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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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SLAP-2 inhibits antigen receptor signaling in B and T lymphocytes. Epitope-tagged SLAP-2 and SLAP cDNAs in the pTRA-IRES.GFP vector or vector alone were infected into tTA-BJAB or Jurkat cells. Surface CD69 expression was analyzed in stimulated GFP-negative (uninfected; dotted line) and GFP-positive (infected; solid line) cells by analytical gating. The geometric means of APC-CD69 fluorescence are shown for GFP-negative and GFP-positive cells. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2 or PMA. Both SLAP-2 and SLAP significantly decrease anti-IgM F(ab′)2-induced but not PMA-induced CD69 expression compared with control. (B) tTA-Jurkat cells stimulated with C305 or PMA. Both SLAP-2 and SLAP significantly decrease C305-induced CD69 expression compared with control. (C) NFAT promoter activation is inhibited by SLAP-2. BJAB or Jurkat-TAg cells were transiently co-transfected with 40 μg pEFBOS-SLAP-2 or vector alone, plus an NFAT-Luciferase reporter construct. Cells were stimulated with anti-IgM F(ab′)2, C305, or PMA plus ionomycin for 12 h, and assayed for luciferase activity in triplicate. Fold induction of luciferase activity over the unstimulated, vector-transfected sample is shown. The basal luciferase activity for this experiment was ∼100 arbitrary light units (AU). The data are representative of several independent experiments. (D) Jurkat-TAg cells were transiently cotransfected with 1, 2, 4, 16, or 32 μg of SLAP-2 DNA and NFAT-Luciferase reporter construct, keeping the total DNA amount constant with empty vector. Cells were stimulated and luciferase assays were performed as above. Equal aliquots of cells were analyzed by anti-FLAG Western blot for SLAP-2 protein expression.
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fig3: SLAP-2 inhibits antigen receptor signaling in B and T lymphocytes. Epitope-tagged SLAP-2 and SLAP cDNAs in the pTRA-IRES.GFP vector or vector alone were infected into tTA-BJAB or Jurkat cells. Surface CD69 expression was analyzed in stimulated GFP-negative (uninfected; dotted line) and GFP-positive (infected; solid line) cells by analytical gating. The geometric means of APC-CD69 fluorescence are shown for GFP-negative and GFP-positive cells. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2 or PMA. Both SLAP-2 and SLAP significantly decrease anti-IgM F(ab′)2-induced but not PMA-induced CD69 expression compared with control. (B) tTA-Jurkat cells stimulated with C305 or PMA. Both SLAP-2 and SLAP significantly decrease C305-induced CD69 expression compared with control. (C) NFAT promoter activation is inhibited by SLAP-2. BJAB or Jurkat-TAg cells were transiently co-transfected with 40 μg pEFBOS-SLAP-2 or vector alone, plus an NFAT-Luciferase reporter construct. Cells were stimulated with anti-IgM F(ab′)2, C305, or PMA plus ionomycin for 12 h, and assayed for luciferase activity in triplicate. Fold induction of luciferase activity over the unstimulated, vector-transfected sample is shown. The basal luciferase activity for this experiment was ∼100 arbitrary light units (AU). The data are representative of several independent experiments. (D) Jurkat-TAg cells were transiently cotransfected with 1, 2, 4, 16, or 32 μg of SLAP-2 DNA and NFAT-Luciferase reporter construct, keeping the total DNA amount constant with empty vector. Cells were stimulated and luciferase assays were performed as above. Equal aliquots of cells were analyzed by anti-FLAG Western blot for SLAP-2 protein expression.

Mentions: To confirm that the inhibitory phenotype observed in clones 584 and 780 was indeed dependent on the presence of the cDNAs, full length SLAP-2 and SLAP cDNAs were subcloned into the pTRA-IRES.GFP retroviral vector (17) and reintroduced into parental tTA-BJAB cells (Fig. 3 A). Coexpression of SLAP-2 or SLAP with GFP allowed for parallel analysis of uninfected and infected cells within the same sample, thus providing an internal control. No significant difference in basal CD69 levels was observed between GFP-positive and GFP-negative cells within samples (data not shown). Cross-linking of the BCR with anti-IgM led to a ∼3.5-fold upregulation of CD69 expression in both uninfected and infected cells in the vector control population (data not shown). However, in GFP-positive cells expressing either SLAP-2 or SLAP, anti-IgM–induced CD69 expression was significantly reduced compared with vector control or uninfected controls (Fig. 3 A) verifying that the inhibitory phenotype could be transferred to naive cells. In contrast, expression of SLAP-2 did not affect CD69 induction in response to PMA, which bypasses early signaling events leading to activation of PKC and downstream signaling molecules Erk 1/2 MAPKs.


