Phospho-Regulation of the Anillin-Related Scaffolding Protein, Mid1 in Fission Yeast Cytokinesis

The anillin-related protein Mid1 plays a critical role in organizing the early steps of contractile ring formation and functions as a scaffold to bridge the cell cortex with the contractile ring. Cells lacking mid1 form off-centered, highly disorganized ring structures and exhibit severe cytokinesis defects. Coincident with its cortical accumulation, Mid1 becomes hyper-phosphorylated. Our previous research demonstrates that cyclin-dependent kinase, Cdc2, and the polo-like kinase, Plo1, directly phosphorylate Mid1. In addition to consensus phosphorylation motifs for Plo1 and Cdc2, Mid1 contains several RXXS motifs, which fits the phosphorylation consensus sequence for Sid2 kinase. Sid2 is the most downstream kinase in the Septation Initiation Network signaling cascade, which signals from the spindle pole body to trigger constriction of the contractile ring. To examine Mid1 phospho-regulation, Sid2 phosphorylation sites were identified and mutated to non-phosphorylatable alanine residues. Interestingly, phospho-site mutants displayed cell division defects, including sensitivity to low dose latrunculin A and disorganized actin localization. While phospho-site mutants maintained the mitotic spindle checkpoint, the majority of cells exhibited severe polarity phenotypes that were not observed in checkpoint activated cells expressing wild-type Mid1. Our results reveal a regulatory role for Sid2 phosphorylation of Mid1 in cytokinesis