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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-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).
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Figure 4: 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).

Mentions: Since PMA and ionomycin treatment bypassed SLAP-dependent inhibition of NFAT induction, but anti-TCR plus PMA stimulation did not, we reasoned that SLAP might suppress the TCR-dependent Ca2+ flux. To examine this, we cotransfected SLAP or an empty vector with a plasmid encoding Tac, and enriched for Tac-expressing cells as described above (populations were >98% Tac+; Fig. 4 A). As shown in Fig. 4 B, cells transfected with SLAP, when compared with the vector-transfected cells, showed a substantially reduced calcium flux in response to anti-TCR stimulation. Importantly, this pattern of responsiveness was reversed upon subsequent stimulation of cells with ionomycin. Note that SLAP-transfected cells responded to the calcium ionophore by mobilizing more intracellular Ca2+ than vector-transfected cells. As a control in this same experiment, we also transfected the NFAT luciferase reporter plasmid to show that SLAP was expressed at levels sufficient to inhibit the TCR signaling pathway leading to NFAT activation (Fig. 4 C). We employed the same approach of cotransfection and enrichment for Tac+ cells to study the effect of SLAP on TCR-dependent increase in protein tyrosine phosphorylation. However, we failed to observe any differences in the pattern of tyrosine phosphorylation in total cell lysates from TCR-stimulated cells when SLAP was expressed (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-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).
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Related In: Results  -  Collection

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Figure 4: 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).
Mentions: Since PMA and ionomycin treatment bypassed SLAP-dependent inhibition of NFAT induction, but anti-TCR plus PMA stimulation did not, we reasoned that SLAP might suppress the TCR-dependent Ca2+ flux. To examine this, we cotransfected SLAP or an empty vector with a plasmid encoding Tac, and enriched for Tac-expressing cells as described above (populations were >98% Tac+; Fig. 4 A). As shown in Fig. 4 B, cells transfected with SLAP, when compared with the vector-transfected cells, showed a substantially reduced calcium flux in response to anti-TCR stimulation. Importantly, this pattern of responsiveness was reversed upon subsequent stimulation of cells with ionomycin. Note that SLAP-transfected cells responded to the calcium ionophore by mobilizing more intracellular Ca2+ than vector-transfected cells. As a control in this same experiment, we also transfected the NFAT luciferase reporter plasmid to show that SLAP was expressed at levels sufficient to inhibit the TCR signaling pathway leading to NFAT activation (Fig. 4 C). We employed the same approach of cotransfection and enrichment for Tac+ cells to study the effect of SLAP on TCR-dependent increase in protein tyrosine phosphorylation. However, we failed to observe any differences in the pattern of tyrosine phosphorylation in total cell lysates from TCR-stimulated cells when SLAP was expressed (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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