Regulation of RB1CC1/FIP200 stability and autophagy function by CREBBP-mediated acetylation in an intrinsically disordered region.

ABSTRACT

RB1CC1/FIP200 is an essential macroautophagy/autophagy protein that plays an important role in a variety of biological and disease processes through its canonical autophagy-dependent and -independent functions. However, it remains largely unknown whether post-translational modifications could regulate RB1CC1 and its associated autophagy functions. Here, we report acetylation of several lysine residues of RB1CC1 by acetyltransferase CREBBP (CREB binding protein), with K276 as the major CREBBP acetylation site. K276 is also identified as a ubiquitination site by mass spectrometry, and acetylation at this site reduces ubiquitination of RB1CC1 to inhibit its ubiquitin-dependent degradation. We also find that RB1CC1 contains an N-terminal intrinsically disordered region (IDR) capable of forming liquid-liquid phase separation (LLPS) in vitro, which may drive formation of RB1CC1 puncta with LLPS properties in cells independent of SQSTM1/p62 and other autophagy receptors CALCOCO2/NDP52, NBR1, TAX1BP1 and OPTN. Mutational analysis shows that both K276 acetylation and the N-terminal IDR containing it are important for maintaining canonical autophagy function of RB1CC1 in breast cancer cells. Our findings demonstrate regulation of RB1CC1 by a new post-translational mechanism and suggest potential therapeutic application of inducing RB1CC1 degradation through blocking K276 acetylation in the treatment of cancer and other diseases.Abbreviations Baf-A1 bafilomycin A1; CREBBP/CBP CREB binding protein; CHX cycloheximide; EP300/p300 E1A binding protein p300; FRAP fluorescence recovery after photobleaching; HADCs histone deacetylases; IDR intrinsically disordered region; LLPS liquid-liquid phase separation; KAT2A/GCN5 lysine acetyltransferase 2A; KAT2B/PCAF lysine acetyltransferase 2B; KAT5/TIP60 lysine acetyltransferase 5; KAT8/MOF lysine acetyltransferase 8; NAM nicotinamide; PAS phagophore assembly site; PEG-8000 polyethylene glycol 8000; RB1CC1/FIP200 RB1 inducible coiled-coil 1; TSA trichostatin A.