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)

SLAP-2 inhibits antigen receptor signaling in B and T lymphocytes. Epitope-tagged SLAP-2 and SLAP cDNAs in the pTRA-IRES.GFP vector or vector alone were infected into tTA-BJAB or Jurkat cells. Surface CD69 expression was analyzed in stimulated GFP-negative (uninfected; dotted line) and GFP-positive (infected; solid line) cells by analytical gating. The geometric means of APC-CD69 fluorescence are shown for GFP-negative and GFP-positive cells. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2 or PMA. Both SLAP-2 and SLAP significantly decrease anti-IgM F(ab′)2-induced but not PMA-induced CD69 expression compared with control. (B) tTA-Jurkat cells stimulated with C305 or PMA. Both SLAP-2 and SLAP significantly decrease C305-induced CD69 expression compared with control. (C) NFAT promoter activation is inhibited by SLAP-2. BJAB or Jurkat-TAg cells were transiently co-transfected with 40 μg pEFBOS-SLAP-2 or vector alone, plus an NFAT-Luciferase reporter construct. Cells were stimulated with anti-IgM F(ab′)2, C305, or PMA plus ionomycin for 12 h, and assayed for luciferase activity in triplicate. Fold induction of luciferase activity over the unstimulated, vector-transfected sample is shown. The basal luciferase activity for this experiment was ∼100 arbitrary light units (AU). The data are representative of several independent experiments. (D) Jurkat-TAg cells were transiently cotransfected with 1, 2, 4, 16, or 32 μg of SLAP-2 DNA and NFAT-Luciferase reporter construct, keeping the total DNA amount constant with empty vector. Cells were stimulated and luciferase assays were performed as above. Equal aliquots of cells were analyzed by anti-FLAG Western blot for SLAP-2 protein expression.
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Related In: Results  -  Collection

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fig3: SLAP-2 inhibits antigen receptor signaling in B and T lymphocytes. Epitope-tagged SLAP-2 and SLAP cDNAs in the pTRA-IRES.GFP vector or vector alone were infected into tTA-BJAB or Jurkat cells. Surface CD69 expression was analyzed in stimulated GFP-negative (uninfected; dotted line) and GFP-positive (infected; solid line) cells by analytical gating. The geometric means of APC-CD69 fluorescence are shown for GFP-negative and GFP-positive cells. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2 or PMA. Both SLAP-2 and SLAP significantly decrease anti-IgM F(ab′)2-induced but not PMA-induced CD69 expression compared with control. (B) tTA-Jurkat cells stimulated with C305 or PMA. Both SLAP-2 and SLAP significantly decrease C305-induced CD69 expression compared with control. (C) NFAT promoter activation is inhibited by SLAP-2. BJAB or Jurkat-TAg cells were transiently co-transfected with 40 μg pEFBOS-SLAP-2 or vector alone, plus an NFAT-Luciferase reporter construct. Cells were stimulated with anti-IgM F(ab′)2, C305, or PMA plus ionomycin for 12 h, and assayed for luciferase activity in triplicate. Fold induction of luciferase activity over the unstimulated, vector-transfected sample is shown. The basal luciferase activity for this experiment was ∼100 arbitrary light units (AU). The data are representative of several independent experiments. (D) Jurkat-TAg cells were transiently cotransfected with 1, 2, 4, 16, or 32 μg of SLAP-2 DNA and NFAT-Luciferase reporter construct, keeping the total DNA amount constant with empty vector. Cells were stimulated and luciferase assays were performed as above. Equal aliquots of cells were analyzed by anti-FLAG Western blot for SLAP-2 protein expression.
Mentions: To confirm that the inhibitory phenotype observed in clones 584 and 780 was indeed dependent on the presence of the cDNAs, full length SLAP-2 and SLAP cDNAs were subcloned into the pTRA-IRES.GFP retroviral vector (17) and reintroduced into parental tTA-BJAB cells (Fig. 3 A). Coexpression of SLAP-2 or SLAP with GFP allowed for parallel analysis of uninfected and infected cells within the same sample, thus providing an internal control. No significant difference in basal CD69 levels was observed between GFP-positive and GFP-negative cells within samples (data not shown). Cross-linking of the BCR with anti-IgM led to a ∼3.5-fold upregulation of CD69 expression in both uninfected and infected cells in the vector control population (data not shown). However, in GFP-positive cells expressing either SLAP-2 or SLAP, anti-IgM–induced CD69 expression was significantly reduced compared with vector control or uninfected controls (Fig. 3 A) verifying that the inhibitory phenotype could be transferred to naive cells. In contrast, expression of SLAP-2 did not affect CD69 induction in response to PMA, which bypasses early signaling events leading to activation of PKC and downstream signaling molecules Erk 1/2 MAPKs.

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