In S. pombe, formation and constriction of the cytokinetic ring requires a GTPase-regulated protein kinase signaling pathway called the septation initiation network (SIN) that is scaffolded at the spindle pole body (SPB; i.e. yeast centrosomal equivalent) (Morrell et al., 2004; Rosenberg et al., 2006). Regulating SIN activation is one way the cell coordinates cytokinesis with chromosome segregation; during a mitotic checkpoint arrest, the S. pombe checkpoint protein Dma1 facilitates ubiquitination of the SIN scaffold Sid4, which impedes Polo-like kinase Plo1 localization to the SPB and thereby inhibits SIN activation and delays cytokinesis (Johnson and Gould, 2011; Johnson et al., 2012).
Dma1 is an FHA-RING E3 ligase that is related to human E3 ligases and tumor suppressors RNF8 and CHFR; these proteins relay checkpoint signals by binding phosphorylated proteins via their FHA domains and promoting ubiquitination of downstream targets. We are currently investigating what precise cellular defects trigger Dma1 activation and how Dma1-mediated signaling is shut off once the mitotic checkpoint is satisfied (see rotation project 1). By studying Dma1, we hope to learn more about its mammalian counterparts.
Dma1 is recruited to its substrate Sid4 by a phospho-priming event mediated by members of the highly conserved CK1 protein kinase family Hhp1 and Hhp2. During a mitotic arrest, Hhp1/2 accumulate at SPBs where they phosphorylate Sid4 to create a docking site for Dma1’s FHA domain (Johnson et al., 2013). We are studying what mechanisms govern the ability of Hhp1/2 to bind the spindle poles. We are also very interested in understanding the evolutionary conservation of these mechanisms and so we are examining how Hhp1/2's mammalian orthologs CK1δ and CK1ε localize to centrosomes and identifying their substrates at this key signaling hub (see rotation project 2).
Our studies of CK1 enzymes in both yeast and mammalian cells have raised many new questions about these poorly understood protein kinases. What controls their enzymatic activity? How do they get to spindle poles and what are they doing there? We are addressing these questions with phosphoproteomic approaches as well as biochemical and genetic studies in yeast and human cells (see rotation project 3 and postdoctoral positions).
For review, see: Johnson AE, McCollum D, Gould KL. Polar opposites: Fine-tuning cytokinesis through SIN asymmetry. Cytoskeleton (Hoboken, N.J.). 2012 Oct;69(10): 686-99. DOI: 10.1002/cm.21044. PMID: 22786806.