Dma1 mediates SIN inhibition by preventing the Polo-like kinase Plo1, an upstream activator of the SIN, from localizing to the SPB (Guertin et al., 2002b
). Sid4 ubiquitination by Dma1 prevents SIN activation and cytokinesis during a mitotic checkpoint arrest by delaying recruitment of Plo1 to SPBs while at the same time prolonging the residence of the SIN inhibitor Byr4 (Johnson and Gould, 2011
). However, Dma1 does not seem to inhibit Plo1 function in early mitotic events such as spindle assembly, suggesting that Dma1 itself may be regulated to limit its ability to antagonize Plo1.
In this study, we identified Dnt1 as a factor that negatively regulates the extent of Dma1 recruitment to SPBs and Dma1's E3 ubiquitin ligase activity in early mitosis, possibly in response to a mitotic checkpoint. Given that we detected a strong interaction between Dma1 and Dnt1 in both metaphase and anaphase, Dnt1 might modulate Dma1 and SIN activity throughout mitosis, with the interaction in early mitosis being important for Plo1 regulation and spindle assembly. Previous studies showed that both Dma1 and Dnt1 are negative regulators of the SIN pathway (Murone and Simanis, 1996
; Guertin et al., 2002b
; Jin et al., 2007
). These seemingly contradictory results could be reconciled if Dnt1 acts to inhibit Dma1 in early mitosis, and then the two proteins function together or separately to inhibit the SIN in anaphase.
Similar to mammalian polo-like kinase 1 (Plk1), Plo1 in fission yeast is critical for proper mitotic progression, and its association with the SPB is important for microtubule nucleation and function (Ohkura et al., 1995
; Bahler et al., 1998a
). Previous studies identified Sid4, Cut12, and Pcp1 as major factors that recruit Plo1 to SPBs in fission yeast (Mulvihill et al., 1999
; Morrell et al., 2004
; Fong et al., 2010
). On the basis of the results from the present study, we propose a model in which Dnt1 regulates Plo1 kinase through inhibition of Dma1 (). Dnt1 might function to fine tune Dma1 activity at the SPB to avoid problematic inhibitory effects on Plo1. However, because the rescue of the growth defects in dnt1Δ plo1
double mutants by removal of Dma1 was not complete ( and Supplemental Figure S3), there might be additional functions for Dnt1 in the regulation of the SPB and mitosis.
FIGURE 6: Model for Dnt1 regulation of Dma1 in early mitosis. Dnt1 is phosphorylated by an unknown kinase in prometaphase, which promotes binding of Dnt1 to Dma1 through Dma1's FHA domain. Dnt1–Dma1 interaction inhibits Dma1, which normally targets Sid4 (more ...)
The mammalian protein Chfr functions in an early mitotic checkpoint that delays the cell cycle in response to microtubule-targeting drugs by inhibiting polo kinase (Plk1) and the Aurora A kinase (Scolnick and Halazonetis, 2000
; Kang et al., 2002
; Yu et al., 2005
). Like Chfr, Dma1 also inhibits the Polo kinase Plo1 to maintain a cell cycle arrest after microtubule depolymerization (Murone and Simanis, 1996
; Guertin et al., 2002b
), although, unlike Chfr, which blocks progression into prophase (Scolnick and Halazonetis, 2000
; Summers et al., 2005
), Dma1 blocks initiation of cytokinesis. Of interest, a recent study identified a cytosolic and centrosomal protein, Stil, as the first negative regulator of the mammalian Chfr in mouse embryonic fibroblasts (Castiel et al., 2011
). Stil limits Chfr's inhibition of Plk1 in early mitosis to allow normal mitotic progression and proper centrosome assembly by affecting Chfr's stability and protein level (Castiel et al., 2011
). Therefore the activities of both Chfr and Dma1 seem to be carefully modulated to keep them from interfering with normal mitotic progression (Castiel et al., 2011
, and this study). Although fission yeast Dnt1 and mammalian Stil do not show any amino acid sequence similarity, they do share similar functions in antagonizing their respective E3 ubiquitin ligase.
It is not known how Dnt1–Dma1 interaction is regulated. However, our results show that the interaction depends on Dnt1 phosphorylation. Because the interaction between the two proteins is strongest in early mitosis, when multiple kinases are active, phosphorylation of Dnt1 would provide a mechanism for the cell cycle–specific interaction between the two proteins. Clearly, finding the kinase(s) responsible for Dnt1 phosphorylation is an important goal for future studies.
In summary, we showed that in the absence of Dnt1, Dma1 can inhibit SPB function and spindle formation much like Chfr in human cells. Thus it is possible that Dma1 acts like Chfr to inhibit mitotic progression in response to certain stimuli. Because the ability of Dma1 to block mitotic progression requires loss of Dnt1, it will be interesting in future studies to determine the conditions that modulate the interaction between Dnt1 and Dma1 to regulate passage through early mitosis.