This study shows that phosphatase inhibitor 2 (I-2) is required for the successful completion of mitosis in human epithelial cells. Knockdown of I-2 by RNAi reduced the I-2 protein levels and resulted in accumulation of cells with multiple nuclei and supernumerary centrosomes. We demonstrate the effects are dependent on progression through the cell cycle, and we show by time-lapse microscopy that the phenotypes arise from failed cytokinesis and subsequent retention of duplicated chromosomes and centrosomes in single cells. Cytokinesis failure and accumulation of multi-nucleated cells have been associated with lagging chromosomes during anaphase and telophase (Shi and King, 2005
). We observed lagging chromosomes in I-2 knockdown cells and faulty chromosome segregation could have caused or contributed to the failure of cytokinesis. Asymmetric kinetochore–microtubule attachments can cause chromosomes to not congress to the metaphase plate in a timely manner. Aurora B kinase is required for correction of asymmetric kinetochore–microtubule attachments and maintenance of the spindle checkpoint (Kallio et al., 2002
; Hauf et al., 2003
). Aurora B is thought to promote resolution of microtubule attachments by phosphorylation of MCAK and Hec1 proteins at kinetochores (Walczak and Heald, 2008
). Aurora B and PP1 form a phosphorylation gradient across the midzone during mitosis to determine the location of the cleavage furrow for cytokinesis (Fuller et al., 2008
). These considerations suggest the mitotic function of I-2 is linked to the regulation and actions of Aurora B.
Consistent with a relationship between I-2 and Aurora B, we found knockdown of I-2 was equivalent to hesperadin partial inhibition of Aurora B. This was in terms of formation of multinucleated cells, override of the spindle checkpoint imposed by taxol, and phosphorylation of histone H3S10. Conversely, I-2 overexpression prevented the formation of multinucleated cells in response to hesperadin without causing cell cycle arrest. We suspect that Aurora B-dependent phosphorylation events were enhanced by I-2 overexpression probably by inhibition of certain PP1 holoenzymes. Knockdown of I-2 could have generated lagging chromosomes by increasing PP1 activity, thereby indirectly reducing phosphorylation of Aurora B substrates at kinetochores. In addition, both I-2 and Aurora B are localized to the midzone in anaphase and to the midbody during cytokinesis. Previous studies have shown that Aurora B regulates cytokinesis by phosphorylation of certain substrates in the midzone and midbody (Delaval et al., 2004
; Guse et al., 2005
). Therefore, we hypothesize that in addition to lagging chromosomes, the failure of cytokinesis in I-2 knockdown cells was due at least partially to reduced Aurora B activity and/or reduced phosphorylation of its substrates in the midzone and midbody, because of enhanced local PP1 activity. I-2 might not regulate the PP1 holoenzyme reactive with Aurora B because we did not observe a change in anti-phospho-T232 staining of Aurora B in the midzone or midbody of I-2 knockdown cells, relative to cells treated with control siRNA. However, there could be other I-2–dependent mechanisms for Aurora B activation in addition to T232 phosphorylation (e.g., other phosphosites in Aurora B) that would not be detected by pT232 immunostaining. Alternatively, I-2 may primarily be regulating the activity of PP1 holoenzymes that dephosphorylate Aurora B substrates. We propose there are one or more I-2 sensitive holoenzyme forms of PP1 that inactivate Aurora B and/or dephosphorylate Aurora B substrates during mitosis to account for the phenotypes ().
Figure 9. Model for regulation of Aurora B by PP1 and I-2 during mitosis. Aurora B is activated by autophosphorylation of T232 in its activation loop, and this reaction can be enhanced by specific binding partners and is reversed by PP1 that inactivates the kinase. (more ...)
Besides Aurora kinases, another Ser/Thr kinase, Nek2A, has been claimed to regulate kinetochore–microtubule attachments and spindle checkpoint signaling (Lou et al., 2004
; Du et al., 2008
). Our previous studies have shown that I-2 binds to Nek2A–PP1 complexes to activate the Nek2A (Eto et al., 2002
; Li et al., 2007
). The Nek2A–PP1 complex involves reciprocal negative feedback and autoactivation, predicted to respond as a bistable switch (Ferrell, 2002
). Therefore, Nek2A is another candidate that might mediate the actions of I-2 and be affected by RNAi knockdown. However, we found knockdown of Nek2A did not mimic the effects of knockdown of I-2. Furthermore, coincident knockdown of Nek2A in I-2 knockdown cells did not enhance or reverse the appearance of abnormal or multiple nuclei (Supplemental Figure S6). This implies that knockdown of I-2 produces phenotypes independent of Nek2A.
