Linker for activation of T cells (LAT) plays a central role in T-cell activation by nucleating signaling complexes that are critical for the propagation of T-cell signals from the plasma membrane to the cellular interior. The role of phosphorylation and palmitoylation in LAT function has been well studied, but not much is known about other strategies by which the cell modulates LAT activity. We have focused on LAT ubiquitylation and have mapped the sites on which LAT is ubiquitylated. To elucidate the biological role of this process, we substituted LAT lysines with arginines. This resulted in a dramatic decrease in overall LAT ubiquitylation. Ubiquitylationresistant mutants of LAT were internalized at rates comparable to wild-type LAT in a mechanism that required Cbl family proteins. However, these mutants displayed a defect in protein turnover rates. T-cell signaling was elevated in cells reconstituted with LAT mutants resistant to ubiquitylation, indicating that inhibition of LAT ubiquitylation enhances T-cell potency. These results support LAT ubiquitylation as a molecular checkpoint for attenuation of T-cell signaling.endocytosis | protein degradation | ubiquitin A ntigen activation of T cells leads to major cellular changes essential to the onset of a productive immune response. The earliest events occur upon T-cell antigen receptor (TCR) engagement and include activation of protein tyrosine kinases (PTKs) and phosphorylation of multiple protein substrates. One critical PTK substrate is the integral membrane adapter molecule linker for activation of T cells (LAT), which upon phosphorylation, serves as a nucleation site for the formation of protein complexes composed of itself, other adapter molecules, and essential signaling enzymes activated at this site (1). A large number of studies have revealed that LAT-based complexes catalyze critical TCR-mediated signaling reactions and lead to the induction of multiple downstream pathways that direct almost all TCRinitiated cellular responses (2).T-cell signaling must be tightly regulated, otherwise autoimmunity or a compromised immune response might ensue. Clearly, signal attenuation occurs at various stages during signal propagation. Proximally, the number and type of inhibitory coreceptors that are engaged on the T cell following TCR engagement greatly influence signal strength (3, 4). In addition, activation of the TCR also triggers mechanisms within the TCR signaling machinery that can decrease TCR-mediated signaling, including dephosphorylation of substrates by phosphatases, as well as phosphorylation on inhibitory residues by kinases (5). Finally, ubiquitylation and activationinduced endocytosis of proteins from the plasma membrane and subsequent degradation may eliminate activated signaling molecules (6, 7). Thus, tuning the excitation threshold of a T cell requires the coordinated regulation of multiple negative regulators.A growing body of evidence supports the concept that clustering of the TCR and downstream signaling proteins is associated with and is ...