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J Cell Biol
2018 Jan 02;2171:107-116. doi: 10.1083/jcb.201706103.
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Aurora A activation in mitosis promoted by BuGZ.
Huang Y
,
Li T
,
Ems-McClung SC
,
Walczak CE
,
Prigent C
,
Zhu X
,
Zhang X
,
Zheng Y
.
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Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly. It remains unclear, however, whether BuGZ could regulate additional proteins to promote spindle assembly. In this study, we report that BuGZ promotes Aurora A (AurA) activation in vitro. Depletion of BuGZ in cells reduces the amount of phosphorylated AurA on spindle MTs. BuGZ also enhances MCAK phosphorylation. The two zinc fingers in BuGZ directly bind to the kinase domain of AurA, which allows AurA to incorporate into the coacervates formed by BuGZ in vitro. Importantly, mutant BuGZ that disrupts the coacervation activity in vitro fails to promote AurA phosphorylation in Xenopus laevis egg extracts. These results suggest that BuGZ coacervation promotes AurA activation in mitosis.
Figure 2. BuGZ contributes to MT assembly and reduces S196 phosphorylated xMCAK. (A) MT asters and spindles assemble around AurA beads at the indicated time points in mock-depleted XEEs or XEEs depleted (dep) of xBuGZ or xTPX2 or treated with MLN8237. Bar, 10 µm. (B and C) MT aster lengths (B) and percentages of bipolar spindles (C) were quantified. Approximately 50 (B) and ∼200 (C) AurA bead structures were quantified in three independent experiments. (D) XEE mock-depleted by unimmunized rabbit IgG, xBuGZ, or xTPX2 antibodies or treated with MLN8237 were analyzed by Western blotting probing with the indicated antibodies. The means and SEM are graphed. Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3. The zinc fingers of BuGZ directly bind to the kinase domain of AurA. (A and B) Immunoprecipitation (IP) using BuGZ (A) or AurA (B) antibodies in mitotic HeLa cell lysates. Equivalent of 1% of the input lysate and 20% of total immunoprecipitates were loaded. (C and D) Immunoprecipitation using xBuGZ (rabbit polyclonal IgG; r IgG; C) or xAurA (mouse polyclonal IgG; m IgG; D) antibodies in XEEs. Equivalent of 0.2% of the input lysate and 30% of total immunoprecipitates were loaded. (E) Schematic of xBuGZ with NLS, zinc fingers (ZnFs), and the C-terminal region as well as YFP-tagged xBuGZ used in the study. (F) Purified YFP, YFP-xBuGZ, YFP-xBuGZΔ7, YFP-xBuGZΔN58, or YFP-xBuGZΔN92 were incubated with purified xAurA, followed by pulldown with antibody to YFP and then Western blotting and Coomassie Brilliant blue (CBB) staining. (G) Purified GST or GST-xBuGZ(8–58) was incubated with purified xAurA in the presence or absence of EDTA followed by GST pulldown, Western blotting, and Coomassie blue staining. (H) Purified GST, GST-xAurA, GST-xAurA(1–25), GST-xAurA(26–126), or GST-xAurA(127–407) were incubated with YFP or YFP-xBuGZ followed by GST pulldown, Western blotting, and Coomassie blue staining. For G and H, 0.1% of total reaction and 10% of total xBuGZ pulldowns were analyzed by Western blotting. 1% of total reaction and 10% of total pulldowns were analyzed by Coomassie blue staining.
Figure 4. Effects of BuGZ phase separation on AurA binding. (A and B) Purified YFP-xBuGZ or YFP-xBuGZΔN58 was incubated to allow coacervation in the presence of purified mCherry-xAurA (A) or mCherry (B). Bars, 10 µm. (C) Purified YFP, YFP-xBuGZ, or YFP-xBuGZ13S were incubated with purified GST or GST-xAurA at RT or 4°C followed by GST pulldown, Western blotting (0.1% of total reaction and 10% of total YFP-xBuGZ and YFP-xBuGZ13S pulldowns were analyzed), and Coomassie Brilliant blue (CBB) staining (1% of the total reaction and 10% of total pulldowns were analyzed). (D) Schematic of droplet spindown assay. (E–G) Quantification of YFP-xBuGZ and xAurA (E), YFP-xBuGZΔN58 and xAurA (F), and YFP-xBuGZ13S and xAurA (G) in the pellets (X axis) against the initial input xBuGZ proteins (Y axis). Error bars indicate SEM.
Figure 5. Effects of BuGZ phase transition and binding to AurA on kinase activity. (A) Purified xAurA (cleaved from mCherry-xAurA) was incubated from purified xBuGZ (cleaved from YFP-xBuGZ) and MBP to assay AurA kinase activity in vitro. CBB, Coomassie Brilliant blue. (B) XEE was mock depleted or depleted (dep) with BuGZ antibody, and the BuGZ-depleted XEE was supplemented with the indicated purified proteins followed by xAurA immunoprecipitation and Western blotting analyses. r IgG, rabbit polyclonal IgG.
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