This study has revealed a novel function of the HECT E3 ligase Smurf2 in mitosis. During unperturbed mitotic progression, Smurf2 dynamically relocalizes from the centrosome to the center of the anaphase spindle midzone and finally to the midbody in the intercellular bridge during cytokinesis. Smurf2 is also observed at unattached centromeres during spindle assembly checkpoint activation. Smurf2 depletion impairs the spindle assembly checkpoint, leading to increased misalignment and missegregation of chromosomes, premature anaphase onset, and defective cytokinesis. The function of Smurf2 in the spindle assembly checkpoint is mediated at least partly by the stability and localization of Mad2 because Smurf2 depletion results in enhanced polyubiquitination and rapid destabilization of Mad2 protein. Mad2 destabilization presumably leads to premature activation of APC/C-Cdc20, which is associated with impaired mitotic accumulation of the APC/C-Cdc20 substrates such as cyclin B1 and securin. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of these proteins, confirming the antagonistic function of Smurf2 against APC/C-Cdc20 activity. Ectopic expression of Mad2 is insufficient to restore the spindle assembly checkpoint in Smurf2-depleted cells, apparently because Mad2 mislocalizes in the absence of Smurf2. Collectively, Smurf2 is a novel regulator of the Mad2-dependent spindle assembly checkpoint.
Mad2 interaction with kinetochore-associated checkpoint proteins, such as Mad1 and Bub1, is required for the inhibition of APC/C-Cdc20 until the proper alignment of all mitotic spindles (Nasmyth, 2005
; Yu, 2006
). Mad2, together with BubR1 and Bub3, exhibits rapid movement on and off kinetochores, transiently interacting with Mad1- and Bub1-associated centromeres (Howell et al., 2004
). This dynamic regulation of Mad2 is critical for the diffusible inhibitory signal against cytoplasmic APC/C-Cdc20. This study presents evidence that Smurf2 activity is required for the normal progression of mitosis as well as for spindle disruption–mediated mitotic arrest. Similarly, cells with Mad2 or BubR1 depletion show various mitotic defects (i.e., failure in chromosome segregation, premature anaphase onset, and cytokinesis defects; Taylor and McKeon, 1997
; Gorbsky et al., 1998
; Meraldi et al., 2004
). Analogous phenotypes have been observed in cells with depletion of USP44, a deubiquitinating enzyme that counteracts disassembly of Mad2–Cdc20 complexes by removing ubiquitin from Cdc20 (Stegmeier et al., 2007
). The binding of Mad2 to Cdc20 requires a substantial conformational change of Mad2, which Mad1 seems to facilitate at the kinetochore (Yu, 2006
). Although it remains to be elucidated how Smurf2 regulates the stability and localization of Mad2, it is tempting to speculate that the E3 ligase activity of Smurf2 is involved in regulating Mad2 conformation. Smurf2 levels are highest before the metaphase–anaphase transition, when Smurf2 localizes predominantly at spindle poles/centrosomes. Mad2 also has been found at spindle poles before anaphase onset, possibly via microtubule-mediated transit between kinetochores and spindle poles (Howell et al., 2000
; Shah et al., 2004
). Depletion of Mad2 and BubR1 results in kinetochore-independent premature anaphase onset, whereas knockdown of other spindle checkpoint components (e.g., Bub3 and Bub1) impairs only kinetochore-dependent checkpoint function, not the timing of anaphase onset (Meraldi et al., 2004
). Our data demonstrating premature anaphase onset in Smurf2-depleted cells support a hypothesis that a functional interaction of Smurf2 and Mad2 that occurs dynamically between spindle poles/centrosome and kinetochores may control the spindle assembly checkpoint. Of course, it is possible that Smurf2 plays multiple roles in mitosis and cytokinesis by affecting multiple downstream targets in addition to Mad2. A previous study showed that Smad2, 3, and 4 bind to microtubules in the absence of TGF-β, colocalizing with tubulin at the bipolar spindle and midzone (Dong et al., 2000
). These Smad proteins can function as a cofactor of multiple E3 ligases, including SCF (skp1-Cul1-F-box), APC/C, and Smurf2 (Fukuchi et al., 2001
; Ogunjimi et al., 2005
; Ray et al., 2005
). Therefore, the cofactor Smad protein might complex with Smurf2 alongside microtubules and modulate Smurf2 activity.
Regulation upstream of the spindle checkpoint components has been understudied. The transcriptional control of Mad2 by E2F and Myc was recently identified (Hernando et al., 2004
); however, it had been unknown whether cellular levels of Mad2 protein are regulated by ubiquitination. To our knowledge, this study is the first to demonstrate that Mad2 undergoes polyubiquitination and proteasomal degradation. The experiments using Smurf2 siRNA and Smurf2(C716A) showed that the E3 ligase activity of Smurf2 is required to prevent Mad2 from polyubiquitination and degradation (). Smurf2 physically associates with Mad2. Although Smurf2 is likely to affect the modification and/or conformation of Mad2 at close proximity, the precise mechanism remains to be determined. A hypothetical mechanism is that Smurf2 directly modifies Mad2 by monoubiquitination or Lys63-linked polyubiquitination (Haglund and Dikic, 2005
), and such a modification somehow alters the conformation of Mad2 to a stable form and promotes its dynamic localization at kinetochores critical for surveillance of spindle integrity. For instance, the chromatin passenger complex protein survivin undergoes Lys63 polyubiquitination, which is required for centromere localization of this protein (Vong et al., 2005
). When the level of active Smurf2 is low, Mad2 undergoes an alternative form of ubiquitination, presumably Lys48-linked polyubiquitination mediated by an undefined E3 ligase, and is recruited to proteasomal degradation. Another possibility is that Smurf2 plays an indirect role in the control of Mad2 stability. Smurf2 may antagonize the action of an unidentified E3 ligase required for proteasomal degradation of Mad2, possibly by ubiquitinating and destabilizing the E3 ligase. In this case, Smurf2 depletion would up-regulate the E3 ligase by stabilization, leading to enhanced Mad2 polyubiquitination and subsequent proteasomal degradation. Further biochemical investigations are needed to evaluate these hypotheses regarding the Mad2 regulatory function of Smurf2.
Putative roles of Smurf2 in regulation of the spindle assembly checkpoint.