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Oncogenes and Tumor Suppressors

Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP

Sukumar Sarkar, David L. Brautigan and James M. Larner
Sukumar Sarkar
Department of Radiation Oncology, University of Virginia School of Medicine, Charlottesville, Virginia.
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David L. Brautigan
Center for Cell Signaling, Department of Microbiology, Immunology & Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia.
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James M. Larner
Department of Radiation Oncology, University of Virginia School of Medicine, Charlottesville, Virginia.
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  • For correspondence: jml2p@virginia.edu
DOI: 10.1158/1541-7786.MCR-17-0062 Published August 2017
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  • Figure 1.
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    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.

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    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.

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    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.

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    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).

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    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.

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    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.

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    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.

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  • Table 1.

    Analysis of CHIP phosphorylation upon 2-ME treatment of prostate cancer cells

    SequencePhosphosite2-ME/DMSO
    LGAGGGsPEKSPSAQELKEQGNRS19−2X
    LGAGGGsPEKsPSAQELKEQGNRS19 and S23+4X
    VGHFDPVTRsPLTQEQLIPNLAMKS273+12X to +15X
    • NOTE: Fold change of CHIP phosphorylation upon 2-ME treatment of LNCaP cells stably expressing FLAG-WT-CHIP.

Additional Files

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  • 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)
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Molecular Cancer Research: 15 (8)
August 2017
Volume 15, Issue 8
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Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP
Sukumar Sarkar, David L. Brautigan and James M. Larner
Mol Cancer Res August 1 2017 (15) (8) 1063-1072; DOI: 10.1158/1541-7786.MCR-17-0062

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Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP
Sukumar Sarkar, David L. Brautigan and James M. Larner
Mol Cancer Res August 1 2017 (15) (8) 1063-1072; DOI: 10.1158/1541-7786.MCR-17-0062
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