FIG 5.

Plk1 phosphorylates axin2 at Ser311. (A to D) Cell cycle-regulated expression of axin2 and Plk1. (A and C) PC3 (A) and LNCaP (C) cells were synchronized with a double-thymidine block (DTB) (16-h thymidine block, 8 h of release, and a second thymidine treatment for 16 h), released for different times, and harvested for IB. (B and D) PC3 (B) and LNCaP (D) cells were treated with or without nocodazole for 12 h and harvested. (E) Axin2 binds to Plk1. PC3 cells were treated with nocodazole and harvested for IP with antibodies against Plk1 or axin2, followed by IB. (F) Plk1 targets N-terminal axin2. Purified Plk1 was incubated with purified GST-axin2 regions (aa 1 to 300, aa 301 to 577, and aa 578 to 840) in the presence of [γ-³²P]ATP. The reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. (G) Plk1 phosphorylates axin2 at Ser311 in vitro. Purified Plk1 was incubated with different recombinant axin2 regions spanning aa 1 to 577 (WT, S311A mutant, or S318A mutant) and analyzed. (H) pSer311-axin2 antibody specifically recognizes the phosphorylated form of axin2. Recombinant axin2 proteins (WT or S311A) were incubated with or without purified Plk1 in the presence of unlabeled ATP and immunoblotted with the pSer311-axin2 antibody. (I) Axin2-Ser311 is phosphorylated in vivo in a Plk1 activity-dependent manner. PC3 cells were cotransfected with pBS/U6-Plk1 and pBabe-puro at a ratio of 8:1. After a 2-day selection of transfection-positive cells with puromycin, attached cells were treated with nocodazole and harvested for IB. shPlk1, short hairpin RNA against Plk1. (J) Inhibition of Plk1 activity downregulates the phosphorylation level of axin2 at S311. HEK293T cells were transfected with Flag-axin2 and treated with BI2536 for 8 h in the presence of nocodazole. (K) Inhibition of Plk1 activity downregulates the phosphorylation level of endogenous axin2 at S311. PC3 or LNCaP cells were treated with 50 nM BI2536 for 8 h in the presence of nocodazole. (L and M) Cell cycle-dependent phosphorylation of axin2-S311. After treatment with nocodazole, PC3 (L) or LNCaP (M) cells were collected with mitotic shake-off, released for different times, and harvested. All data shown are representative of results from three or more experiments.