T Cell Antigen Receptor Ubiquitination Is a Consequence of Receptor-mediated Tyrosine Kinase Activation

Abstract

Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptormediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56 lck and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56 lck . The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors.For many transmembrane receptors, including the multisubunit TCR, 1 signaling is initiated by ligand-induced aggregation (1, 2). The earliest obligate intracellular event following TCR aggregation is the activation of the src-family protein tyrosine kinases, Lck (p56 lck ) and Fyn (p59 fyn ). Lck and/or Fyn phosphorylate TCR subunits resulting in the association of a third tyrosine kinase, ZAP-70 (70-kDa -associated protein), with the TCR and to subsequent activation events (3-5). CD45, a tyrosine phosphatase that dephosphorylates key regulatory residues on Lck and Fyn, is also implicated in the initiation of TCR-mediated signaling (6).TCRs consist of six different polypeptides, these include the antigen-recognition element, in most cells an ␣-␤ heterodimer, and a set of invariant signal transducing subunits. The invariant subunits include CD3-␦, -⑀, and -␥ and the structurally distinct TCR-subunit, which exists within the TCR as a disulfide-linked homodimer (4). The minimal signal transducing element of the TCR is the immunoreceptor tyrosine-based activation motif (ITAM) (7). monomers have three ITAMs, and each CD3 subunit has one. ITAMs include two tyrosine residues 10 or 11 amino acids apart that are potential phosphorylation sites. The subunit is a particularly prominent substrate for tyrosine phosphorylation; up to 5% of subunits are phosphorylated on multiple tyrosines upon TCR engagement (8 -11).In addition to being a substrate for tyrosine phosphorylation when cross-linked by antibody or mitogen (11), TCRs also are ubiquitinated. The covalent modification of proteins with chains of ubiquitin, a highly conserved 76-amino acid polypeptide, plays a central role in the targeting of abnormal proteins and a number of regulatory cytosolic and nuclear proteins for degradation in the 26 S proteasome (12-16). Ubiquitination occurs via a multienzyme process involving families of enzymes ter...

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

“…Among the immunoreceptors, the T cell receptor undergoes phosphory- lation-dependent ubiquitination (19), possibly also involving Cbl. Interestingly, expression of a constitutively active form of p56lck causes internalization and lysosomal degradation of the assembled cell surface T cell receptors (20).…”

Allelic Exclusion of the TCR α-Chain Is an Active Process Requiring TCR-Mediated Signaling and c-Cbl

“…Lck is required for the TCR to be ubiquitinated and degraded following TCR engagement (27). Because c-Cbl functions as a ubiquitin ligase, resulting in targeting of tyrosine-phosphorylated receptors for endocytosis and degradation (23,24), and inactivation of c-Cbl results in up-regulation of TCR expression in immature DP thymocytes that normally have a TCR low phenotype (21,22), we hypothesized that c-Cbl could be involved in maintenance of ␣-chain phenotypic allelic exclusion.…”

“…Ubiquitination of cell surface receptors is an endocytotic signal that can target them for degradation in proteosomes or by lysosomes (25). This suggested that c-Cbl could be involved in endocytosis and/or degradation of TCR after activation, because ubiquitination of TCR occurs after TCR-mediated stimulation and requires Lck activity (26,27). Recent data confirm that c-Cbl and its close relative Cbl-b are closely involved in the down-modulation and degradation of TCR after activation (28).…”

Endoplasmic Reticulum (ER)-associated Degradation of T Cell Receptor Subunits

“…Anti-CD3-␦ (R9) (17); antiubiquitin (18); and anti-TCR-␣, H28 (19) have all been described. Anti-HA (12CA5) and anti-Myc (9E10) monoclonal antibodies were from culture supernatants.…”