Cell Cycle. 2016 Dec;15(23):3296-3305.

Bcl2l10, a new Tpx2 binding partner, is a master regulator of Aurora kinase A in mouse oocytes. 

Su-Yeon Lee, Eun-Young Kim, Kyeoung-Hwa Kim, Kyung-Ah Lee.

Institute of Reproductive Medicine, Department of Biomedical Science, College of Life Science, CHA University, Pan-Gyo, Korea.

 

Abstract

Previously, we demonstrated that Bcl-2-like 10 (Bcl2l10) is associated with meiotic spindle assembly and that the gene that is most strongly down-regulated by Bcl2l10 RNAi is targeting protein for Xklp2 (Tpx2). Tpx2 is a well-known cofactor that controls the activity and localization of Aurora kinase A (Aurka) during mitotic spindle assembly. Therefore, this study was conducted (1) to identify the associations among Bcl2l10, Tpx2, and Aurka and (2) to understand how Bcl2l10 regulates meiotic spindle assembly in mouse oocytes. Bcl2l10, Tpx2, and Aurka co-localized on the meiotic spindles, and Bcl2l10 was present in the same complex with Tpx2. Tpx2 and Aurka expression decreased, whereas phospho-Aurka increased, in Bcl2l10 RNAi-treated oocytes. Counterbalancing changes in the levels of these 2 activators, Tpx2 and phospho-Aurka, resulted in decreased Aurka catalytic activity after Bcl2l10 RNAi treatment. Bcl2l10 RNAi decreased the expression of microtubule organizing center (MTOC)-related proteins, disturbed MTOC formation and disrupted meiotic spindle assembly. Our data demonstrate that Bcl2l10 is a binding partner of Tpx2 and a new regulator of the complex controlling the organization of microtubules and MTOC biogenesis in meiotic spindle assembly. The discovery of Bcl2l10 as a new effector of Aurka suggests that Bcl2l10 may have diverse functions in mitotic cells.

Keywords: Aurka; Bcl2l10; MTOC; Tpx2; mouse oocyte maturation

PMID: 27753540, PMCID: PMC5176144 [Available on 2017-10-18],

DOI: 10.1080/15384101.2016.1243630

 

 

Supplement

In our previous Cell Cycle paper, we reported that 1) Bcl2l10 knockdown induced a concurrent decrease in Tpx2 as well as Aurka protein expression in mouse oocytes and 2) that Bcl2l10 forms a protein-protein complex with Tpx2 but not with Aurka. However, Bcl2l10 seems to be a master regulator that has various effects on the mRNA expression, protein expression, auto-phosphorylation, and kinase activity of Aurka in mouse oocytes.

 

It is well known that Aurka has a crucial role in spindle assembly during mitosis and that abnormally increased expression of Aurka is associated with oncogenic transformation [1]. Therefore, the discovery of Bcl2l10 as a new effector of Aurka during meiosis is an important reason that we decided to research the diverse functions of Bcl2l10 in mitosis.

 

It has been reported that Tpx2 is required for the localization of Aurka in mitotic cells [2] and for regulating the stability of Aurka in human mitotic cells [3]. Thus, based upon our new findings regarding the relationship between Tpx2 and Aurka in oocytes and meiotic cells, and the previous findings from mitotic cells, we questioned whether Bcl2l10 regulation of Aurka protein expression is mediated by Tpx2.

 

To evaluate the role of Tpx2 in Bcl2l10-regulated Aurka protein expression, we rescued Tpx2 protein expression in Bcl2l10-silenced oocytes by injecting Tpx2 mRNA simultaneously with Bcl2l10 dsRNA into the cytoplasm of germinal vesicle (GV) oocytes. To achieve sufficient knockdown of Bcl2l10 and/or translation of Tpx2, oocytes were incubated for specific periods (8-24 hours) in IBMX-supplemented M16 medium after injection followed by further culture for 16 hours in plain M16 medium to induce in vitro oocyte maturation (Figure 1A). In the case of Bcl2l10 knockdown, we incubated oocytes for 8 hours, while for the case of Tpx2 translation, we incubated oocytes for 24 hours after co-injection of Bcl2l10 dsRNA and Tpx2 mRNA.

