Phosphatase-Regulated Recruitment of the Spindle and Kinetochore Associated (SKA) Complex to Kinetochores

Kinetochores move chromosomes on dynamic spindle microtubules and regulate cell cycle progression by signaling the spindle checkpoint. The Spindle and Kinetochore-Associated (Ska) Complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, participates in both roles. The mitotic kinases, Cdk1, Aurora B, Plk1, Mps1 and Bub1 play key, overlapping tasks in regulating chromosome movement and checkpoint signaling. However, roles for the phosphatases that oppose these kinases are more poorly defined. Recently, we showed that Ska1 is important for recruiting protein phosphatase 1 (PP1) to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A) both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes also seen after depletion of Ska. Tethering of PP1 to the kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and reduces mitotic defects seen after Ska depletion. We propose that kinetochore-associated phosphatases generate a positive feedback cycle to reinforce Ska complex accumulation and function at kinetochores. SUMMARY STATEMENT Phosphatases reinforce recruitment of the Ska complex at kinetochores to stabilize microtubule attachment and oppose spindle checkpoint signaling.


INTRODUCTION
At mitotic entry, protein kinases phosphorylate numerous substrates to trigger important events such as chromosome condensation, nuclear envelope breakdown, mitotic spindle assembly, and proper kinetochore-microtubule interactions (Bollen et al., 2009;Wurzenberger and Gerlich, 2011). Throughout mitosis and particularly at mitotic exit, these phosphorylations are opposed by phosphatases. In mammalian cells, PP1 and PP2A, in association with specific regulatory and targeting subunits, are thought to dephosphorylate many of the substrates targeted by mitotic kinases (Bollen et al., 2009).
Phosphorylation of Ska by Aurora B kinase inhibits its binding to kinetochores, but paradoxically, a recent study reported that Ska also promotes Aurora B accumulation on kinetochores and increases its kinase activity in vivo and in vitro (Chan et al., 2012;Redli et al., 2016).
In this study, we show that PP1 and PP2A phosphatases promote Ska recruitment to kinetochores. These results corroborate and extend previous work (Redli et al., 2016).
Forced targeting of PP1 to kinetochores partially rescues defects caused by Ska3 depletion. We propose a feedback mechanism in which the Ska complex recruits PP1 to kinetochores at metaphase which further recruits Ska to stabilize kinetochore-microtubule attachments and initiate anaphase.

