Abstract
Cell division-related proteins are essential for the normal development and differentiation of cells and may be related to the occurrence of cancer and the drug resistance mechanism of cancer cells. The mitotic kinesin-like protein 1 (MKLP1) is a kinesin protein that has been involved in the assembly of the midzone/midbody during mitosis and cytokinesis. In this study, we found that the tail domain of MKLP1 exhibited an autoinhibitory effect on its motor activity. Overexpression of the tail domain in HEK293 cells blocked cytokinesis and caused bi-/multinucleation. It is possible that protein binding to the MKLP1 tail relieves this autoinhibition and induces the motility of MKLP1. We used the GST pull-down assay followed by the LC-MS/MS analysis and identified 54 MKLP1 tail domain-specific binding proteins. Further, we confirmed the MS result by coimmunoprecipitation and FRET that a serine/threonine kinase, p21-activated kinase 2 (PAK2), binding to MKLP1. Endogenous PAK2 expression was found to be identical to that of MKLP1 in HEK293 cells during cytokinesis. Finally, functional studies indicated that when PAK2 expression was downregulated by siRNA, MKLP1 underwent a change in its localization away from the midbody, and cell cytokinesis was subsequently impeded. This study presents a novel regulatory mechanism that PAK2 promotes the activation of MKLP1 and contributes to complete cell cytokinesis.
Highlights
Proper cell division requires spatiotemporal coordination of cytoplasmic division and nuclear division to ensure that each daughter cell receives a complete chromosome and proper cytoplasmic and organelle components
We found that overexpression of the tail domain of mitotic kinesin-like protein 1 (MKLP1) in HEK293 cells inhibits the completion of cytokinesis and causes bi-/multinucleation
MKLP1 can move along microtubules to the spindle midzone and the cleavage furrow during anaphase, concentrating at the midbody during cytokinesis and executing its normal function during cell division [11]
Summary
Proper cell division requires spatiotemporal coordination of cytoplasmic division (cytokinesis) and nuclear division (mitosis) to ensure that each daughter cell receives a complete chromosome and proper cytoplasmic and organelle components. Overexpression of an ATP-binding mutant of MKLP1 inhibits the completion of cytokinesis and caused bi-/multinucleation [1]. These results indicate that the motor activity of MKLP1 is necessary for the completion of cytokinesis. We found that overexpression of the tail domain of MKLP1 in HEK293 cells inhibits the completion of cytokinesis and causes bi-/multinucleation. This finding suggests that the tail domain of MKLP1 exhibited autoinhibition on its motor activity like the other kinesin proteins [3,4,5,6]. By RNA-interference-mediated knockdown of PAK2 expression, we proved that PAK2 was involved in cell cytokinesis, perhaps through its regulatory effect on MKLP1 localization
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