c-Cbl-Mediated Regulation of LAT-Nucleated Signaling Complexes

Abstract

The engagement of the T-cell receptor (TCR) causes the rapid recruitment of multiple signaling molecules into clusters with the TCR. Upon receptor activation, the adapters LAT and SLP-76, visualized as chimeric proteins tagged with yellow fluorescent protein, transiently associate with and then rapidly dissociate from the TCR. Previously, we demonstrated that after recruitment into signaling clusters, SLP-76 is endocytosed in vesicles via a lipid raft-dependent pathway that requires the interaction of the endocytic machinery with ubiquitylated proteins. In this study, we focus on LAT and demonstrate that signaling clusters containing this adapter are internalized into distinct intracellular compartments and dissipate rapidly upon TCR activation. The internalization of LAT was inhibited in cells expressing versions of the ubiquitin ligase c-Cbl mutated in the RING domain and in T cells from mice lacking c-Cbl. Moreover, c-Cbl RING mutant forms suppressed LAT ubiquitylation and caused an increase in cellular LAT levels, as well as basal and TCR-induced levels of phosphorylated LAT. Collectively, these data indicate that following the rapid formation of signaling complexes upon TCR stimulation, c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…In addition, consistent with its role as an E3-ubiquitin ligase in several signaling systems, c-Cbl has been implicated in ubiquitylation of the TCRz and CD3d chains of the TCR as well as the signaling proteins ZAP-70, LAT, PLC-g1, PI3K, Vav, and PKC-u in T cells (Weissman 2001;Duan et al 2004;Balagopalan et al 2007). The effects of LATubiquitylation on controlling internalization and cellular LAT levels are discussed in the imaging section later.…”

“…Microclusters play a predominant role in the generation of sustained signals. Current models also indicate that cluster centralization plays an important role in signal termination and that the cSMAC is a site both for TCR down-regulation yet also for amplification of weak signals (Lee et al 2003;Mossman et al 2005;Varma et al 2006;Balagopalan et al 2007;Cemerski et al 2008;Nguyen et al 2008). A recent study using a photoactivatable agonist in a lipid bilayer system has provided even greater precision in the temporal resolution of these events and demonstrated that adapters are recruited to microclusters within four seconds (Huse et al 2007).…”

“…The adapters LAT and SLP-76 departed the initial complexes in what appeared to be small vesicular intermediates and dissipated soon thereafter (Bunnell et al 2002). Recent studies have shown that microclusters containing LAT and SLP-76 undergo endocytosis upon TCR stimulation Balagopalan et al 2007). SLP-76 is endocytosed in a lipid-raft dependent pathway that requires the association of the endocytic machinery with ubiquitylated proteins.…”

“…SLP-76 is endocytosed in a lipid-raft dependent pathway that requires the association of the endocytic machinery with ubiquitylated proteins. LAT endocytosis is a complex process and internalized LAT is found in several intracellular compartments (Bonello et al 2004;Brignatz et al 2005;Balagopalan et al 2007). Insights into the molecular mechanisms that regulate endocytosis of microcluster-associated proteins came from studies in which internalization of the microclusters was inhibited.…”

“…Palmitoylation of LAT is necessary for LAT function (Lin et al 1999;Zhang et al 1999a), but the role of palmitoylation in specific localization of LAT within the plasma membrane has been controversial and is discussed below. More recently, studies have shown that LAT is also subject to ubiquitylation (Brignatz et al 2005;Balagopalan et al 2007), a modification that might be involved in activation-induced internalization of LAT complexes and regulation of LAT protein levels. Inspection of LAT amino acid sequence reveals two lysines (K52 and K204 in human LAT), which might serve as potential sites for ubiquitylation.…”