Abstract
The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.