Dlg1 associates with membrane actin after receptor engagement in T cells and relocalizes after T cell receptor activation by antibody- conjugated beads. (A). Jurkat cells were allowed to settle in complete medium on coverslips coated with polylysine, anti-CD3 antibody, or anti-CD3 plus anti-CD28 antibodies. Cells were fixed and permeabilized and stained with phallotoxin conjugated with Alexa Fluor 594 (for visualization of actin) and polyclonal rabbit anti-Dlg1. (B) Images acquired 5 min after exposure of cells to beads bearing anti-CD3 and anti-CD28 antibody. Dlg1 and actin were visualized with anti-Dlg1 antibody and fluorescent phallotoxin, respectively. Dlg1 does not localize at the contact region between Jurkat cells and beads conjugated with anti-CD19 antibody. (C) Images acquired 15 min after exposure of cells to beads bearing anti-CD3 and anti-CD28 antibody. Four representative images demonstrate the redistribution of endogenous Dlg1 away from the contact surface.

Proteins coordinated by Dlg1. (A) Multiple signaling molecules associate with Dlg1 in T cells. Membrane and cytosolic fractions from unstimulated and anti-CD3 plus CD28-stimulated Jurkat cells were immunoprecipitated with Dlg1 antibody, resolved by SDS-PAGE and immunoblotted with antibodies recognizing the proteins shown. TL represents 10 μl of non-immunoprecipitated total lysate loaded with each blot to confirm antibody specificity. Iso represents the result of immunoblotting lysates prepared by immunoprecipitation with an isotype-matched control antibody. (B) Phospho-ζ (p23ζ) association with Dlg1. Dlg1 immunoprecipitates from cytosol, membrane fractions, and nucleus fractions were probed with anti-TCRζ (left) or with anti-phosphotyrosine antibody 4G10 (right). The top bands in the immunoblots represent immunoglobulin light chain. White lines indicate that intervening lanes have been spliced out.

Stable RNA interference of Dlg1 in Jurkat cells enhances NFAT activity. (A) Cells expressing shRNA were lysed in 1% Triton X-100 buffer and an equal amount protein from each cell line was resolved on a 7.5% SDS gel and blotted with antibodies recognizing Dlg1, actin, GSK, and Lck. (B) Activation of NFAT luciferase reporter in Jurkat cells stably transfected to express shRNA targeted against the Dlg1 SH3 domain (shRNA1) or GK domain (shRNA2). Cells were transfected with NFAT-luciferase reporter. 24 h after transfection, cells were stimulated with anti-CD3 (5 μg/ml) for 8 h and lysed for assay. (C) Activation of NFAT luciferase reporter in Jurkat cells stably transfected to express shRNA targeted against the Dlg1 SH3 domain (shRNA1) or GK domain (shRNA2). Pooled stable cells were transfected with NFAT-luciferase reporter construct. 20 h after transfection, cells were stimulated with Raji cells pulsed with SEE for 8 h and lysed for luciferase assay. (Bottom) Immunoblot analysis confirms shRNA suppression of Dlg1 expression. White lines indicate that intervening lanes have been spliced out. Error bars represent SEM.

APC-T cell activation induces Dlg1 relocalization. (A) Dlg1 accumulates at the T cell–B cell contact site. Jurkat cells were incubated with Raji cells that had been exposed to medium with or without SEE for 5, 10, 15, and 20 min. Raji cells were labeled with CMAC cell tracker blue. F-actin and Dlg1 accumulation were induced by SEE pulsed Raji cells at 5 and 10 min (not depicted). Representative images show no increase in Dlg1 accumulation at cell–cell contact site at 20 min. (B) Codistribution of CD3ζ and Dlg1 after activation of Jurkat cells with Raji cells loaded with SEE. CD3ζ and Dlg1 colocalize at the cell–cell contact site at early time points (5 min) after activation. Raji cells are labeled with CMAC tracker blue (CT blue). (C) Dlg1 localizes transiently at the immune synapse after antigenic stimulation of T cells. LPS-activated B cells (labeled blue) were pulsed with ova-peptide and mixed with DO11.10 transgenic T cells. F-actin and Dlg1 were recruited to the contact area at 5 min.

Effects of Dlg1 expression on transcriptional activity in Jurkat cells. (A) Overexpression of Dlg1 attenuates Vav1-induced NFAT transcriptional activity. NFAT activity was measured after cotransfection of Jurkat cells with the indicated plasmids and an NFAT-luciferase reporter construct. After stimulation, cells were lysed and luciferase activity was measured. Relative activity is corrected for transfection efficiency. (Bottom) Immunoblot analysis confirms expression of constructs used in the reporter assay. (B) Effects of Dlg1 on Vav1 basal and anti-CD3 stimulated NFAT activation in Jurkat cells. Overexpression of Dlg1 inhibits Vav1 induction of NFAT activity, whereas overexpression of Dlg1 carboxy-terminal fragment potentiates the Vav1 effect. (Bottom) Immunoblot illustrates relative expression of various constructs used in reporter assay. (C) Overexpression of Dlg1 suppresses NFAT activation by coexpressed Vav1 in Jurkat cells stimulated by superantigen pulsed Raji cells. Jurkat T cells were transfected with NFAT-luciferase reporter, Vav1 and Dlg1 constructs. 20 h after transfection, cells were stimulated with Raji cells pulsed with SEE for 8 h and lysed for luciferase assay. (Bottom) Immunoblot analysis confirms expression of constructs used in the reporter assay. White lines indicate that intervening lanes have been spliced out. Error bars represent SEM.