Fig. 4. Contribution of Mps1p to cell-cycle-dependent phosphorylation of Spc110p. (A) Cell-cycle-dependent phosphorylation of Spc110p following inactivation of Mps1p. The indicated strains were synchronised by -factor-induced block at 23°C and released from G1 at 34°C. The residual cell-cycle-dependent pattern of phosphorylation was dependent on the two CDK consensus sites (and independent of Mps1p). Numbers indicate time in minutes following release from G1. Cell-cycle progression until metaphase was comparable for all strains analysed (not shown). (B) Western blot analysis of extracts from the same synchrony experiment shown in A corresponding to all the strains analysed (and including MPS1⁺ controls not shown in A) at the 50-minute time point, rearranged for comparison. (C) Cell-cycle-dependent profile of phosphorylation in Spc110p mutants lacking Mps1p phosphorylation sites (mps^-) in combination with S36A or S91A substitutions. Extracts prepared from the indicated strains following release from a G1 block. Numbers indicate time in minutes following release from the -factor-induced arrest. Again, phosphorylation was dependent on the two CDK sites in the absence of the previously characterised Mps1p phosphorylation sites (Friedman et al., 2001). a, extract from asynchronous cells. For analysis of cell-cycle progression accompanying this experiment see supplementary material Fig. S3.