Aurora B kinase phosphorylates and instigates degradation of

Chris P. Gullya,b,c, Guermarie Velazquez-Torresa,c,d,1, Ji-Hyun Shina,c,d,1, Enrique Fuentes-Matteic,1, Edward Wanga,c,d, Colin Carlocka,b,c, Jian Chenc, Daniel Rothenbergc, Henry P. Adamse, Hyun Ho Choia,c,d, Sergei Gumaa,c,d, Liem Phana,c, Ping-Chieh Choua,c,d, Chun-Hui Sua,b,c, Fanmao Zhanga,c, Jiun-Sheng Chenc, Tsung-Ying Yangc, Sai-Ching J. Yeunga,f,g,2, and Mong-Hong Leea,b,c,d,2 aGraduate School of Biomedical Sciences, University of Texas, Houston, TX 77030; bThe M. D. Anderson Cancer Center, Program in Genes and Development, University of Texas, Houston, TX 77330; cDepartment of Molecular and Cellular Oncology, The M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030; dThe M. D. Anderson Cancer Center, Program in Cancer , University of Texas, Houston, TX 77330; eDepartment of Genetics, The M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030; fDepartment of Emergency Medicine, The M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030; and gDepartment of Endocrine Neoplasia and Hormonal Disorders, The M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030 AUTHOR SUMMARY

Aurora B, a kinase, To answer this question, we plays a key role in by Aurora B used bimolecular fluorescence maintaining correct chromo- complementation, which allows some segregation and pro- Interphase Mitosis direct observation of the in- gression of cells through mitosis. teraction between two Ub Cancer cells frequently express Ub in living cells, and showed that

Aurora B at unusually high lev- Ub Aurora B and p53 interact els, leading to dysregulated mi- Ub during interphase and mitosis tosis and therefore, causing MDM2 Ub Ub (Fig. P1). Immunofluorescence Aurora B p53 unequal segrega- p53 studies showed that, during mi- Aurora B tion, which may confer a growth P P P tosis, p53 associates with Aurora advantage. The tumor suppres- B located at centromeres at sor protein p53 guards the ge- or the middle nome by delaying or arresting zone of the cleavage furrow at fi –

the cycle at speci c check- chromosome the border. points (G1/S) when DNA is p53 degradation We were also able to show that damaged and prevents damaged Survivin, a protein component cells from entering mitosis (G2/ of CPC localized at the cen- M checkpoint). Despite in- , PUMA spindle checkpoint regulation tromeres to activate Aurora B tensive studies on p53 for more Fig. P1. Functional consequences of the interaction between p53 and (3), colocalized with Aurora B than three decades, the exact Aurora B. In interphase, Aurora B phosphorylates p53, leading and p53 to the DNA of prom- mechanism by which it regulates to its degradation by MDM2-mediated ubiquitination, which down- etaphase cells. We also showed mitotic checkpoint is unknown. regulates the expression of / regulators such as p21 that Aurora B and p53 could be Whether Aurora B and p53 are and PUMA. In , Aurora B associates with p53 at the coimmunoprecipitated with spe- coordinately regulated during centromere region to regulate the spindle checkpoint. cific antibodies. Using the tech- the cell cycle remains to be de- nique, we were also able to show termined, and there is no report to our knowledge suggesting the Aurora B–p53 association in every phase of the cell cycle that Aurora B functions in processes other than mitosis. By except late M phase, when Aurora B is known to be degraded. studying cells synchronized to divide at the same time, we show The interaction in interphase is quite surprising, because Aurora here that Aurora B and p53 interact during all stages of the B has been shown to function exclusively during mitosis. We then cell cycle except during late M phase, when Aurora B is de- showed that Aurora B phosphorylates p53 at three predicted graded. Moreover, we show that Aurora B is a negative regulator Aurora B sites (S183, T211, and S215). We also showed that these of p53 and that, when overexpressed, it affects the ability of p53 phosphorylations lead to enhanced degradation of p53 through to mitigate the detrimental consequences of DNA damage and ubiquitination, which is mediated by the murine double minute 2 control the mitotic checkpoint. (MDM2) regulatory protein, an E3 ubiquitin ligase of p53. The regulation of mitosis ensures the equal segregation of Moreover, AZD1152-hydroxyquinazoline pyrazole anilide to daughter cells, and Aurora B plays a critical role in this process as a component of the chromosomal passenger protein complex (CPC). CPC is located on the chromosome arms Author contributions: C.P.G., S.-C.J.Y., and M.-H.L. designed research; C.P.G., G.V.-T., during and at the centromeres during prometaphase J.-H.S., E.F.-M., E.W., C.C., J.C., D.R., H.P.A., H.H.C., S.G., L.P., P.-C.C., C.-H.S., F.Z., J.-S.C., and metaphase (1). Aurora B subsequently localizes to the and T.-Y.Y. performed research; C.P.G. and M.-H.L. analyzed data; and C.P.G., S.-C.J.Y., midbody during and participates in ensuring the and M.-H.L. wrote the paper. correct attachment of chromosomes to spindle The authors declare no conflict of interest. (spindle checkpoint) and equal distribution of chromosomes. This article is a PNAS Direct Submission. The effects of Aurora B are exerted by its phosphorylation on Freely available online through the PNAS open access option. specific proteins of the mitotic apparatus. The p53 protein is also 1G.V.-T., J.-H.S., and E.F.-M. contributed equally to this work. involved in facilitating chromosome segregation to ensure the 2To whom correspondence may be addressed. E-mail: [email protected] or maintenance of diploid cells because cells deficient in p53 [email protected]. expression become tetraploid (2). Thus, it raises the question of See full research article on page E1513 of www.pnas.org. whether Aurora B and p53 are functionally related. Cite this Author Summary as: PNAS 10.1073/pnas.1110287109.

