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Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling.

Sosinowski T, Pandey A, Dixit VM, Weiss A - J. Exp. Med. (2000)

Bottom Line: SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents.In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT).These results suggest that SLAP is a negative regulator of TCR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0795, USA.

ABSTRACT
Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

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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.
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Figure 3: 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.

Mentions: This suppressive effect was not limited to the NFAT element, since SLAP could also inhibit TCR-dependent activation of AP-1 and IL-2 luciferase reporters (Fig. 3a and Fig. c). To stimulate the IL-2 luciferase reporter, we used a combination of PHA and PMA, an effective stimulus known to induce transcriptional activity of the IL-2 promoter in a TCR-dependent manner 34. SLAP did not interfere with the general transcriptional machinery in a nonspecific manner, since it had no effect on NFAT, AP-1, or the IL-2 promoter in cells stimulated with PMA and the calcium ionophore (ionomycin; Fig. 2 B and Fig. 3b and Fig. d), nor did it suppress the luciferase activity under the control of the constitutive Rous sarcoma virus (RSV) promoter (data not shown). The ability of PMA and ionomycin—pharmacological agents that bypass proximal TCR signaling by activating Ras and increasing intracellular calcium, respectively—to activate transcription in the presence of SLAP places the SLAP-imposed blockade more proximal in the TCR signaling pathway than Ras activation and calcium increases. Moreover, TCR-dependent activation of both the Ras and Ca2+ pathways is inhibited by SLAP, since stimulation of cells with anti-TCR Ab and either PMA alone or ionomycin alone did not bypass the blockade (data not shown).


Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling.

Sosinowski T, Pandey A, Dixit VM, Weiss A - J. Exp. Med. (2000)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195826&req=5

Figure 3: 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.
Mentions: This suppressive effect was not limited to the NFAT element, since SLAP could also inhibit TCR-dependent activation of AP-1 and IL-2 luciferase reporters (Fig. 3a and Fig. c). To stimulate the IL-2 luciferase reporter, we used a combination of PHA and PMA, an effective stimulus known to induce transcriptional activity of the IL-2 promoter in a TCR-dependent manner 34. SLAP did not interfere with the general transcriptional machinery in a nonspecific manner, since it had no effect on NFAT, AP-1, or the IL-2 promoter in cells stimulated with PMA and the calcium ionophore (ionomycin; Fig. 2 B and Fig. 3b and Fig. d), nor did it suppress the luciferase activity under the control of the constitutive Rous sarcoma virus (RSV) promoter (data not shown). The ability of PMA and ionomycin—pharmacological agents that bypass proximal TCR signaling by activating Ras and increasing intracellular calcium, respectively—to activate transcription in the presence of SLAP places the SLAP-imposed blockade more proximal in the TCR signaling pathway than Ras activation and calcium increases. Moreover, TCR-dependent activation of both the Ras and Ca2+ pathways is inhibited by SLAP, since stimulation of cells with anti-TCR Ab and either PMA alone or ionomycin alone did not bypass the blockade (data not shown).

Bottom Line: SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents.In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT).These results suggest that SLAP is a negative regulator of TCR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0795, USA.

ABSTRACT
Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

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