The interaction profile between CDC20 and the components of the anaphase promoting complex or cyclosome (APC/C) in human Hela cells

Abstract

The mis-segregation of chromosomes during mitosis can lead to genetic disorders, like Down syndrome, birth defects and even cancers (5-9). The spindle assembly checkpoint (SAC) is the most important mechanism in mitosis (3, 10). The SAC functions to prevent premature sister-chromatid segregation - at anaphase onset by inhibiting the premature activation of the anaphase promoting complex/cyclosome (APC/C) by its coactivator CDC20 (cell division cycle protein 20) (10, 12). The APC/C is a large multi-subunit protein complex which functions as an E3 ubiquitin ligase and targets substrates by ubiquitination and consequently destruction by the proteasome throughout the cell cycle (reviewed in 61). It contains three functional subdomains: the scaffolding platform consists of APC1, APC4, and APC5; the catalytic domain consists of APC2 (a Cullin family related protein), APC10 (Doc1) and APC11 (RING finger protein); and the TPR (tetratricopeptide repeat) lobe domain consists of APC3, APC6, APC7, APC8, APC13, APC16 and Cdc26 (Reviewed in 63). The spatiotemporal activation of the APC/C is primarily achieved by sequential and regulated binding to its two co-activators, CDC20 and Cdh1 leading to the formation of APC/CCDC20 and APC/CCdh1 which are two E3 ligase complexes (Reviewed in 61). The APC/CCDC20 primarily controls the metaphase/anaphase transition and mitotic exit by targeting and destroying Cyclin B1 and securin through regulation by the SAC (Reviewed in 61). More recently, it has been shown that the APC/C can interact with a second CDC20 and target other substrates, such as Nek2A and Cyclin A which are degraded in prometaphase independently of the SAC (64). As the interaction between CDC20 and the APC/C, or the CDC20MCC and the APC/C has never been seen in vivo, it will be important to understand the timing of these interactions and the part that they play in regulating the APC/C (50, 52). Therefore, the overall aim of this project is using PLA to investigate the protein-protein interactions between the components of the APC/C and its co-activator, CDC20; and the interactions among the subunits of the APC/C to provide insights into the regulation of the APC/C. We have studied the in vivo protein-protein interactions between APC3-CDC20, APC8-CDC20, and APC11- CDC20 and intended to examine the interactions between CDC20 and the APC/C. We have also examined the dynamic assembly of the APC/C by looking at APC3-APC6, and APC3- APC10 complexes. Our results suggest that APC11-CDC20APC8, and APC11-CDC20APC3 interacted at the same time, and we favour the interpretation that there is only one APC/C. The interaction profiles of the APC3-APC6 and APC3-APC10 suggest that the assembly of the APC/C is cell cycle regulated.