Control of Centrin Stability by Aurora A Kara B. Lukasiewicz1¤, Tammy M. Greenwood1, Vivian C. Negron2, Amy K. Bruzek2, Jeffrey L. Salisbury1*, Wilma L. Lingle1,2* 1 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America, 2 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, United States of America Abstract Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over- expression of Aurora A results in excess phospho-centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of centrin phosphorylation. Finally, a recombinant centrin mutation that mimics centrin phosphorylation increases centrin’s stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of centrin is regulated in part by Aurora A, and that excess phosphorylated centrin may promote centrosome amplification in cancer. Citation: Lukasiewicz KB, Greenwood TM, Negron VC, Bruzek AK, Salisbury JL, et al. (2011) Control of Centrin Stability by Aurora A. PLoS ONE 6(6): e21291. doi:10.1371/journal.pone.0021291 Editor: Daniela Cimini, Virginia Tech, United States of America Received September 24, 2010; Accepted May 28, 2011; Published June 23, 2011 Copyright: 2011 Lukasiewicz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was through the following grants: Mayo Clinic Breast Cancer SPORE NIH CA116201 (W.L. Lingle, Core Leader and James Ingle, PI) (http://www.nih.gov/); NIH R01 CA072836 (J.L. Salisbury, PI) (http://www.nih.gov/); The Breast Cancer Research Foundation FNDTBCRF-16, 17 (W.L. Lingle, Project Leader and J.N. Ingle, PI), (http://www.bcrfcure.org/); and Predoctoral Fellowship from the Mayo Foundation (http://www.mayo.edu/), (K.B. Lukasiewicz). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lingle.wilma@mayo.edu (WLL); Salisbury@mayo.edu (JLS) ¤ Current address: Section on Cell Cycle Regulation, Program for Cell Regulation and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America Introduction The centrosome aids in the assembly of the bipolar mitotic spindle and in maintenance of cell polarity. Centrosome abnormalities are observed in many cancers and have been shown to drive chromosomal instability (CIN) and aneuploidy. Several key mitotic kinases, including the Plk, NEK, and Aurora families [1,2,3,4,5,6], and elevated levels of phosphorylated centrosomal proteins, including centrin [7], have been implicated in centro- some amplification in cancer. Aurora A is frequently over-expressed in breast and bladder cancer and its ectopic expression causes centrosome amplification and CIN in cell lines and in vivo models [8,9]. Studies using rat and mouse mammary cancer models demonstrate that Aurora A over- expression and genomic instability are early events in tumor progression [10,11,12]. Both Aurora A and the tumor suppressor p53 [13,14] have been implicated in control of genomic stability and centrosome amplification. Interestingly, phosphorylation of p53 by Aurora A leads to its inactivation and degradation [15,16]. Centrin, a small EF-hand phospho-protein, is located in the centrosome, pericentriolar material, throughout the cytoplasm, and at times, in the nucleus [17,18,19]. Despite its ubiquity, centrin is a reliable marker for centrioles because of its highly focal centriolar concentration [20]. Centrin is essential to centriole duplication, as demonstrated by centriole loss and ultimately cell death when centrin is knocked down [21]. Centrin is phosphor- ylated at G2/M [17], yet little is known about the regulation of centrin stability and abundance during the cell cycle. Because both Aurora A and centrin have been implicated in regulating centrosome structure and function, we hypothesized that post- translational centrin modifications driven by Aurora A regulate its stability and abundance. Given that centrin is required for centriole duplication, we also investigated whether alterations in centrin stability lead to centrosome amplification. Results We performed immunofluorescence confocal microscopy on HeLa cells stained with antibodies directed against Aurora A and total and phosphorylated-S170 centrin (p-S170 centrin) to determine the localization of p-S170 centrin and Aurora A in intact cells. As demonstrated in Figure 1A, both Aurora A and p- S170 centrin localize at the centrosome from prophase through metaphase. Phospho-centrin, while faintly detectable at some interphase centrosomes (Fig. S4), was most abundant in mitotic cytoplasm and at mitotic spindle poles (Fig. 1A, fourth column). A robust increase in Aurora A at mitotic spindle poles compared to interphase cells (Figures S4+S6) in prophase was accompanied by markedly intense p-S170 centrin staining (Fig. 1A; prophase). This dramatic and specific appearance of p-S170 centrin co-localizing with Aurora A in early prophase cells persisted throughout metaphase. Subsequently p-S170 centrin diminished during PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21291