Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1

Abstract

SUMMARY In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister-chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN-box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

“…Intriguingly, the Phe box in Bub1 is located only two residues N-terminal to its first KEN box (KEN1) and ϳ100 residues N-terminal to the second KEN box (KEN2) (47). The two KEN boxes of Bub1 have been previously shown to bind cooperatively to Cdc20 (48). Because Cdc20 only has one KEN boxbinding site, this cooperativity remained unexplained.…”

The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

“…The stronger effect of KNL1 RNAi could point to a role of Bub1 as an additional binding partner for Cdc20 at kinetochores and indeed human Bub1 has been shown to bind Cdc20 (ref. 38). Further experiments are needed to address the role of Bub1 in BubR1 and Cdc20 kinetochore localization.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Consistent with these data, a Bub1 checkpoint substrate in yeast has not been identified. Although Bub1 phosphorylation of Cdc20 in mammalian cells has been implicated in the checkpoint (Tang et al 2004;Kang et al 2008), its kinase activity is more important for segregation than the checkpoint (Sharp-Baker and Chen 2001; Klebig et al 2009). As mentioned above, Aurora B phosphorylation in yeast has also been implicated in the checkpoint because it phosphorylates Mad3 to generate a response to defects in tension (Rancati et al 2005;King et al 2007).…”