Figure 2. Engineered mutations at S170 affect centrin protein stability and cell proliferation rates in HeLa. (A) Western blotting of whole cell lysates of cells expressing inducible HA-tagged mutant centrin constructs (bHA) at time points following doxycycline washout reveals that the truncated centrin mutant has the shortest half-life, the S170D mutant that mimics phosphorylated centrin has the longest half- life, while endogenous wildtype centrin (bE) remains fairly constant. (B) The half-lives of the centrin mutants were determined by plotting the changes in optical density plots of the Western blots shown in (A) for 5 independent experiments. The S170A and wildtype half-lives are virtually the same at 12.2 and 10.9 hr respectively (p = 0.942). The S170D half-life, at 20.7 hr, is significantly longer than either S170A or wildtype (p,0.027 and 0.013, respectively). In 3 of 5 experiments, the half-life of the truncation mutant could not be determined because the time points measured were not early enough. Results of the 2 Control of Centrin Stability by Aurora A experiments in which it could be analyzed indicate the half-life of the truncation mutant is approximately 4.1 hr. (C) Proliferation rates of cells, normalized to HA-tagged wildtype centrin, in which centrin has been knocked down by shRNA and then induced to express only engineered centrin mutants reveal that the truncation mutation greatly reduces the growth rate less than 30% of cells expressing wildtype centrin by 48 hr. Likewise, the S170A mutant (KKTALY) reduces growth rate to approximately 40% at 72 hr. The S170D mutant (KKTDLY), which mimics centrin phosphorylated at serine 170, rescues the centrin depletion phenotype to a slightly greater extent than does wildtype centrin. The error bars represent standard deviation of three independent experiments. * indicates p values#0.03 when compared to wildtype or the S170D centrin construct. doi:10.1371/journal.pone.0021291.g002 In contrast, the wildtype, S170A, and S170D mutant centrins greatly increased in cells treated with either proteasome inhibitor, indicating that these centrins are degraded via the proteasome (Fig. 3D). Western blots of lysates from HeLa Tet-On cells expressing the S170A centrin mutant treated with the proteasome inhibitor ALLnL demonstrated a laddering of approximately 7 kDa increments when probed with an anti-centrin antibody, consistent with ubiquitination of centrin (Fig. 3E). This laddering, the canonical D-box, and the physical interaction of centrin in a complex containing cdc20, cdc27, and cdc16 suggest that centrin is degraded by ubiquitin-mediated mitosis-specific APC/C activities. To investigate the consequences of centrin phosphorylation in cells, endogenous centrin was knocked down using a plasmid based shRNA directed against the 39 UTR of centrin 2 in HeLa Tet-On cells left untreated (Fig. 4B) or treated with doxycycline to induce expression of the S170D centrin mutant (Fig. 4A). Expression of the S170D mutant resulted in a dramatic increase in HA-tagged centrin staining as compared to untreated cells, and this was accentuated in cells when endogenous centrin was knocked down (Fig. 4A). Detailed electron microscopic studies of the S170D centrin expressing mutant, described elsewhere (Salis- bury et al, in preparation), demonstrate that these cells exhibit aberrant centriole-like structures, which may explain the excess centrin staining we observe in Figure 4A. Because this accumu- lation of centrin resembles centrosome aberrations seen in breast cancer [26] and human breast tumors frequently over-express Aurora A, we extended our studies of phospho-centrin to human breast tumors. Examination of frozen sections of normal breast epithelium adjacent to tumor tissue stained with antibodies directed against Aurora A, gamma-tubulin, and p-S170 centrin show low levels of Aurora A and p-S170 centrin staining at the centrosome (Fig. 4G) consistent with the low proliferative rate of normal breast tissue. In contrast, ductal carcinoma in situ (Fig. 4H) and invasive breast tumor tissues (Fig. 4I) displayed both increased size [27] and number of centrosomes (amplified centrosomes or more than two centrosomes per cell) [28], as marked by gamma- tubulin, with striking co-localization of Aurora A and p-S170 centrin. These observations are consistent with what we observed in normal and cancerous breast tissues in our previous study [27]. This is consistent with our observation that expression of the S170D phospho-centrin mutant results in increased centrin-foci in cultured human cells and that Aurora A and phospho-centrin co- localize at amplified centrosomes in human breast tumors. To determine if the phosphorylation of centrin at serine 170 is necessary for Aurora A-mediated centrosome amplification, we utilized two of the doxycycline HA-tagged centrin mutant expressing HeLa Tet-On cell lines: HA-S170A centrin and HA- WT centrin with the expectation that cells expressing only non- phosphorylatable centrin would not develop amplified centro- PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21291