Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells

Abstract

Self-regulated movement of Polo-like kinase 1 to the midzone of the mitotic spindle initiates a local signaling cascade that activates the cell division machinery at the cell's equator.

Abstract

Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.

Author Summary

During mitosis, the separation of duplicated chromosomes and subsequent cytokinesis (cell division) are tightly coupled processes. Cytokinesis must occur not only after chromosomes have separated but also in the physical space between the chromosomes, so that each daughter cell inherits the appropriate genetic material. The mechanisms responsible for this cellular choreography are poorly understood, however. We used chemical genetics to dissect the role of a key regulator of cell division, Polo-like kinase 1 (Plk1) in human cells. We show that, contrary to previous models, the ability of Plk1 to seek out microtubules that lie between the separated chromosomes (so-called midzone microtubules) provides the cell with an affirmative command to divide. Once assembled at this landmark, Plk1 phosphorylates HsCYK-4, a component of the centralspindlin complex (so named because it assembles at the spindle midzone) and enables binding between HsCYK-4 and Ect2, another regulator of cell division. Bound Ect2 then communicates with the machinery that assembles the actin- and myosin-based contractile ring, leading to division of the cell into two daughters. Our work therefore reveals new insights into how Plk1 temporally and spatially orchestrates division of human cells.