Abstract
Dlk/ZIP kinase is a serine/threonine kinase highly homologous to DAP kinase. We have reported that HeLa ZIP kinase (hZIPK) phosphorylated the regulatory light chain of myosin II (MRLC) at both Ser19 and Thr18 in vitro. In this study, we demonstrate that hZIPK also induces the diphosphorylation of MRLC in nonmuscle cells. Peptide mapping revealed that transient transfection of hZIPK into HeLa cells caused diphosphorylation of MRLC. In contrast, transfection of the kinase inactive mutant of hZIPK did not induce any phosphorylation of MRLC. Using antibodies specific for mono- or diphosphorylated MRLC, we showed that diphosphorylated MRLC induced by the overexpression of hZIPK was concentrated in striking aggregates or bundles of actin filaments in HeLa cells, while monophosphorylated MRLC showed no prominent localization to these aggregates. Overexpression of hZIPK also induced dramatic changes in cell shape and disruption of nuclear morphology reminiscent of changes during apoptosis. These effects of hZIPK were suppressed by the coexpression of a mutant MRLC where both phosphorylation sites were replaced with alanine, indicating that the changes in actin organization were a consequence of MRLC diphosphorylation. These results suggested that hZIPK plays a role in regulating actin organization and cell morphology in non-muscles and at least part of its effects are mediated through the diphosphorylation of MRLC.
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Acknowledgements
We are grateful to Drs K Katoh (National Cardiovascular Center Research Institute) and K Fujiwara (University of Rochester) for providing anti-pan myosin antibody. We thank A Kikuchi (Hiroshima University) for preparation of MLCK. We also thank Dr Yu-Li Wang (University of Massachusetts) for critical reading of this manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to M Murata-Hori and H Hosoya and by the Hayashi Memorial Foundation for Female Natural Scientists to M Murata-Hori.
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Murata-Hori, M., Fukuta, Y., Ueda, K. et al. HeLa ZIP kinase induces diphosphorylation of myosin II regulatory light chain and reorganization of actin filaments in nonmuscle cells. Oncogene 20, 8175–8183 (2001). https://doi.org/10.1038/sj.onc.1205055
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DOI: https://doi.org/10.1038/sj.onc.1205055
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