 

Prior to the rescue experiment, we synthesized Tpx2 mRNA by in vitro transcription and verified it by measuring protein levels after injection into GV oocytes. Figure 1B shows the confirmation of Tpx2 protein overexpression as a result of the Tpx2 mRNA injection into the oocytes. After verifying the activity of Tpx2 mRNA, we rescued Tpx2 protein expression by injecting this Tpx2 mRNA into the oocytes concurrent with Bcl2l10 silencing. As a result, despite of the presence of Tpx2 protein expression in Bcl2l10-silenced oocytes, Aurka protein expression was not rescued and was still reduced (Figure 1C). This suggests that Bcl2l10 regulation for Aurka protein expression does not require the presence of Tpx2.

 

Unexpectedly, however, Tpx2-rescued oocytes did not resume meiosis and were arrested at the GV stage, in contrast to the MI arrest of the Bcl2l10 RNAi-treated oocytes. Currently, we cannot explain this oocyte arrest at the GV stage, but we anticipate that it is a result of the imbalance of these 3 important proteins, Bcl2l10, Tpx2, and Aurka, because of untimely biosynthesis and/or degradation.

 

Almost complete reduction of Aurka protein expression in Bcl2l10-silenced but Tpx2-rescued GV oocytes (Figure 1C, 3rd lane) was mainly due to the decreased Bcl2l10 and not an effect of the oocytes’ maturity. In other words, undetectable levels of Aurka expression in the Bcl2l10-silenced but Tpx2-rescued GV oocytes was not because they were in GV stage but was rather the result of decreased Bcl2l10 levels. We did not measure Aurka expression during oocyte maturation in this study because it has already been reported that mouse oocytes in all stages of maturity (GV, GVBD, MI, and MII) express Aurka protein [4, 5].

 

In addition to the major findings reported in the Cell Cycle paper, we add the important finding that Bcl2l10-dependent regulation of Aurka protein expression does not require mediation via Tpx2. To our knowledge, there is no report on the regulation of Aurka protein expression via Bcl2l10 in mitotic cells, including cancer cells. Further studies on the regulatory role of Bcl2l10 in mitotic cells in relation with Aurka and Tpx2 are necessary for developing therapeutic strategies for cancer treatment.

 

 

Figure. Rescued Tpx2 expression in Bcl2l10-silenced oocytes did not affect Aurka protein expression.

(A) Schematic diagram depicting the experimental design. GV oocytes were microinjected with Bcl2l10 dsRNA without or with Tpx2 mRNA and cultured in M16 medium containing IBMX for 8 and 24 hours, respectively. Then, oocytes were washed and transferred to plain M16 medium for an additional 16 hours to induce in vitro maturation.

(B) In vitro synthesized Tpx2 mRNA was validated by microinjection into GV oocytes. After injection, oocytes were incubated for 24 hours in IBMX-supplemented M16 medium for sufficient translation of Tpx2 and collected for western blot analysis. The protein lysates of 150 oocytes were loaded into each lane. α-Tubulin was used as a loading control.

(C) Co-injection of Bcl2l10 dsRNA and Tpx2 mRNA rescued Tpx2 expression to a degree but did not affect Aurka expression, indicating that the Bcl2l10-dependent regulation of Aurka protein expression does not require Tpx2 in mouse oocytes. The protein lysates of 200 oocytes were loaded into each lane. α-Tubulin was used as a loading control. The lanes contain the following samples (from left): non-injected MI oocytes, MI-arrested oocytes after Bcl2l10 RNAi (8 hours incubation), and GV-arrested oocytes after co-injection of Bcl2l10 dsRNA and Tpx2 mRNA (24 hours incubation).

 

References

  1. Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PN Jr., Gandra DR. Aurora kinases as anticancer drug targets. Clin Cancer Res 2008; 14:1639-1648.
  2. De Luca, M, Lavia P, Guarguaglini G. A functional interplay between Aurora-A, Plk1 and TPX2 at spindle poles. Cell Cycle 2006; 5, 296-303.
  3. Giubettini M, Asteriti IA, Scrofani J, De Luca M, Lindon C, Lavia P, Guarguaglini G. Control of Aurora-A Stability through interaction with TPX2. J Cell Sci 2010; 124:113-122.
  4. Yao LJ, Zhong ZS, Zhang LS, Chen DY, Schatten H, Sun QY. Aurora-A iis critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos. Biol Reprod 2004, 70:1392-1399.
  5. Saskova A, Solc P, Baran V, Kubelka M, Schultz RM, Motlik J. Aurora kinase A controls meiosis I progression in mouse oocytes. Cell Cycle 2008;7:2368-2376.