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Molecular and Cellular Biology, December 2007, p. 8622-8636, Vol. 27, No. 24
0270-7306/07/$08.00+0     doi:10.1128/MCB.00467-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Lakshmi Balagopalan, Valarie A. Barr, Connie L. Sommers, Mira Barda-Saad, Amrita Goyal, Matthew S. Isakowitz, and Lawrence E. Samelson^*

Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Received 19 March 2007/ Returned for modification 8 May 2007/ Accepted 27 September 2007

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.

Published ahead of print on 15 October 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.