The J domain of Tpr2 regulates its interaction with the proapoptotic and cell-cycle checkpoint protein, Rad9

Human Rad9 is a key cell-cycle checkpoint protein that is postulated to fun ction in the early phase of cell-cycle checkpoint control through complex f ormation with Rad1 and Hus1. Rad9 is also thought to be involved in control ling apoptosis through its interaction with Bcl-2. To explore the biochemic al functions of Rad9 in these cellular control mechanisms, we performed two -hybrid screening and identified Tetratricopeptide repeat protein 2 (Tpr2) as a novel Rad9-binding protein. We found that Tpr2 binds not only to Rad9, but also to Rad1 and Hus1, through its N-terminal tetratricopeptide repeat region, as assessed by in vivo and in vitro binding assays. However, the i n vivo and in vitro interactions of Tpr2 with Rad9 were greatly enhanced by the deletion of its C-terminal J domain or by a point mutation in the cons erved HPD motif in the J domain, though the binding of Tpr2 to Rad1 and Hus 1 was not influenced by these J-domain mutations. We further found: (1) Rad 9 transiently dissociates from Tpr2 following heat-shock or UV treatments, but the mutation of the J domain abrogates this transient dissociation of t he Tpr2/Rad9 complex; and (2) the J domain of Tpr2 modulates the cellular l ocalization of both Tpr2 itself and Rad9. These results indicate that the J domain of Tpr2 plays a critical role in the regulation of both physical an d functional interactions between Tpr2 and Rad9. (C) 2001 Academic Press.