Characterization of the S. cerevisiae Mps1 protein kinase

The S. cerevisiae MPS1 gene encodes an essential dual-specificity protein kinase that has been implicated in spindle pole body (SPB) duplication and cell cycle control. Kinase-defective mutants fail to duplicate the SPB, and also fail to arrest the cell cycle in response to the defective mitotic spindle. Overexpression of MPS1 arrests cells at the mitotic checkpoint with a morphologically normal spindle. Our conclusion from this data was that Mps1p protein kinase activity is required for SPB duplication in the G1 phase of the cell cycle, but aberrant activity in the G2/M phase triggers the mitotic checkpoint.; In order to better understand how Mps1p's kinase activity controls these functions, I have tracked the activity of the enzyme through the cell cycle, in the absence and presence of factors that would trigger the mitotic checkpoint. The kinase activity of Mps1p is increased transiently at the G1/S transition in normal cell cycles, correlating with SPB maturation and possibly separation. The activity then returns to basal levels for the remainder of the cell cycle. If the mitotic checkpoint is activated, Mps1p's kinase activity is increased in S phase, and remains high as long as the arrest is maintained. While this profile of activity is consistent with the genetic requirements of MPS1, the timing of the peaks of activity is surprising. Triggering the mitotic checkpoint also leads to a loss of the transient G1/S peak associated with SPB duplication, which is also unexpected.; I have attempted to understand how these changes in Mps1p kinase activity occur. To this end, I performed mass spectrometry on the overexpressed protein to determine which amino acids are phosphorylated. Those amino acids are candidates for regulatory sites by phosphorylation. Three candidate sites have been mutagenized, and one of those mutants is incapable of complementing kinase-defective alleles of MPS1, or the mps1{dollar}Delta.{dollar}; Finally, I have used indirect immunofluorescence to determine the localization of the Mps1 protein. The kinase is localized to the SPB, as determined by overlap with a known SPB component, but the localization is lost from one SPB as the organelles migrate apart. This asymmetric localization is uncommon in SPB components, and raises interesting questions as to the timing and biological relevance of the loss of Mps1p on one SPB.