More than 30 y ago, I-2 was discovered as a heat-stable protein inhibitor of PP1 (Huang and Glinsmann, 1976
) and has been used over the years as a selective inhibitor to implicate PP1 in various biological processes, and to differentiate between PP2A, PP2C, and other phosphatases (Ingebritsen and Cohen, 1983
; Cohen, 1991
). The approach depended on the assumption that I-2 inhibited all the PP1 present. More recent comparison of I-2 with another PP1-specific inhibitor protein, CPI-17, used affinity chromatography on immobilized inhibitor protein to separate from cell extracts-specific pools of PP1 holoenzymes with different regulatory subunits (Eto et al., 2004
). Thus, one needs to consider that actions of I-2 are mediated through selective inhibition of only a fraction of PP1 in cells. The specificity of PP1 inhibitor proteins for different PP1 holoenzymes accounts for the inability of PHI-1 knockdown to mimic knockdown of I-2. Knockdown of PHI-1 reduces the rates of cell spreading and migration (Tountas and Brautigan, 2004
; Tountas et al., 2004
), probably through deregulation of a juxtamembrane form of PP1 that is responsible for PHI-1 localization in cells and tissues. Our view is that different PP1 inhibitor proteins regulate specific PP1 holoenzymes to regulate discrete cellular processes. To fully understand the basis for I-2 function in mitosis will require identification of the mitotic PP1 complex(es) inhibited by I-2, a future goal of investigations.
Although thought to be a simple protein inhibitor for PP1, I-2 functions in a network of biochemical reactions during mitosis and has several possible actions in cells that might be compromised by knockdown. I-2 is specifically phosphorylated at a conserved PXTP site during early mitosis by the kinase cdc2-cyclinB (Leach et al., 2003
; Li et al., 2006
). Biochemical studies of PP1::I-2 heterodimer showed years ago that phosphorylation-dephosphorylation of this PXTP site in I-2 causes conformational activation of bound PP1 (Ballou et al., 1985
). Therefore, mitotic phosphorylation of I-2 might conceivably involve activation of specific PP1 holoenzymes. In yeast, either deletion or overexpression of the I-2 homologue GLC8 suppresses mutations in Ipl1
(Aurora kinase), suggesting possible activation or inhibition of GLC7 (PP1). Treatment of mitotic cells with high-dose hesperadin (500 nM) did not interfere with mitotic phosphorylation of I-2 (data not shown), indicating that Aurora B was not required. Phosphorylation of the PXTP site in I-2 also effectively eliminates its binding to the Pin1 prolyl isomerase (Li et al., 2008
), an enzyme that reacts with a host of mitotic phosphoproteins (Shen et al., 1998
). Binding of I-2 to Pin1 drastically alters substrate specificity of Pin1 (Li et al., 2008
), providing another possible mechanism for I-2 to control events during mitosis. Thus, during mitosis I-2 regulates kinases, phosphatases and Pin1 prolyl isomerase to coordinate events in a complex network of regulatory reactions.
Control of chromosome segregation is a conserved function of I-2. In Drosophila
, I-2 is a maternal function gene and the I-2 protein is loaded into the oocyte for early rounds of mitosis within the syncytial embryo (Wang et al., 2008
). Embryos of mutant mothers with reduced levels of I-2 exhibit defects in chromosome segregation, with DNA bridges linking adjacent nuclei, and they show loss of synchrony of mitosis across the embryo (Wang et al., 2008
). Reduction of maternal I-2 levels results in a profound loss of viability in terms of hatching rate and survival of the larvae, effects that are partially rescued by dose-dependent transgenic expression of Drosophila
I-2. These in vivo results provide strong support for our conclusions based on experiments in ARPE-19 cells. Furthermore, we now have evidence from two organisms in addition to yeast that I-2 plays a critical role in mitosis.
Aurora B kinase is over expressed in a variety of human tumors (Bischoff et al., 1998
; Vischioni et al., 2006
), and is implicated in tumorigenesis (Sorrentino et al., 2005
). Aurora B kinase has been highlighted as a target for anticancer drug development, small molecule inhibitors have been generated, and these already show promising antitumor activity (Keen and Taylor, 2004
). Our discovery that I-2 is highly expressed in several cancer cell lines, and ectopic I-2 overexpression makes cells resistant to hesperadin, suggest that I-2 levels may at least influence the sensitivity of tumors to Aurora B inhibition. Further, I-2 itself might be a target for anti-tumor drug development, and blocking its action or suppressing its levels may increase the sensitivity of tumor cells to drug inhibition of Aurora B kinase.