WT centrin in the absence of endogenous centrin also displayed centrosome amplification in the great majority of cells (84% of cells; Fig. 4D, n = 32). Figure S3 shows the expression of HA- centrin while endogenous centrin is knocked down. Approximately half of the cells expressing S170A centrin in the presence of endogenous wildtype centrin exhibited centrosome amplification (56%; Fig. 4E, n = 32), while cells expressing only S170A centrin demonstrated a dramatic decreased in centrosome amplification to 7% (Fig 4F, n = 30). Together with the S170D centrin experiments (Fig. 4A, B), these experiments clearly demonstrate that Aurora A- dependent phosphorylation of centrin is sufficient to cause centrosome amplification and the extent of amplification directly correlates with the expression levels of S170-phosphorylatable centrin. Discussion We previously demonstrated phosphorylation of centrin at serine 170 by PKA [17]. Here we show that Aurora A is another such kinase, which we demonstrate is the relevant mitotic kinase that mediates centrin stability at the time of cell division. This does not preclude a role for PKA in centrin phosphorylation under other physiological circumstances. The precise manner in which excess phosphorylated centrin causes centrosome amplification is not known. One possibility is that the increased stability of centrin directly affects centriole formation through mass action equilibri- um binding due to centrin accumulation. Alternatively, p-S170 centrin may adopt a conformation that affects its interaction with other centriole proteins leading to nascent centriole-like structure assembly. It is also worth noting that centrin’s stability is severely affected when the six carboxy-terminal amino acids are deleted (centrin truncation mutant kktsly). These observations coupled with the analysis of the phosphorylation status within this C-terminal tail suggest that these amino acids are essential for proper centrin stability. It is likely that centrin is misfolded without its C-terminal 6 amino acids. Hydrophobic residues are often exposed when proteins are incorrectly folded and this can lead to aggregation of the misfolded proteins. Cells have two main mechanisms for dealing with misfolded proteins: repair and proteolysis [29,30,31]. These mechanisms involve both chaperones and the proteasome, but the details of cytosolic protein ‘‘quality control’’, as compared to endoplasmic reticulum (ER) mechanisms, are currently poorly understood [29,30,31]. If refolding of truncated centrin by molecular chaperones, like the heat shock proteins, is not successful, then the truncated centrin would likely be degraded as part of the cytosolic quality control mechanism. Interestingly, the chaperones, Hsp90 and Hsp70, localize to the centrosome and interact with centrin in Xenopus [32]. Several published accounts describe the proteasome at the centrosome lending credence to the idea that truncated centrin could be degraded at the centrosome [33,34]. While it is possible that a truncation mutation of centrin could occur naturally, we chose to focus on the phosphorylation status because we observed centrin phosphorylation in unper- turbed cells. Because both Aurora A and centrin are located at the centrosome and manipulation of either protein can alter the centrosome cycle, we investigated the association between these two proteins. We found that Aurora A and pS170-centrin interact with one another and are both located at the centrosome throughout the cell cycle. Importantly, we show that Aurora A is necessary for the focal localization of phospho-S170-centrin at mitotic spindle poles. Our studies show that Aurora A phosphor- ylates centrin at serine 170 in vitro and that the serine 170 Control of Centrin Stability by Aurora A phosphorylation affects the stability of centrin by regulating its interaction with APC/C. Furthermore, we found that over- expression of a centrin mutant that mimics phosphorylation results in accumulation of centriole-like structures in cultured cells and that excess Aurora A and phospho-centrin accumulate at amplified centrosomes of human breast tumors. Expression of the phospho-centrin mimicking mutant can rescue the centrin depletion phenotype, whereas expression of the non-phosphor- ylatable mutant and the truncation mutant lacking the phosphor- ylation site cannot rescue the phenotype. Finally we demonstrated that phosphorylation of centrin serine 170 is an absolute requirement for Aurora A-mediated centriole amplification. Our studies demonstrate that Aurora A mediates phosphory- lation of centrin at serine 170 in a cell cycle dependent fashion. Strikingly, the pattern of accumulation and then decrease of phospho-centrin during the course of mitosis is consistent with that observed for many other centrosome-associated proteins. Pro- nounced accumulation of phosphorylated centrosomal proteins at the onset of prophase has been described previously [35]. Loss of phospho-proteins can be due to the dephosphorylation and/or degradation of phosphorylated proteins. We propose that Aurora A-mediated phosphorylation of centrin at late G2/M transiently stabilizes the protein until its dephosphorylation at the metaphase- anaphase transition, allowing for its recognition by the APC/C- machinery, ubiquitination, and consequent proteasomal degrada- tion. Aurora A can stabilize various proteins via kinase activity- independent and -dependent mechanisms. Both E2F1 and N-myc proteins are stabilized by Aurora A independent of its kinase activity [36,37]. Aurora A’s ability to phosphorylate GSK3beta indirectly protects beta-catenin from GSK3beta-mediated phos- phorylation and subsequent proteasomal degradation [38]. Interestingly, Aurora A expression stabilizes ASAP protein levels by preventing its proteasomal degradation, and although, Aurora A directly phosphorylates ASAP, it is unclear if this phosphory- lation is responsible for ASAP protein stabilization [39]. Additionally, Aurora A’s kinase activity is required for stabilization of cyclin B and its APC/C-mediated proteasomal degradation. But again it is unclear if Aurora A must directly phosphorylate cyclin B for this to occur [40]. Phosphorylation is known to impact the stability of a number of APC/C substrates including cdc6 and Aurora A [41,42,43]. Here we show that non-phosphorylated, endogenous centrin interacts with APC/C components cdc20, cdc16, and cdc27, that specific proteasome inhibitors block the degradation of centrin, and that centrin mutated to resemble constitutive phosphorylation at serine 170 (KKTDLY, S170D) is more stable than either wildtype or non-phosphorylatable centrin (KKTALY, S170A). Furthermore, kinetic analysis of the degradation of specific centrin mutants shows that wildtype or non-phosphorylatable centrins have a half-life of approximately 11 hours, which is consistent with its degradation once during cell cycle. Interestingly for Aurora A, phosphorylation also plays a protective role against proteasomal degradation [41]. Phosphorylation of Aurora A at serine 51 (S51) protects it from APC/Ccdh1-mediated ubiquitination and subse- quent proteasomal degradation [41]. Similar to centrin, a non- phosphorylatable mutation in Aurora A (S51A) has a shorter protein half-life than wildtype Aurora A [41]. Centrin therefore joins Aurora A and a growing list of cell cycle proteins whose abundance is regulated by phosphorylation and APC/C-mediated destruction. Proteasomal degradation plays a critical role in regulation of a number of diverse cell cycle components. Among those associated with centrosome behavior are the degradation of cyclin B during PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e21291