The availability of a cell surface receptor can be modified through regulation of its endo-lysosomal degradation rate. This has been well established for growth-factor receptors, which are ubiquitylated and internalised into endosomal compartments upon activation. Interaction with the ESCRT (endosomal sorting complexes required for transport) machinery seals their commitment to the lysosome for degradation. Much less is known about the trafficking itinerary of the immune checkpoint regulator and immunotherapy target, CTLA4.
Here, we show that CTLA4 is an unusually short-lived membrane protein (half-life <2 h), which is constitutively expressed, not just in T cells, but also in a selection of cancer cell lines. Its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213 and K203/213R mutated CTLA4 accumulates in early endosomal compartments. Inhibition of the vacuolar H+-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal DUB, USP8, interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells and in cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype, but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive, nor a localisation-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4, or counteract its accumulation in clustered early and late endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.