FLAG-PCNT9A-Myc (9A) during the forced mitotic exit. (f,g) Specific cleavage of ectopic FLAG-PCNT-Myc (WT) and FLAG-PCNT9D-Myc (9D) was determined in the presence of BI2536 (f) and in the separase-depleted cells (g). (h,i) Immunoblot analysis was carried out to determine the specific cleavage of the FLAG-mCherry-PCNT2059–2398-GFP-PACT fusion proteins with indicated phospho- (9A and 13A) and cleavage-resistant (RA) point mutations (h), and with phospho-mimetic mutation (9D) in the presence of BI2536 (i). The experiments were independently repeated at least twice. Asterisk, a non-specific band.