Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anti-cancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is most frequently mutated in prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a common degron motif. In contrast, prostate cancer-associated SPOP mutants impair binding and proteasomal degradation of BET proteins, thus inducing their accumulation in prostate cancer cells and patient specimens. Transcriptome and BRD4 cistrome analyses reveal that SPOP mutation enhances BRD4-dependent expression of GTPase RAC1 and cholesterol biosynthesis genes and AKT-mTORC1 activation. SPOP mutant expression confers BET inhibitor resistance and this effect can be overcome by AKT inhibitors. Thus, SPOP mutations promote AKT-mTORC1 activation and intrinsic BET inhibitor resistance by stabilizing BET proteins, suggesting that SPOP mutation can be an effective biomarker to guide BET inhibitor-oriented therapy of prostate cancer.

Thioredoxin interacting protein (TXNIP) was originally characterized as an endogenous inhibitor of thioredoxin, a key regulator in cellular redox homeostasis. TXNIP is also known to play important roles in tumor growth and metastasis, glucose and lipid metabolism. TXNIP expression is induced by various stress stimuli. However, it has been unclear how TXNIP is down-regulated. Here, we report that TXNIP undergoes proteasomal degradation in cells. We identify Itch as the E3 ubiquitin ligase for TXNIP. We demonstrate that Itch mediates polyubiquitination of TXNIP both in vitro and in vivo. Overexpression of Itch leads to TXNIP proteasomal degradation. Knockdown of Itch by small interfering RNA causes an accumulation of the steady-state level of TXNIP. We also show that the PPXY motifs of TXNIP and the WW domains of Itch mediate their interaction. Furthermore, the Itch-TXNIP interaction regulates intracellular reactive oxygen species levels and apoptosis. These findings establish a new mechanism for the negative regulation of TXNIP by Itch and shed new light on the regulation of cellular redox homeostasis.

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AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.

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