nocodazole arrested HeLa cell lysates. Both recombinant and immunoprecipitated Aurora A phosphorylate centrin in vitro, resulting in a gel shift as detected using a monoclonal antibody against total centrin and using an antibody specific for centrin phosphorylated at S170 (Fig. 1C, D). As a control, we performed an in vitro kinase assay solely with centrin and ATP to demonstrate that centrin cannot phosphorylate itself. Mass spectrometry of the excised gel-shifted band from the lane containing: Aurora A, centrin, and ATP confirmed that centrin was phosphorylated at serine 170, as well as at serine 122, in the presence of Aurora A and ATP (data not shown). Both of these sites, (120KISF123 and 168KTSL171) match the Aurora A substrate consensus sequence (R/K-x-T/S-I/L/V/F) [24,25]; in this study we focused on the serine 170 phosphorylation site, which occurs near the carboxy- terminus of centrin. To further confirm that Aurora A mediates phosphorylation of centrin in cells, we knocked down Aurora A using a plasmid based shRNA directed against the 39 UTR of the Aurora A mRNA. A Western blot of cell lysates from transfections of the vector control and Aurora A shRNA vector is shown in Figure S2. The mitotic index was reduced to 20% of vector only controls following knock down of Aurora A. In experiments where endogenous Aurora A was knocked down, expression of shRNA-resistant recombinant wildtype Aurora A, but not kinase dead (K162R) Aurora A, rescued the bipolar spindle phenotype and showed intense focal staining with antibodies against gamma-tubulin and p-S170 centrin at the poles of mitotic cells (Fig. 1E). Although bipolar spindles still formed in the presence of kinase dead Aurora A, we noted that the spindle poles lacked focal p-S170 centrin when compared to cells expressing shRNA-resistant-GFP-WT Aurora A. These experiments demonstrate that Aurora A is a physiolog- ically relevant kinase that localizes at the centrosome with centrin and phosphorylates centrin at mitotic spindle poles. In addition, cells over-expressing Aurora A have higher level of centrin and phosphorylated centrin than do cells expressing empty vector (Fig. S5). Because the level of phospho-centrin remains high from prophase through metaphase but abruptly decreases at the onset of anaphase (Fig. 1A, anaphase), we sought to determine if the phosphorylation of centrin may play a role in the regulation of centrin stability during the cell cycle. To accomplish this, we established stable HeLa Tet- On cell lines expressing different recombinant HA-tagged centrin mutants that alter the serine residue 170 phosphorylation site: 1) wildtype centrin (167KKTSLY172, WT); 2) a truncation mutant in which the C-terminal six amino acids of centrin were deleted (167kktsly172, trunc); 3) a non-phosphorylatable mutant centrin (167KKTALY172, S170A); and 4) a mutant that mimics constitutive centrin phosphorylation (167KKTDLY172, S170D). Unless other- wise stated, all experiments were performed in the presence of endogenous centrin. In order to assess the half-life of these HA- tagged centrin recombinant products, we performed a ‘‘pulse/wash- out’’ experiment by inducing their expression with doxycycline for 12 hours, followed by washing with and incubation in doxycycline- free medium over a time-course. The half-life of the wildtype HA- centrin (WT), 167KKTSLY172, and the non-phosphorylatable centrin, 167KKTALY172 (S170A), were approximately 11 and 12 hours respectively, indicating a near complete turnover once per approximately 24-hour cell cycle period (Fig. 2A, B). The lack of significant differences between the WT and S170A mutant half-lives is likely because only a small pool of centrin (the centrosomal pool) is phosphorylated in a cell cycle dependent fashion. Conversely, the truncation mutant (trunc), 167kktsly172, was strikingly unstable with a protein half-life of approximately 4.1 hours. Notably, a dramatic increase in half-life of the 167KKTDLY172 (S170D) mutant protein Control of Centrin Stability by Aurora A (approximately 21 hours) was observed (Fig. 2A, B). Due to the striking differences in the half-lives of the wildtype and mutant centrins, we performed cell counts to determine if these mutants affect cell proliferation when endogenous centrin is knocked down and the HA-tagged centrins were expressed. As demonstrated by graph in Figure 2C, the S170D centrin expressing cells rescued the centrin depletion phenotype [17] at least as well as, if not better than, the wildtype centrin expressing cells did. On the other hand, the two centrin mutants that were non-phosphorylatable either due to point mutation (S170A) or deletion (kktsly) were not able to rescue the centrin depletion phenotype and proliferated at a much slower rate similar to the rate seen when Aurora A is knocked down (Fig. 2C). Due to the abrupt loss of phospho-centrin after anaphase (Fig. 1A), we performed double thymidine/nocodazole-synchro- nization, shake-off, and release experiments to determine if the total level of centrin fluctuates during cell cycle. Figure 3A illustrates that both Aurora A and centrin abundance peak in G2/M (nocodazole) compared to cycling cells and remain high for 60–90 minutes after release from nocodazole treatment. At 120 minutes post-release, Aurora A levels begin to drop; this occurs prior to significant decreases in centrin although there is a slight transient decrease in centrin at 60 minutes post-release. Nonetheless, by 150 minutes both Aurora A and centrin have reached levels comparable to or lower than cycling cells. Cyclin B degradation, which begins 60 minutes after nocodazole release, indicates the physiological onset of metaphase (Fig. 3A). FACS analysis confirms that cell cycles were synchronized and shows cells entering G1 as early as 60 minutes after nocodazole release (Fig. S1). This experiment demonstrates temporally coincident expression, accumulation, and post- metaphase decrease of both Aurora A and centrin during the cell cycle. We reasoned that the decrease in centrin levels could be due to proteasomal degradation because, after peaking in early mitosis, centrin appears to decrease as mitosis progresses to anaphase, when Aurora A and cyclin B also decrease due to proteasome-mediated degradation. The timing of mitotic decrease in centrin protein suggested that centrin could be an anaphase promoting complex/cyclosome (APC/C) substrate. Consistent with this hypothesis, we identified a destruction box (D-box) (151RDgdGEVSe159) within centrin’s protein sequence that closely matches the D-box of the canonical mitotic APC/C substrate cyclin B (http://bioinfo2.weizmann.ac.il/,danag/ dbox/form.html). As shown in Figure 3B, centrin immunopre- cipitates with cdc20, an activator and substrate binding subunit of the APC/C. Cdc20 immunoprecipitations demonstrate that it interacts with other units of the APC/C, cdc16 and cdc27 (Fig. 3B). Cdc20 immunoprecipitations from asynchronous HeLa cell lysates illustrate that centrin, but not p-S170 centrin, forms a complex with cdc20 (Fig. 3C). These data support the notion that phosphorylated centrin is more stable than unpho- sphorylated centrin perhaps because the phosphorylated form does not interact with the APC/C protein degradation machinery. These results led us to further investigate centrin degradation by treating HeLa Tet-On centrin cell lines overnight with a serine protease inhibitor (leupeptin, L), a lysosomal inhibitor (ammonium chloride, N), and two proteasome inhibitors (ALLnL, A and MG132, M), to determine the mechanism of centrin degradation. The lysosome and serine protease inhibitors did not alter the level of centrin in any of the mutants; although the centrin truncation mutant appears to be intrinsically unstable and is likely degraded quickly via an alternative constitutive mechanism (see Discussion). PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21291