SLAP is predominantly expressed in the lymphoid tissues. Northern blot analysis was performed on (A) multiple tissues, (B) various cell lines (fibroblast [NIH 3T3], macrophage [Raw264.7], mastocytoma [P815], B cell lines, and T cell lines), and enriched splenic B and T cells (>92% and 98% pure, respectively). A ³²P-labeled DNA fragment from the unique COOH terminus of SLAP cDNA was used to detect SLAP mRNA present in 10 μg of total RNA. Equal loading and complete transfer of RNA were confirmed by ethidium bromide staining and quantitation of ribosomal 28S and 18S RNA using the Bio-Rad Gel Doc 1000 system (bottom panels).

SLAP inhibits TCR induction of NFAT-driven transcription in a dose-dependent manner. Jurkat TAg cells were cotransfected with four different plasmids: a luciferase reporter under the transcriptional control of a multimerized NFAT element (NFAT luciferase); a transfection efficiency indicator (pRc/βgal); a transfection tag (pEF-TacT); and an effector plasmid (either pEF-SLAP or an empty vector, pEF-BOS). The total amount of DNA was kept constant by supplementing the various amounts of pEF-SLAP plasmid with an empty vector. After 16–24 h, cells from each transfection were tested for the transfection efficiency by β-galactosidase assay (data not shown), or incubated for an additional 8 h with medium alone (Media), soluble anti-TCR mAb (C305), or PMA and ionomycin (PMA+Ion), and were assayed for luciferase activity. Luciferase activity was normalized to β-galactosidase and is presented in relative light units (RLU; A and B). Cells were also tested for the level of SLAP protein: transfections containing 0.25, 0.5, or 1.0 μg of pEF-SLAP were enriched for Tac⁺ cells by magnetic beads (D) and used for IPs of the ectopically expressed SLAP (C; see Materials and Methods). As positive and negative controls for the presence of murine SLAP, EL-4 and nontransfected Jurkat TAg cells were used, respectively.

SLAP inhibits TCR-dependent Ca²⁺ mobilization in Jurkat cells. Jurkat TAg cells were transiently cotransfected with three plasmids: NFAT luciferase, pEF-TacT, and pEF-SLAP or an empty vector. 16 h later, transfected cells were enriched based on expression of Tac on the cell surface (see Materials and Methods). Highly enriched populations (A) were loaded with the fluorescent dye indicator Indo-1, then stimulated with soluble anti-TCR mAb (C305) followed by ionomycin (arrows indicate the time of stimulation), and the change in intracellular Ca²⁺ concentration was measured as a function of time (B). Cells from the same experiment were also tested for TCR-dependent NFAT induction (C).

SLAP inhibits TCR induction of both AP-1– and IL-2 promoter–driven transcription in a dose-dependent manner. Cells were cotransfected with three different plasmids: a luciferase reporter under the transcriptional control of either AP-1 element (A and B) or IL-2 promoter (C and D); a transfection efficiency indicator (pRc/βgal); and an effector plasmid (either pEF-SLAP or an empty vector, pEF-BOS). After 16–24 h, transfected cells were assayed for β-galactosidase activity, and stimulated with either C305 (A), PMA (B), PHA and PMA (C), or a combination of PMA and ionomycin (PMA+Ion, D), as indicated (see Materials and Methods). The activity of each reporter is presented as described in the legend to Fig. 2.

Both SH2 and SH3 domains of SLAP participate in inhibition of anti-TCR–induced NFAT activity. Jurkat TAg cells were cotransfected with an NFAT luciferase reporter and various amounts of one of the following effector plasmids: a WT SLAP (WT), an SH2 domain point mutant (SH2*), an SH3 domain point mutant (SH3*), an SH2 and SH3 domain double point mutant (SH2*SH3*), and a mutant with deleted COOH terminus (ΔC). An empty vector was used as a control, and the total amount of DNA was kept constant by adding empty vector DNA. Individual transfections were matched based on the expression level of the various SLAP proteins (bottom). TCR-dependent increase in NFAT activity was calculated as a fold induction, and represents a ratio of luciferase counts of anti-TCR over media-treated cells. Fold induction obtained for vector-transfected cells was defined as maximum (100% MAX).

The SLAP SH2 domain is required for its colocalization with endosomes. Jurkat (A, B, and C) and HeLa cells (D–I) were transiently transfected with pEF-SLAP WT plasmid (A–F) or with pEF-SLAP SH2* mutant (G–I), and the indirect immunofluorescence analysis was performed (see Materials and Methods). The endogenous endosomal marker, CI-M6PR, is present in all cells (green); however, SLAP is present only in transiently transfected cells (red). To depict the degree of colocalization of SLAP and CI-M6PR, the red and green channels were merged (C, F, and I, OVERLAY). Each image represents a single section through a field of cells.

The unique COOH terminus of SLAP determines its insolubility in mild detergents. Jurkat cells were transiently transfected with an expression plasmid encoding either WT SLAP or the indicated mutants. After 24 h, cells were lysed in 1% NP-40 lysis buffer, and the detergent-soluble (S) and the pellet (P) fractions were separated by standard centrifugation procedure (see Materials and Methods). Pellet fractions were solubilized in the SDS-PAGE loading buffer, and cell-equivalent amounts were run on SDS-PAGE. SLAP protein was detected by Western blotting using rabbit anti-SLAP C1661 Ab. EXP, experiment; MWM, molecular weight marker.

SLAP interacts in vitro with a specific subset of phosphoproteins. Lysates from unstimulated (−) or stimulated (+) Jurkat TAg cells were used in the GST pulldown experiment with the indicated fusion proteins (see Materials and Methods). Tyrosine-phosphorylated proteins that interacted with the fusion proteins were resolved by SDS-PAGE and detected with the anti-PTyr mAb (4G10). Soluble WCLs were used as a control for stimulation. Molecular weight markers are indicated on the left (MWM), and the phosphoproteins consistently present in GST-SLAP pulldowns are marked by arrowheads and a bracket on the right.

SLAP interacts in vivo with a specific subset of phosphoproteins. Jurkat TAg cells were transiently transfected with either an empty vector or pEF-SLAP ΔC. After 12–16 h, cells were either left unstimulated (−) or stimulated with soluble anti-TCR Ab (C305) for 2 min (+), and lysed in 1% NP-40 lysis buffer. Equivalent amounts of lysates were used in anti-Lck and anti-SLAP IPs (see Materials and Methods). Subsequently, WCLs or washed immunoprecipitates were separated by SDS-PAGE and probed with anti-PTyr mAb (4G10). MWM, molecular weight marker.