Identification and characterization of critical phosphorylation required for Bub1 function

Bub1 is a multifunctional kinase that plays important roles in a number of critical cellular processes. Classically, Bub1 is a key component of the spindle assembly checkpoint activation complex that ensures the fidelity of chromosome segregation during mitosis. The most well-defined activity of Bub1 involves the direct phosphorylation and inhibition of the anaphase-promoting complex (APC) activator Cdc20 to prevent premature sister chromatid separation in response to spindle checkpoint activation. However, we have demonstrated a new role for Bub1 in the induction of premature cellular senescence. Loss of Bub1 in normal human diploid fibroblasts leads to increased senescence that depends on the presence of functional p53, as loss of Bub1 in a p53 dominant-negative background blocks the senescence induced by Bub1 knockdown. Surprisingly, overexpression of Bub1 also leads to increased senescence. Here we show that the kinase activity of Bub1 is required for the senescence induction caused by Bub1 overexpression.Because so little was known regarding the basic requirements governing the function of Bub1's kinase activity, we mapped 18 phosphorylation sites within the kinase domain of Bub1. We found that the senescence resulting from Bub1 overexpression depends on the phosphorylation of Bub1 at three critical residues situated within its activation loop, at human residues T960, T968, and S969. Mutational analysis of these three sites confirms that they are required for in vitro substrate phosphorylation. Phospho-specific antibodies raised against these sites demonstrate that they are phosphorylated in vivo during checkpoint activation in human cells. This study, therefore, describes the first in vivo verified phosphorylation sites for human Bub1 and establishes an important functional role for its kinase activity. Mutations that render Bub1 kinase-inactive or hinder the activation of Bub1 could potentially lead to a block in cellular senescence under critical conditions, possibly playing a role in cellular immortality and tumorigenesis.