Ligand of Numb proteins LNX1p80 and LNX2 interact with the human glycoprotein CD8α and promote its ubiquitylation and endocytosis

Massimo D'Agostino,Giovanna Mottola,Stefano Bonatti,Eric Ghigo,Maria Vittoria Barone,Maria Gabriella Caporaso,Giusy Tornillo

doi:10.1242/jcs.081224

Abstract

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 α-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 CD8α 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 CD8α. Moreover, CD8α redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8α 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 CD8α localization at the plasma membrane by ubiquitylation and endocytosis.

Highlights

The localization of transmembrane receptors at the plasma membrane (PM) is dynamically controlled by both the rate of delivery via the exocytic pathway and the rate of internalization and transport to the endosomes via endocytosis
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

Summary

Introduction

The localization of transmembrane receptors at the plasma membrane (PM) is dynamically controlled by both the rate of delivery via the exocytic pathway and the rate of internalization and transport to the endosomes via endocytosis. Once thought to only mediate proteasomal degradation in the cytosol (Pickart and Fushman, 2004), ubiquitylation might occur at the PM where it regulates protein internalization, at the trans-Golgi complex where it directs proteins to the late endosomes, and in endosomes to sort proteins to the multivesicular bodies (reviewed in Mukhopadhyay and Riezman, 2007; Piper and Luzio, 2007). In all these cases, it results in protein degradation into the lysosomes. More than 600 E3 ligases were found in the human proteome and were classified into two major groups, defined by the presence of either a HECT (homologous to the E6 associated protein C terminus) or a RING (really interesting new gene) domain as catalytic domain

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