Ligand of Numb proteins LNX1p80 and LNX2 interactwith the human glycoprotein CD8a and promote itsubiquitylation and endocytosis

E3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown. In this study, we identified the human T-cell co-receptor CD8 a-chain as binding partner of the ligand of Numb proteins X1 (LNX1p80 isoform) and X2 (LNX2). Both LNX mRNAs were found expressed in T cells purified from human blood, and both proteins interacted with CD8a in human HPB-ALL T cells. By using an in vitro assay and a heterologous expression system we showed that the interaction is mediated by the PDZ (PSD95-DlgA-ZO-1) domains of LNX proteins and the cytosolic C-terminal valine motif of CD8a. Moreover, CD8a redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8a ubiquitylation, downregulation from the plasma membrane, transport to the lysosomes, and degradation. Our findings highlight the function of LNX proteins as E3 ligases and suggest a mechanism of regulation for CD8a localization at the plasma membrane by ubiquitylation and endocytosis.