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Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

Holland SJ, Liao XC, Mendenhall MK, Zhou X, Pardo J, Chu P, Spencer C, Fu A, Sheng N, Yu P, Pali E, Nagin A, Shen M, Yu S, Chan E, Wu X, Li C, Woisetschlager M, Aversa G, Kolbinger F, Bennett MK, Molineaux S, Luo Y, Payan DG, Mancebo HS, Wu J - J. Exp. Med. (2001)

Bottom Line: Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade.In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl.Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

View Article: PubMed Central - PubMed

Affiliation: Rigel, Incorporated, South San Francisco, CA 94080, USA. sholland@rigel.com

ABSTRACT
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

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CD69 cell surface marker screen in BJAB cells. (A) Characterization of the tTA-BJAB cell line using dominant-negative ΔSyk. The tTA-BJAB cell line was infected with pTRA-IRES.GFP vector or a truncated, dominant negative version of Syk (ΔSyk) in the same vector. Cells were stimulated (stim.) with anti-IgM F(ab′)2 and stained for surface CD69 expression. In GFP-gated control cells, fivefold antigen-induced upregulation of CD69 expression over the basal level was achieved. Infection of ΔSyk reduced anti-IgM F(ab′)2-induced CD69 expression to 38% of the control value and also slightly depressed basal CD69 expression. Geometric mean values of CD69 fluorescence are shown in brackets. (B) Screening strategy. tTA BJAB cells stably infected with pTRA cDNA libraries were stimulated with anti-IgM F(ab′)2 and stained for CD69 surface expression. Cells expressing the lowest levels of antigen-induced CD69 were sorted, expanded, and resorted until significant enrichment of nonresponsive cells was observed. Single cell clones were deposited from several sorts and analyzed for anti-IgM F(ab′)2-induced CD69 upregulation in the absence (cDNA on) and presence (cDNA off) of dox. (C) Cell surface CD69 expression in representative positive cell clones. Unstimulated cells (dotted line) and cells stimulated with anti-IgM F(ab′)2 (solid line) were stained with APC-conjugated anti-CD69 antibody. Gray line: stimulated cells stained with isotype control-APC antibody. Inhibition of anti-IgMF(ab′)2-induced CD69 expression (top panel) by cDNAs in clones 584, 780, and G18 (containing spiked control Δsyk) is reversed when cells are grown in the presence of dox (cDNA off; bottom panel).
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fig1: CD69 cell surface marker screen in BJAB cells. (A) Characterization of the tTA-BJAB cell line using dominant-negative ΔSyk. The tTA-BJAB cell line was infected with pTRA-IRES.GFP vector or a truncated, dominant negative version of Syk (ΔSyk) in the same vector. Cells were stimulated (stim.) with anti-IgM F(ab′)2 and stained for surface CD69 expression. In GFP-gated control cells, fivefold antigen-induced upregulation of CD69 expression over the basal level was achieved. Infection of ΔSyk reduced anti-IgM F(ab′)2-induced CD69 expression to 38% of the control value and also slightly depressed basal CD69 expression. Geometric mean values of CD69 fluorescence are shown in brackets. (B) Screening strategy. tTA BJAB cells stably infected with pTRA cDNA libraries were stimulated with anti-IgM F(ab′)2 and stained for CD69 surface expression. Cells expressing the lowest levels of antigen-induced CD69 were sorted, expanded, and resorted until significant enrichment of nonresponsive cells was observed. Single cell clones were deposited from several sorts and analyzed for anti-IgM F(ab′)2-induced CD69 upregulation in the absence (cDNA on) and presence (cDNA off) of dox. (C) Cell surface CD69 expression in representative positive cell clones. Unstimulated cells (dotted line) and cells stimulated with anti-IgM F(ab′)2 (solid line) were stained with APC-conjugated anti-CD69 antibody. Gray line: stimulated cells stained with isotype control-APC antibody. Inhibition of anti-IgMF(ab′)2-induced CD69 expression (top panel) by cDNAs in clones 584, 780, and G18 (containing spiked control Δsyk) is reversed when cells are grown in the presence of dox (cDNA off; bottom panel).

Mentions: The optimized tTA-BJAB cells were further characterized by expressing a dominant negative kinase-deleted version of the B cell signaling protein Syk (ΔSyk). When overexpressed from the TRE enhancer element, ΔSyk significantly reduced anti-IgM–induced CD69 expression (Fig. 1 A). This reduction was sensitive to dox, which suppresses the expression of ΔSyk (data not shown).


Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

Holland SJ, Liao XC, Mendenhall MK, Zhou X, Pardo J, Chu P, Spencer C, Fu A, Sheng N, Yu P, Pali E, Nagin A, Shen M, Yu S, Chan E, Wu X, Li C, Woisetschlager M, Aversa G, Kolbinger F, Bennett MK, Molineaux S, Luo Y, Payan DG, Mancebo HS, Wu J - J. Exp. Med. (2001)

CD69 cell surface marker screen in BJAB cells. (A) Characterization of the tTA-BJAB cell line using dominant-negative ΔSyk. The tTA-BJAB cell line was infected with pTRA-IRES.GFP vector or a truncated, dominant negative version of Syk (ΔSyk) in the same vector. Cells were stimulated (stim.) with anti-IgM F(ab′)2 and stained for surface CD69 expression. In GFP-gated control cells, fivefold antigen-induced upregulation of CD69 expression over the basal level was achieved. Infection of ΔSyk reduced anti-IgM F(ab′)2-induced CD69 expression to 38% of the control value and also slightly depressed basal CD69 expression. Geometric mean values of CD69 fluorescence are shown in brackets. (B) Screening strategy. tTA BJAB cells stably infected with pTRA cDNA libraries were stimulated with anti-IgM F(ab′)2 and stained for CD69 surface expression. Cells expressing the lowest levels of antigen-induced CD69 were sorted, expanded, and resorted until significant enrichment of nonresponsive cells was observed. Single cell clones were deposited from several sorts and analyzed for anti-IgM F(ab′)2-induced CD69 upregulation in the absence (cDNA on) and presence (cDNA off) of dox. (C) Cell surface CD69 expression in representative positive cell clones. Unstimulated cells (dotted line) and cells stimulated with anti-IgM F(ab′)2 (solid line) were stained with APC-conjugated anti-CD69 antibody. Gray line: stimulated cells stained with isotype control-APC antibody. Inhibition of anti-IgMF(ab′)2-induced CD69 expression (top panel) by cDNAs in clones 584, 780, and G18 (containing spiked control Δsyk) is reversed when cells are grown in the presence of dox (cDNA off; bottom panel).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195979&req=5

fig1: CD69 cell surface marker screen in BJAB cells. (A) Characterization of the tTA-BJAB cell line using dominant-negative ΔSyk. The tTA-BJAB cell line was infected with pTRA-IRES.GFP vector or a truncated, dominant negative version of Syk (ΔSyk) in the same vector. Cells were stimulated (stim.) with anti-IgM F(ab′)2 and stained for surface CD69 expression. In GFP-gated control cells, fivefold antigen-induced upregulation of CD69 expression over the basal level was achieved. Infection of ΔSyk reduced anti-IgM F(ab′)2-induced CD69 expression to 38% of the control value and also slightly depressed basal CD69 expression. Geometric mean values of CD69 fluorescence are shown in brackets. (B) Screening strategy. tTA BJAB cells stably infected with pTRA cDNA libraries were stimulated with anti-IgM F(ab′)2 and stained for CD69 surface expression. Cells expressing the lowest levels of antigen-induced CD69 were sorted, expanded, and resorted until significant enrichment of nonresponsive cells was observed. Single cell clones were deposited from several sorts and analyzed for anti-IgM F(ab′)2-induced CD69 upregulation in the absence (cDNA on) and presence (cDNA off) of dox. (C) Cell surface CD69 expression in representative positive cell clones. Unstimulated cells (dotted line) and cells stimulated with anti-IgM F(ab′)2 (solid line) were stained with APC-conjugated anti-CD69 antibody. Gray line: stimulated cells stained with isotype control-APC antibody. Inhibition of anti-IgMF(ab′)2-induced CD69 expression (top panel) by cDNAs in clones 584, 780, and G18 (containing spiked control Δsyk) is reversed when cells are grown in the presence of dox (cDNA off; bottom panel).
Mentions: The optimized tTA-BJAB cells were further characterized by expressing a dominant negative kinase-deleted version of the B cell signaling protein Syk (ΔSyk). When overexpressed from the TRE enhancer element, ΔSyk significantly reduced anti-IgM–induced CD69 expression (Fig. 1 A). This reduction was sensitive to dox, which suppresses the expression of ΔSyk (data not shown).

Bottom Line: Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade.In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl.Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

View Article: PubMed Central - PubMed

Affiliation: Rigel, Incorporated, South San Francisco, CA 94080, USA. sholland@rigel.com

ABSTRACT
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Show MeSH
Related in: MedlinePlus