Abstract
Antigen recognition triggers the recruitment of the critical adaptor protein SLP-76 to small macromolecular clusters nucleated by the T-cell receptor (TCR). These structures develop rapidly, in parallel with TCR-induced increases in tyrosine phosphorylation and cytosolic calcium, and are likely to contribute to TCR-proximal signaling. Previously, we demonstrated that these SLP-76-containing clusters segregate from the TCR and move towards the center of the contact interface. Neither the function of these clusters nor the structural requirements governing their persistence have been examined extensively. Here we demonstrate that defects in cluster assembly and persistence are associated with defects in T-cell activation in the absence of Lck, ZAP-70, or LAT. Clusters persist normally in the absence of phospholipase C-γ1, indicating that in the absence of a critical effector, these structures are insufficient to drive T-cell activation. Furthermore, we show that the critical adaptors LAT and Gads localize with SLP-76 in persistent clusters. Mutational analyses of LAT, Gads, and SLP-76 indicated that multiple domains within each of these proteins contribute to cluster persistence. These data indicate that multivalent cooperative interactions stabilize these persistent signaling clusters, which may correspond to the functional complexes predicted by kinetic proofreading models of T-cell activation.
Supplemental material for this article may be found at http://mcb.asm.org/.
We thank Arthur Weiss, Robert Abraham, and David Strauss for various Jurkat cell lines, C. Jane McGlade for Gads expression constructs, and Jon Houtman for many helpful discussions.
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and by grants from the National Institutes of Health (F32-DK09969 to A.L.S. and P01-CA93615 to M.S.J. and G.A.K.). S.C.B. was a fellow of the Cancer Research Institute. D.I.H. was a Howard Hughes Medical Institute-National Institutes of Health research scholar.