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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 associates with tyrosine phosphorylated proteins including Cbl after antigen stimulation. (A) SLAP-2 associates with tyrosine phosphorylated proteins in B cells. Sorted tTA-BJAB cells infected with epitope-tagged wild-type (WT) SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were stimulated with anti-IgM F(ab′)2 for 2 min, lysed, and SLAP-2 was immunoprecipitated using anti-FLAG agarose. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies. SLAP-2 associates with tyrosine phosphorylated proteins of ∼110 and 70 kD after antigen stimulation. The ΔC mutant lacks the 110 kD SLAP-2-associated phosphoprotein. Bottom panel: reprobe with anti-FLAG. (B) SLAP-2 interacts with Cbl in B cells. Wild-type SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were immunoprecipitated as in (A) and immunoblotted with anti-Cbl antibodies. Wild-type SLAP-2 and SLAP-2-myr but not SLAP-2-ΔC associate with Cbl after antigen stimulation.
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fig6: SLAP-2 associates with tyrosine phosphorylated proteins including Cbl after antigen stimulation. (A) SLAP-2 associates with tyrosine phosphorylated proteins in B cells. Sorted tTA-BJAB cells infected with epitope-tagged wild-type (WT) SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were stimulated with anti-IgM F(ab′)2 for 2 min, lysed, and SLAP-2 was immunoprecipitated using anti-FLAG agarose. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies. SLAP-2 associates with tyrosine phosphorylated proteins of ∼110 and 70 kD after antigen stimulation. The ΔC mutant lacks the 110 kD SLAP-2-associated phosphoprotein. Bottom panel: reprobe with anti-FLAG. (B) SLAP-2 interacts with Cbl in B cells. Wild-type SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were immunoprecipitated as in (A) and immunoblotted with anti-Cbl antibodies. Wild-type SLAP-2 and SLAP-2-myr but not SLAP-2-ΔC associate with Cbl after antigen stimulation.

Mentions: Signaling through the TCR or BCR results in a rapid increase in tyrosine phosphorylation of numerous intracellular proteins, initiated by Src family and Syk/ZAP70 kinase activation (1, 3). These early signaling events ultimately result in transcriptional activation, upregulation of surface antigens, and other lymphocyte effector functions. Adaptor proteins play an important intermediary role in integrating upstream signals to produce biological function (6). Although upon initial evaluation, no changes were discernable in patterns of tyrosine phosphorylation in total cell lysates of BJAB cells overexpressing SLAP-2 (data not shown) we further investigated the nature of SLAP-2 signaling complexes. Epitope-tagged versions of SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were immunoprecipitated from lysates of stably expressing BJAB cells using the anti-FLAG antibody. All three proteins became associated with a number of tyrosine phosphorylated proteins after BCR stimulation (Fig. 6 A), indicating that SLAP-2 indeed participates in BCR signaling pathways. Interestingly, a prominent phosphoprotein of ∼110 kD was absent in the immunoprecipitates of SLAP-2-ΔC (Fig. 6 A), which we subsequently identified as the RING finger ubiquitin ligase Cbl (Fig. 6 B). Cbl has been previously shown to be a negative regulator of TCR and BCR signaling pathways (31–33). An NH2-terminal fragment of Cbl constitutively interacts with the COOH-terminal region of SLAP as demonstrated in both the yeast two hybrid system and Cos-7 cells (30). In contrast, the association between SLAP-2 and full-length Cbl in B cells was inducible after antigen receptor stimulation. Whether SLAP-2 functions as an inhibitory adaptor by recruiting a negative regulator such as Cbl into the signaling complex or by competing with a positive regulator such as a Src-family kinase remains to be determined.


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 associates with tyrosine phosphorylated proteins including Cbl after antigen stimulation. (A) SLAP-2 associates with tyrosine phosphorylated proteins in B cells. Sorted tTA-BJAB cells infected with epitope-tagged wild-type (WT) SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were stimulated with anti-IgM F(ab′)2 for 2 min, lysed, and SLAP-2 was immunoprecipitated using anti-FLAG agarose. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies. SLAP-2 associates with tyrosine phosphorylated proteins of ∼110 and 70 kD after antigen stimulation. The ΔC mutant lacks the 110 kD SLAP-2-associated phosphoprotein. Bottom panel: reprobe with anti-FLAG. (B) SLAP-2 interacts with Cbl in B cells. Wild-type SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were immunoprecipitated as in (A) and immunoblotted with anti-Cbl antibodies. Wild-type SLAP-2 and SLAP-2-myr but not SLAP-2-ΔC associate with Cbl after antigen stimulation.
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fig6: SLAP-2 associates with tyrosine phosphorylated proteins including Cbl after antigen stimulation. (A) SLAP-2 associates with tyrosine phosphorylated proteins in B cells. Sorted tTA-BJAB cells infected with epitope-tagged wild-type (WT) SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were stimulated with anti-IgM F(ab′)2 for 2 min, lysed, and SLAP-2 was immunoprecipitated using anti-FLAG agarose. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies. SLAP-2 associates with tyrosine phosphorylated proteins of ∼110 and 70 kD after antigen stimulation. The ΔC mutant lacks the 110 kD SLAP-2-associated phosphoprotein. Bottom panel: reprobe with anti-FLAG. (B) SLAP-2 interacts with Cbl in B cells. Wild-type SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were immunoprecipitated as in (A) and immunoblotted with anti-Cbl antibodies. Wild-type SLAP-2 and SLAP-2-myr but not SLAP-2-ΔC associate with Cbl after antigen stimulation.
Mentions: Signaling through the TCR or BCR results in a rapid increase in tyrosine phosphorylation of numerous intracellular proteins, initiated by Src family and Syk/ZAP70 kinase activation (1, 3). These early signaling events ultimately result in transcriptional activation, upregulation of surface antigens, and other lymphocyte effector functions. Adaptor proteins play an important intermediary role in integrating upstream signals to produce biological function (6). Although upon initial evaluation, no changes were discernable in patterns of tyrosine phosphorylation in total cell lysates of BJAB cells overexpressing SLAP-2 (data not shown) we further investigated the nature of SLAP-2 signaling complexes. Epitope-tagged versions of SLAP-2, SLAP-2-myr, or SLAP-2-ΔC were immunoprecipitated from lysates of stably expressing BJAB cells using the anti-FLAG antibody. All three proteins became associated with a number of tyrosine phosphorylated proteins after BCR stimulation (Fig. 6 A), indicating that SLAP-2 indeed participates in BCR signaling pathways. Interestingly, a prominent phosphoprotein of ∼110 kD was absent in the immunoprecipitates of SLAP-2-ΔC (Fig. 6 A), which we subsequently identified as the RING finger ubiquitin ligase Cbl (Fig. 6 B). Cbl has been previously shown to be a negative regulator of TCR and BCR signaling pathways (31–33). An NH2-terminal fragment of Cbl constitutively interacts with the COOH-terminal region of SLAP as demonstrated in both the yeast two hybrid system and Cos-7 cells (30). In contrast, the association between SLAP-2 and full-length Cbl in B cells was inducible after antigen receptor stimulation. Whether SLAP-2 functions as an inhibitory adaptor by recruiting a negative regulator such as Cbl into the signaling complex or by competing with a positive regulator such as a Src-family kinase remains to be determined.

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