Cdc20 associates with the kinase aurora2/Aik

Abstract

Cdc20/fizzy family proteins are involved in activation of the anaphase-promoting complex/cyclosome, which catalyzes the ubiquitin-dependent proteolysis of cell cycle regulatory proteins such as anaphase inhibitors and mitotic cyclins, leading to chromosome segregation and exit from mitosis. Previous work has shown that human Cdc20 (hCdc20/p55CDC) associates with one or more kinases. We report here that Cdc20-associated myelin basic protein kinase activity peaks sharply in early M phase (embryonic cells) or in G2 phase (somatic cells). In HeLa cells, Cdc20 is associated with the kinase aurora2/Aik. Aurora2/Aik is a member of the aurora/Ipl1 family of kinases that, like Cdc20, previously has been shown to be localized at mitotic spindle poles and is involved in regulating chromosome segregation and maintaining genomic stability. The demonstration that Cdc20 is associated with aurora2/Aik suggests that some function of Cdc20 is carried out or regulated through its association with aurora2/Aik.

Figure 1

Clam Cdc20 protein levels are relatively constant across the meiotic and mitotic cell cycles of clam embryos. Oocytes were fertilized at zero min. Aliquots taken at 5-min intervals were pelleted and resuspended in sample buffer. Five microliters (corresponding to ≈1,200 oocytes) of each sample was analyzed by SDS/PAGE, followed by immunoblotting with α-clam Cdc20 antibodies. GVBD is a visual indication of the progression past the G₂/M border of meiosis I. Small variations in Cdc20 levels at different time points were not reproducible between sample sets. The arrow indicates the position of clam Cdc20.

Figure 2

Clam Cdc20-associated MBP and α-casein kinase activities vary across the cell cycle. (A) Extracts were prepared from: unfertilized oocytes (arrested at G₂ of meiosis I), GVBD (meiosis I), interphase 1, early mitosis 1 (preanaphase), late mitosis 1 (anaphase underway), colchicine-arrested mitosis 1, or emetine-arrested interphase 2. Thirty micrograms of each clam extract was analyzed by immunoblotting with anti-clam Cdc20 polyclonal antibodies. The arrow indicates the position of clam Cdc20. (B) Extracts were incubated with preimmune (−) or anti-clam Cdc20 antibodies (+). Immunoprecipitates were assayed for associated kinase activities toward MBP (Upper) or α-casein (Lower). Samples were analyzed by SDS/PAGE followed by autoradiography or PhosphorImaging. Arrows indicate the positions of MBP (Upper) and α-casein (Lower).

Figure 3

Clam Cdc20 sediments as part of a large complex in both mitotic and interphase cells. Extracts of interphase 1 or early mitosis 1 clam oocytes were separated on 15%–40% sucrose gradients. Gradient fractions were analyzed by Western blotting with antibodies raised against clam Cdc20 or human Cdc16 (49). Cdc16 is a component of the APC/C, and thus indicates the position of the 1,500-kDa clam oocyte APC/C (4). T is a sample of the total extract loaded on the gradient, and numbers indicate gradient fractions (top to bottom). Size markers are BSA (B; 65 kDa, 4.5 S), catalase (C; 248 kDa, 11.3 S), and thyroglobulin (Th; 670 kDa, 19 S) (4, 12, 17).

Figure 4

Human Cdc20 and aurora2/Aik associate in HeLa cells. (A) HeLa cells were synchronized at the indicated stages and extracts produced as described in the text. Thirty micrograms of each extract was analyzed by Western blotting for cell cycle regulated proteins cyclin A, cyclin B1, hCdc20, or aurora2/Aik. As indicates asynchronously dividing cells. (B) For immunoprecipitate kinase assays, extracts were incubated with either preimmune serum (−) or with antibodies to hCdc20 (+, Upper) or aurora2/Aik (+, Lower); immune complexes were recovered and assayed for kinase activity toward MBP, as described in the text. Phosphorylated MBP was detected by SDS/PAGE followed by autoradiography or PhosphorImaging. (C) Cdc20 or aurora2/Aik immunoprecipitates were prepared as in B, eluted from washed beads, precipitated, and analyzed by SDS/PAGE followed by immunoblotting. Immunoprecipitates were probed for human Cdc20 (Upper) or aurora2/Aik (Lower). Arrows indicate the positions of human Cdc20 and aurora2/Aik.

Figure 5

Sucrose gradient sedimentation of HeLa cell Cdc20 and aurora2/Aik. Cell lysates from a synchronized population of HeLa G₂-phase cells were separated by sedimentation on 15–50% sucrose gradients. Fractions were analyzed by Western blotting as indicated. T is a sample of the total extract loaded on the gradient, and numbers indicate gradient fractions (top to bottom). Molecular weight markers are as in Fig. 3.