A Nucleolar Control Of Cell-cycle Progression And P53 Degradation By Def Phosphorylation

Nucleolar factor Def is a cross-kingdom conserved component of the ribosome small subunit (SSU) processome and also functions together with the cysteine protease Capn3 to mediate protein degradation specifically in the nucleolus with p53 being one of the substrates. However, little is known about how Def’s function is regulated.We report here that Def is modified by phosphorylation and that five serine residues, namely S50, S58, S62, S87 and S92, appear to serve as the phosphorylation sites. Our results reveal that phosphorylation is not only important for Def to promote liver development through controlling the G1 to S and G2 to M transitions but is also essential for Def to mediate p53 degradation. Our findings demonstrate that the Def-Capn3b nucleolar protein degradation pathway controls cell cycle progression during organ growth and this function is tunable by Def phosphorylation modification.We also found that Def shows an SDS PAGE-displayed molecular weight (MW) approximately 13 kDa larger than its predicted MW. The first 188 AA in Def is defined by a glutamate-rich region containing～35.6% of acidic AA. We analyzed the relationship between the SDS PAGE-displayed MW of thirteen peptides derived from Def and the AA composition in each peptide. We found that the difference between the predicted and SDS PAGE-displayed MW showed a linear correlation with the percentage of acidic AA that fits the equation y=276.5x-31.33 (x represents the percentage of acidic AA; y represents the average AMW per AA). We demonstrated that this equation could be applied to predict the SDS PAGE-displayed MW for other thirteen acidic proteins.