Regulation of the key mitotic checkpoint protein Dma1 through post-translational modifications

Abstract

Proper cell division to yield two daughter cells with identical complements of genomic material requires coordination between mitosis and cytokinesis. In the event of a mitotic error, checkpoint mechanisms must inhibit both mitotic exit and cytokinesis to ensure accurate segregation of chromosomes. In response to mitotic spindle errors, the spindle checkpoint delays cytokinesis by inhibiting the septation initiation network (SIN), a GTPase-driven signaling cascade. Specifically, Hhp1 and Hhp2 phosphorylate the SIN scaffold protein, Sid4, recruiting dimeric the E3 ligase Dma1. Dma1 ubiquitinates Sid4, antagonizing the localization of the Polo-like kinase Plo1, and preventing phosphorylation of its downstream target Byr4. Consequently, the SIN kinase cascade and cytokinesis are delayed. Upon resolution of the mitotic spindle error, the Dma1 checkpoint signal must be withdrawn to allow continuation of the cell division cycle. However, the mechanism by which Dma1 ubiquitination of Sid4 is stopped is not known. Furthermore, whether Dma1 itself is regulated by the checkpoint remains unclear. In this work, I show that Dma1 is post-translationally modified by auto-ubiquitination and phosphorylation. Dma1 exhibits previously unreported localization dynamics that are dependent on its catalytic activity. In addition to ubiquitinating Sid4, Dma1 demonstrates promiscuous Dma1 auto-ubiquitination in vivo, and evidence indicates that its localization dynamics are impaired in the absence of auto-ubiquitination. I also determined that Dma1 is phosphorylated in vivo throughout the cell cycle and that this phosphorylation occurs on seven sites. Furthermore, I demonstrated that Cdk1 and Plo1 can phosphorylate Dma1 in vitro. The Cdk1/Plo1 phospho-mimetic inhibits Dma1 auto-ubiquitination while maintaining checkpoint activity. Overall, the current data point to a model wherein Dma1 auto-ubiquitination decreases its SPB binding and therefore access to its checkpoint substrate. Dma1 phosphorylation at Cdk1 and Plo1 sites prevents auto-ubiquitination, enabling increased SPB localization and maintenance of the checkpoint.