PHOSPHATASES PROMOTE ACCUMULATION OF SKA AT KINETOCHORES
We and others have shown that Ska binds to kinetochores at prometaphase and maximally accumulates there at metaphase (Chan et al., 2012;Redli et al., 2016;Sivakumar et al., 2014). Inhibition of Aurora B kinase increased Ska accumulation on kinetochores lacking microtubule attachment (Chan et al., 2012). Correspondingly, expression of phosphomimetic mutants of Ska inhibited recruitment (Chan et al., 2012).
These findings and recent data from Redli et al (2016) indicate phosphatases likely regulate Ska binding to kinetochores. PP1 and PP2A are the major phosphatases implicated in mitotic transitions. PP1, principally the PP1γ isoform, localizes to kinetochores and is implicated in spindle checkpoint inactivation Trinkle-Mulcahy et al., 2003). PP2A also accumulates at kinetochores and plays a role in promoting kinetochore-microtubule attachment in prometaphase (Foley et al., 2011). To test the role of the phosphatases in Ska recruitment, we depleted PP1γ or PP2A Aα subunit ( Figure S1A, S1B). We analyzed recruitment of Ska to kinetochores using immunofluorescence with antibody to Ska3. To normalize for the effect of microtubule binding, we performed these experiments in nocodazole-treated cells. Depletion of PP1 or PP2A phosphatase significantly decreased Ska3 accumulation at kinetochores ( Figure   1A, 1B). Plk1 and BubR1 promote PP2A recruitment to kinetochores (Foley et al., 2011;Suijkerbuijk et al., 2012). Depletion of Plk1 or BubR1 with siRNA caused the expected reduction of PP2A at kinetochores in asynchronous cells (Figure S1C-E) and also resulted in lower levels of kinetochore-associated Ska3 in nocodazole ( Figure 1C, 1D).
Since PP1 and PP2A promote Ska kinetochore binding we tested if depletion of the phosphatases in our hands generated phenotypes similar to Ska depletion. Depletion of PP1γ caused small delays in chromosome alignment ( Figure 1E). As reported previously (Foley et al., 2011;Tang et al., 2006), depletion of PP2A Aα resulted in delayed chromosome alignment ( Figure 1E). Combined depletions of both PP1γ and PP2A Aα/β delayed chromosome alignment yet more ( Figure 1E). Depletion of PP1γ by RNAi delayed the metaphase-anaphase transition modestly, confirming its previously demonstrated role in opposing spindle checkpoint signaling . Depletion of PP2A Aα/β also delayed the metaphase-anaphase transition, but co-depletion of PP1γ and PP2A Aα/β showed the strongest delay, consistent with both PP1 and PP2A promoting the metaphase-anaphase transition ( Figure 1F) (Grallert et al., 2015;Lee et al., 2017).
As a second approach to test the roles of phosphatases and kinases in Ska kinetochore recruitment we used small molecule kinase and phosphatase inhibitors to examine effects on Ska recruitment to kinetochores. HeLa cells were treated in the presence of nocodazole to depolymerize microtubules and the proteasome inhibitor MG132 to block mitotic exit. As reported previously, inhibition of Aurora kinase increased recruitment of Ska to kinetochores (Chan et al., 2012). However, in contrast to previous findings we found that treatment of cells with an inhibitor of Mps1 also increased Ska recruitment (Figure 2A, 2B) (Chan et al., 2012). This finding is consistent with roles for Mps1 in recruitment of Aurora B and BubR1 to kinetochores (Krenn et al., 2014;Overlack et al., 2015;van der Waal et al., 2012;Zhang et al., 2014). As also reported in a recent study (Redli et al., 2016), we found that treatment of cells with the phosphatase inhibitor, okadaic acid, decreased Ska accumulation at kinetochores (Figure 2A, 2B), consistent with results from RNAi depletion of phosphatases described above. Previously we found that Ska complex promotes APC/C accumulation on mitotic chromosomes (Sivakumar et al., 2014). We tested if kinase or phosphatase inhibitors that affected kinetochore concentration of Ska had similar effects on chromosome-bound APC/C. Indeed we found that inhibitors of Aurora B or Mps1 increased APC/C on chromosomes while the phosphatase inhibitor, okadaic acid, decreased APC/C on chromosomes ( Figure 2C, 2D).

RECRUITMENT
We previously demonstrated that an important function of the C-terminus of Ska1 is to recruit PP1 to kinetochores (Sivakumar et al., 2016). In those experiments, Ska2, Ska3 and the N terminus of Ska1 and their potential separate functions were retained. We constructed a fusion of the outer kinetochore protein, Nuf2, to PP1. Nuf2 is a component of the Ndc80 complex, which is implicated in recruiting Ska to kinetochores (Chan et al., 2012;Zhang et al., 2012). We reasoned that the Nuf2PP1 fusion would target PP1 in close proximity to the normal Ska-recruited PP1. Expression of the Nuf2PP1 fusion induced a significant increase in kinetochore-associated PP1 both in cells arrested in a prometaphase-like state in nocodazole and at metaphase in MG132 ( Figure 3A, 3B, 3C) even though the Nuf2PPI fusion was expressed at lower levels than unfused Nuf2 or PP1 in controls ( Figure S2A). We then analyzed Ska recruitment. Interestingly, in nocodazole- Previously we showed that Ska promotes binding of the APC/C to mitotic chromosomes (Sivakumar et al., 2014). Similar to the findings described above regarding Ska recruitment to kinetochores, we found that expression of Nuf2PP1 did not impact chromosome-bound APC/C in nocodazole-treated cells, but did increase it in cells arrested at metaphase with MG132 ( Figure 3G, 3H, S2C).

