ZIP kinase phosphorylated and activated by Rho kinase/ROCK contributes to cytokinesis in mammalian cultured cells.

Cytokinesis of animal cells requires contraction of a contractile ring, composed of actin filaments and myosin II filaments. Phosphorylation of myosin II regulatory light chain (MRLC) promotes contraction of the actomyosin ring by activating myosin II motor activity. Both Rho-associated coiled-coil kinase (Rho kinase/ROCK) and Zipper-interacting protein kinase (ZIP kinase/ZIPK) have been reported to phosphorylate MRLC at the contractile ring. However, it remains unclear whether these kinases function independently of each other. Here, we clarified that ROCK colocalizes and forms a complex with ZIPK at telophase. As ROCK is reported to phosphorylate and activate ZIPK in vitro, we hypothesized that ZIPK phosphorylated by ROCK contributes to control cytokinesis. To address this, we expressed EGFP-ZIPK wild type (WT), a non-phosphorylatable mutant (T265A) or a phosphorylation-mimicking mutant (T265D) in HeLa cells and treated these cells with a ROCK inhibitor. Decrease in phosphorylated MRLC and a delay of furrow ingression by the ROCK inhibitor were rescued by the expression of EGFP-ZIPK-T265D, but not EGFP-ZIPK-WT or -T265A. This suggests that ROCK regulates MRLC phosphorylation followed by furrow ingression, through ZIPK phosphorylation.

Impairment of cytokinesis by cancer-associated DAPK3 mutations.

Death-associated protein kinase 3 (DAPK3), a member of the DAPK family, contributes to cytokinesis by phosphorylating myosin II regulatory light chain (MRLC). Missense mutations in DAPK3, T112M, D161N, and P216S, were observed in the lung, colon, and cervical cancers, respectively, but the effects of these mutations on cytokinesis remain unclear. Here, we show that cells expressing EGFP-DAPK3-T112M, -D161N, or -P216S exhibited reduced rates of cytokinesis, with an increased ratio of multinucleated cells. In addition, these cells exhibited reduced levels of phosphorylated MRLC at the contractile ring. Collectively, our data demonstrates that cancer-associated DAPK3 mutations impair cytokinesis by reducing phosphorylated MRLC.