Bromodomain Protein Brd4 Binds to GTPase-Activating SPA-1, Modulating Its Activity and Subcellular Localization

Abstract

Brd4 is a mammalian protein that contains a double bromodomain. It binds to chromatin and regulates cell cycle progression at multiple stages. By immunopurification and mass spectrometry, we identified a Rap GTPase-activating protein (GAP), signal-induced proliferation-associated protein 1 (SPA-1), as a factor that interacts with Brd4. SPA-1 localizes to the cytoplasm and to a lesser degree in the nucleus, while Brd4 resides in the nucleus. Bifluorescence complementation revealed that Brd4 and SPA-1 interact with each other in the nucleus of living cells. Supporting the functional importance of the interaction, Brd4 enhanced Rap GAP activity of SPA-1. Furthermore ectopic expression of SPA-1 and Brd4 redirected subcellular localization of the partner and disrupted normal cell cycle progression. These effects were, however, reversed by coexpression of the two proteins, indicating that a proper balance between Brd4 and SPA-1 in G₂ is required for cell division. This work reveals a novel link between Brd4 and a GTPase-dependent mitogenic signaling pathway.

We thank Anup Dey for helpful discussions, D. Altschuler, J. L. Bos for reagents, T. K. Kerppola for advice on BiFC, G. Humphrey for suggestions on gel filtration, and J. McCormack for technical assistance.