Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.
The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state.