SKA3-DEPLETED CELLS
Depletion of any Ska component results in delayed alignment followed by a robust mitotic delay or arrest at metaphase (Daum et al., 2009;Redli et al., 2016;Schmidt et al., 2012;Sivakumar et al., 2014). The metaphase delay often results in cohesion fatigue, asynchronous separation of chromatids without mitotic exit (Daum et al., 2011;Sivakumar et al., 2014;Stevens et al., 2011). We tested if Nuf2PP1 expression rescued mitotic defects in Ska-depleted cells. In control cells, expression of Nuf2, PP1 or Nuf2PP1 did not alter mitosis ( Figure S2D). In Ska3-depleted cells, expression of the Nuf2PP1, but not Nuf2 or PP1 alone, improved alignment ( Figure 4A, 4B). In addition, while 63% of Skadepleted cells expressing Nuf2 or PP1 arrested at metaphase and underwent cohesion fatigue, only 22% of cells expressing the Nuf2PP1 fusion did so ( Figure 4C). However, the rescue was incomplete, most Ska3-depleted cells expressing the Nuf2PP1 fusion still exhibited a significant delay before entering anaphase ( Figure 4A). There are potential technical reasons why the rescues were incomplete. Both PP1 and Ska have been shown to exhibit rapid turnover at kinetochores (Raaijmakers et al., 2009;Trinkle-Mulcahy et al., 2001). In contrast the Ndc80 complex, of which Nuf2 is a component, is stable (Hori et al., 2003). Thus, Nuf2PP1 likely fails to exhibit the normal dynamics of PP1 and this limitation possibly prevents complete rescue of Ska depletion phenotypes.

CONCLUSIONS
Here we show that PP1 and PP2A are required for full accumulation of Ska on kinetochores. Though cells depleted of PP1γ and PP2A Aα/β show alignment defects and metaphase delays, they rarely exhibit the strong phenotypes characteristic of Ska depletions ( Figure 1G). There are possible technical explanations for this observation.
PP1 and PP2A proteins may be long-lived or be sufficient at low protein concentration.
If PP1 recruitment were the primary function of Ska, expression of Nuf2PP1 in Skadepleted cells might be expected to completely alleviate phenotypes caused by reduction of Ska levels. We found only partial rescue. Most likely, PP1 recruitment, a function of the C terminus of Ska1, is only one function of the full Ska complex. Additional roles such as microtubule tracking may also promote alignment and timely metaphase-anaphase transition (Abad et al., 2014;Abad et al., 2016;Chan et al., 2012;Gaitanos et al., 2009;Raaijmakers et al., 2009;Redli et al., 2016;Schmidt et al., 2012;Welburn et al., 2009). PP1 has been shown to be integral in opposing spindle checkpoint signaling and is required to promote normal anaphase onset Sivakumar et al., 2016). PP1 levels at kinetochores increase from prometaphase to metaphase. We speculate that low levels of PP1 and Ska at kinetochores and APC/C on chromosomes in prometaphase prevent premature anaphase onset and mitotic exit. Then, at metaphase Ska binding to microtubules increases its kinetochore concentration creating a positive feedback loop whereby PP1 accumulation at kinetochores further increases Ska leading to accumulation of kinetochore PP1 and chromosome APC/C. This feedback strengthens microtubule attachment, opposes spindle checkpoint signaling and promotes the rapid and irreversible transition to anaphase and mitotic exit.

CELL CULTURE
HeLa cells stably transfected with GFP fused to Histone 2B (HeLa H2B-GFP) was used in this study. HeLa cell lines were grown in culture flasks or chambered coverslips in DMEM-based media with 10% FBS supplemented with penicillin and streptomycin in 5% CO 2 at 37 o C. Cell lines were routinely tested for mycoplasma contamination and only used if not contaminated. (CGAGUGACCGAUUAUGCUU and GUCUGAGGAGUAAGUGUAC) were obtained from Bioneer Inc. and these were used at 50-100nM final concentration. siRNA against Ska3 was obtained from Dharmacon and used at 50nM final concentration (Daum et al., 2009).

PLASMIDS
Nuf2, PP1 or Nuf2PP1-mCherry plasmids were constructed by inserting respective cDNA in mCherry-N1 vector. mCherry tag was inserted upstream or downstream of transgene and similar results were obtained with proteins tagged in N or C termini.

LIVE CELL IMAGING
HeLa H2B-GFP cells were grown in Nunc chambered coverslips (Thermo Sci. Inc.

IMMUNOFLUORESCENCE AND QUANTIFICATION
HeLa cells were grown on glass coverslips and treated as detailed in the figure legends.
Cells were pre-extracted in PHEM/1% triton solution for 5 minutes before fixing with 1.