9232–9233 | PNAS | June 12, 2012 | vol. 109 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1110287109 Downloaded by guest on October 3, 2021 (HQPA), which specifically inhibits the kinase activity of Aurora potentially prevents p53 from arresting the cell cycle at G1 by PNAS PLUS B, was able to block p53 ubiquitination in a dose-dependent maintaining a negative impact on p53. manner. These data provide insights into the contribution of Also, we previously showed that specific inhibition of Aurora Aurora B-mediated p53 phosphorylation in regulating B expression is sufficient to inhibit tumor growth and induce the p53 stability. regression of tumors. Significantly, we here show that a specific The enhanced degradation of phosphorylated p53 leads to the inhibitor (AZD1152) of Aurora B can cause p53 elevation, finding that its transcriptional activity on cell cycle control gene thereby increasing p53 target gene expression in a cancer was diminished (such as p21 CDK inhibitor) when Aurora B xenograft model. As a result, inhibitor of Aurora B can reduce was overexpressed. Moreover, we were able to show that cell survival through increasing the p53 up-regulated mediator of p53-mediated gene transcription was enhanced when Aurora B apoptosis p53 up-regulated modulator of apoptosis (PUMA) and expression was inhibited. These data provide a rationale for the cause cell cycle inhibition through inducing CDK inhibitor p21. role of Aurora B in interphase, which is to antagonize the in- These data are consistent with biochemical studies in cell lines. hibition of cell cycle progression by p53 and allow entry of cells Our present study defines the molecular mechanisms involved in into mitosis. inhibition of tumor growth by an Aurora B inhibitor. General suppression of transcription is observed during Our studies have revealed an unrecognized mechanism for mitosis (4); therefore, Aurora B-mediated p53 transcriptional regulating the activity of p53 during the cell cycle. We show, using suppression will not play a role during mitosis. The work by multiple approaches, that p53 is degraded as a consequence of its Cross et al. (2) shows that p53 is involved in facilitating chro- phosphorylation by Aurora B. This degradation ultimately pre- mosome segregation to ensure the maintenance of diploid cells. vents p53 from performing its role as a guardian of the genome. Aurora B may coordinate with p53 to mediate the spindle Taken together with our earlier study on the effects of the Aurora checkpoint (2) and aid progression through mitosis. Given that B kinase inhibitor AZD1152 on breast cancer cells and preclinical Aurora B deregulation also results in polyploidy, the interplay evaluations of this drug for treating diverse human tumors, we between p53 and Aurora B is conceivably important for spindle conclude that Aurora B inhibitors may prove to be effective in fi – checkpoint. The functional signi cance of Aurora B p53 treating cancer cells expressing functional p53 molecules. interaction during different stages of mitosis remains to be

investigated, but p53 is involved in spindle checkpoint (2); our 1. Carmena M, Earnshaw WC (2003) The cellular geography of aurora kinases. Nat Rev data serve to confirm its presence in the spindle checkpoint Mol Cell Biol 4:842–854.

machinery. It is important to point out that the binding between 2. Cross SM, et al. (1995) A p53-dependent mouse spindle checkpoint. Science 267: BIOCHEMISTRY Aurora B and p53 decreases after the end of mitosis because 1353–1356. of the down-regulation of Aurora B by E3 ubiquitin ligase 3. Wang F, et al. (2010) H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science 330:231–235. anaphase promoting complex. After mitosis, Aurora B is re- 4. Gottesfeld JM, Forbes DJ (1997) Mitotic repression of the transcriptional machinery. covering from degradation and again binding p53, and thus, it Trends Biochem Sci 22:197–202.

Gully et al. PNAS | June 12, 2012 | vol. 109 | no. 24 | 9233 Downloaded by guest on October 3, 2021