Article Figures & Data
Figures
- Figure 1.
Aurora A kinase inhibitors attenuate 2-ME–induced AR degradation. A–D, LNCaP and C4-2 (A and B), 22RV1 (C), and LAPC4 cells (D) were treated with or without 2-ME in the presence or absence of Aurora A kinase inhibitor MLN8054 and/or VX-680 for 24 hours. Cells were harvested, lysed, and extracts were immunoblotted for indicated proteins. Tubulin served as loading control.
- Figure 2.
Knockdown of Aurora A attenuates 2-ME–induced AR degradation. A and B, LNCaP (A) or C4-2 cells (B) were knocked down for Aurora A, B, or C individually or in combination, treated with or without 2-ME, harvested, and extracts were immunoblotted for AR, CHIP, Aurora A, Aurora B, and GAPDH as a control. Remaining amounts of AR presented as a ratio of the band intensity of 2-ME treated versus corresponding DMSO-treated cells, LNCaP (top right) and C4-2 (bottom right). Mean ± SD of at least three independent experiments. C, *Due to lack of availability of an effective antibody against Aurora C, knockdown was monitored by qRT-PCR. mRNA levels are shown in LNCaP and C4-2 cells.
- Figure 3.
2-ME activates Aurora A kinase in a dose- and time-dependent manner. A, LNCaP or C4-2 cells were treated with increasing concentrations of 2-ME for 24 hours. B, mRNA levels of LNCaP cells from A and C with 2 μmol/L of 2-ME were harvested at indicated times. Extracts were immunoblotted for AR, CHIP, Aurora A, or pThr288-Aurora A and tubulin as control.
- Figure 4.
2-ME activates Aurora A Kinase in cells arrested in the S-phase. A, Flow cytometry analysis of unsynchronized LNCaP and C4-2 treated with 2-ME for 24 hours (top left). Immunoblots for indicated proteins and tubulin as a control (top right). B, LNCaP or C4-2 cells were held in G1–S by adding aphidicolin for 24 hours. Cells were treated with or without 2-ME (2 μmol/L) for additional 24 hours in continued presence of aphidicolin. DNA content of LNCaP and C4-2 (bottom left) and immunoblotted for indicated proteins and tubulin serves as control (bottom right).
- Figure 5.
Knockdown of TPX2 attenuates 2-ME–induced AR degradation. A, LNCaP and C4-2 cells were depleted of TPX2 and treated with or without 2-ME for 24 hours. Cells were harvested and extracts were immunoblotted for indicated proteins. Tubulin serves as control. B, Extracts from Fig. 4 were immunoblotted for TPX2; tubulin serves as control.
- Figure 6.
Aurora A phosphorylates CHIP at S273. A, Cells stably expressing FLAG-WT-CHIP were treated with or without 2-ME for 24 hours, harvested, and immunoblotted using anti-FLAG antibody (top). Immunoblotting for tubulin serves as loading control (bottom). B, FLAG-WT-CHIP and FLAG-CHIP S273A were pulled down with M2 beads and used as substrates in kinase assays performed using bacterially expressed recombinant purified Aurora A and radiolabeled γ-ATP. Top, autoradiogram of 32P incorporated into FLAG-CHIP. Coomassie brilliant blue (CBB) staining of gel shows protein bands.
- Figure 7.
S273A substitution in CHIP attenuates 2-ME–induced AR degradation. A, C4-2 cells stably expressing empty vector, WT CHIP, or CHIP S273 mutants were depleted of endogenous CHIP using siRNA against 3′-UTR, and treated with or without 2-ME for 24 hours, then harvested and immunoblotted for AR, CHIP, and tubulin as a control. B, Quantification of remaining AR expressed as a ratio of the band intensity of 2-ME–treated versus corresponding DMSO-treated cells. The fold change was calculated relative to AR level in 2-ME–treated control cells stably expressing empty vector. Mean ± SD of at least three independent experiments. Statistical significance presented as- ns = not significant; *, P < 0.01; **, P < 0.001; and ***, P < 0.0001.
Tables
- Table 1.
Analysis of CHIP phosphorylation upon 2-ME treatment of prostate cancer cells
Sequence Phosphosite 2-ME/DMSO LGAGGGsPEKSPSAQELKEQGNR S19 −2X LGAGGGsPEKsPSAQELKEQGNR S19 and S23 +4X VGHFDPVTRsPLTQEQLIPNLAMK S273 +12X to +15X NOTE: Fold change of CHIP phosphorylation upon 2-ME treatment of LNCaP cells stably expressing FLAG-WT-CHIP.
Supplementary Data
- Supplementary Documents - Table S1: Summarized data of kinase inhibitor screenings. Figure S1: Aurora A phosphorylates CHIP in Vivo Figure S2: CHIP phosphorylation is independent Cdk(s).
- Supplementary Table 1 - Summarized data of kinase inhibitor screenings.
- Supplementary Figure 1 - Aurora A phosphorylates CHIP in vivo.
- Supplementary Figure 2 - CHIP phosphorylation is independent Cdk(s)
Volume 15, Issue 8
