Lysosomal ubiquitin ligase RNF182 regulates mTORC1 signaling and neuronal differentiation

抄録

Background: We identified 37 ubiquitin ligases containing RING finger and transmembrane domains. Of these, we found that RNF182, a central nervous system-specific gene, is the most highly expressed during neuronal differentiation in P19 cells and that it is predominantly expressed in lysosomes and late endosomes. In the present study, we investigated the role of RNF182 in neural differentiation.

Results: We performed shotgun proteomics techniques and identified lysosomal-associated transmembrane protein (LAPTM) 4A and 4B as its substrates. We found that RNF182 preferentially poly-ubiquitylates LAPTMs via K63-linked chains, suggesting that RNF182 does not promote the K48 chain-mediated degradation of LAPTMs. LAPTM4B reportedly participates in the recruitment of the large neutral amino acid transporter LAT1 to lysosomes, leading to leucine uptake into lysosomes and mammalian target of rapamycin complex 1 (mTORC1) activation. We showed that the RNF182-mediated poly-ubiquitylation of LAPTMs facilitated the interaction of LAPTMs with LAT1 and the recruitment of LAT1 to lysosomes. Furthermore, RNF182-deficient P19 cells exhibited abnormal neurite outgrowth. RNF182-knocked down P19 cells displayed decreased mTORC1 signaling. In contrast, we found that RNF182 expression was induced by the lysosome inhibitor chloroquine and the transcription factor TFEB, which is activated by lysosomal stress.

Conclusions: Based on these results, we speculate that RNF182 upregulates mTORC1 signaling via LAPTM ubiquitination during neuronal differentiation. As TFEB is inactivated by mTORC1, RNF182 may reciprocally regulate the mTORC1 pathway.