Scientific Report 2007

Scripps Florida

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Cancer Biology

Novel Regulators of the Anaphase-Promoting Complex

N. Ayad, D. Harmey, S. Simanksi, L. Owens

The anaphase-promoting complex (APC) is an essential regulator of the eukaryotic cell cycle. New findings suggest that the APC integrates signals from multiple pathways to induce particular cell-cycle transitions. However, no unbiased genome-wide approach has been used to determine the signaling pathways that regulate APC activity. We used a cell-based screening method to identify APC regulators that are potential mediators of signaling pathways. We cotransfected 14,000 cDNAs with the N terminus of the APC substrate cyclin B fused with luciferase and identified several novel regulators of the APC (Fig. 1).

Fig. 1. The N terminus of the APC substrate cyclin B was fused to luciferase and used as a measure of APC activity. Cotransfection of 14,000 cDNAs with the fusion protein or luciferase alone allowed us to identify APC regulators. The known APC regulator Cdh1 was identified as a protein that reduced the steady-state levels of the fusion protein relative to luciferase alone. An uncharacterized F box protein also reduced levels of the fusion protein specifically. Heat map key: green is 2-fold lower than control plasmid transfection (Sport6); red is 2-fold or higher than control plasmid. The known APC inhibitor Emi-1 was used as a control on each plate.

One protein we have extensively characterized is an F box protein that stimulates APC activity. Expression of this protein is highest during exit from mitosis, suggesting that the protein may play a role in regulating APC activity at that time. Furthermore, depletion of this protein by transfection of short interfering RNA induced a delay in mitotic exit. Currently, we are analyzing the mechanism by which this protein regulates APC activity in vitro and in vivo. These studies suggest that cell-based screening is an effective tool for determining the signaling networks that regulate ubiquitin ligases and cell-cycle transitions.

Publications

Smith, A., Simanski, S., Fallahi, M., Ayad, N.G. Redundant ubiquitin ligase activities regulate wee1 degradation and mitotic entry. Cell Cycle, in press.