Fig 4. Bub1^kinase possesses conserved residues that recognize a substrate consensus sequence.

(A) Alignment of phosphorylation sites found in Bub1-dependent phosphorylation reactions using the ClustalX algorithm in Jalview [54]. Numbers indicate boundaries of the protein segments shown; phosphorylated residues are denoted with an asterisk, the conservation of residues is highlighted using the ClustalX coloring scheme. (B) The structure of Bub1^kinase (PDB ID 4QPM) was superimposed onto the structure of PKA bound to a pseudo-substrate inhibitor [PDB ID 1APM, reference [55]]. The image shows only Bub1 and the position of the pseudo-substrate peptide (yellow) after alignment of PKA to Bub1. Bub1 residues putatively involved in substrate recognition are depicted as sticks. (C) Bub1^kinase sequence and structure conservation. The same residues as in (B) are shown in sticks. Conservation was determined by aligning Bub1^kinase from 14 organisms with ConSurf [56], the scoring legend is depicted on the upper left. Conservation scores obtained for positions in the alignment that had less than 6 un-gapped amino acids were considered to be unreliable and colored light yellow in the graphic visualization output. Images were created with CCP4MG [53] and Pymol (Schrödinger LLC, Portland, OR). (D) Bub1^kinase-dependent H2A phosphorylation can be reduced by mutating V115D, L116N, L117N on H2A. GST-H2A constructs were incubated with Bub1^kinase and ATP, then analyzed by SDS PAGE, and phosphorylated proteins were specifically stained using Pro-Q® Diamond Phosphoprotein Gel Stain.