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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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Cloning of a novel inhibitory adaptor, SLAP-2. (A) Human SLAP-2 cDNA sequence. The putative myristoylation site is shown in bold. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF326353. (B) Alignment of human SLAP-2 and SLAP. Identical amino acids are boxed and highlighted. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. The overall amino acid similarity between SLAP-2 and SLAP is 36%. In the SH2 and SH3 domains alone the similarity between SLAP-2 and SLAP is 48% and between SLAP-2 and the Src family kinase Hck is 45%. (C) Northern blot analysis of SLAP-2 expression. Left: human tissues and right: tumor cell lines including promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogeneous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. Blots were stripped and reprobed with an actin probe (bottom panel). (D) RT-PCR of SLAP-2 from purified human primary cells. Specific SLAP-2 primers or control GAPDH primers were used in a standard PCR reaction with cDNA templates obtained from resting (rest.) and activated (act.) CD4+ T cells (stimulated with PHA), CD19+ B cells (stimulated with pokeweed mitogen), or CD14+ monocytes (human blood fractions MTC panel [CLONTECH Laboratories, Inc.]).
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fig2: Cloning of a novel inhibitory adaptor, SLAP-2. (A) Human SLAP-2 cDNA sequence. The putative myristoylation site is shown in bold. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF326353. (B) Alignment of human SLAP-2 and SLAP. Identical amino acids are boxed and highlighted. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. The overall amino acid similarity between SLAP-2 and SLAP is 36%. In the SH2 and SH3 domains alone the similarity between SLAP-2 and SLAP is 48% and between SLAP-2 and the Src family kinase Hck is 45%. (C) Northern blot analysis of SLAP-2 expression. Left: human tissues and right: tumor cell lines including promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogeneous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. Blots were stripped and reprobed with an actin probe (bottom panel). (D) RT-PCR of SLAP-2 from purified human primary cells. Specific SLAP-2 primers or control GAPDH primers were used in a standard PCR reaction with cDNA templates obtained from resting (rest.) and activated (act.) CD4+ T cells (stimulated with PHA), CD19+ B cells (stimulated with pokeweed mitogen), or CD14+ monocytes (human blood fractions MTC panel [CLONTECH Laboratories, Inc.]).

Mentions: Sequencing of retroviral library cDNA inserts recovered from clone 780 identified the Src-like adaptor protein, SLAP (21). One function previously attributed to SLAP is that of a negative regulator of TCR signaling, although SLAP is also expressed in B cells (22). The isolation of the SLAP cDNA from our screen not only validated the efficacy of the functional selection used, but also established its role in the BCR signaling pathway. Clone 584 contained a cDNA encoding a novel protein of 261 a.a. (Fig. 2 A). Sequence analysis revealed that it shares structural homology with both SLAP and Src-family kinases, and contains an NH2-terminal myristoylation consensus sequence (MGX1–4 T/S), one SH2- and one SH3 domain, and a unique COOH terminus, but lacks an enzymatic domain (Fig. 2 B). We therefore named it SLAP-2 for Src-like adapter protein-2. Subsequent analyses of the remaining positive clones identified seven other SLAP-2–containing clones and two more SLAP-containing clones. All of the SLAP-2 and SLAP inserts contained the entire open reading frame and were transcribed in the sense orientation, suggesting that SLAP-2 is a negative regulator.


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)

Cloning of a novel inhibitory adaptor, SLAP-2. (A) Human SLAP-2 cDNA sequence. The putative myristoylation site is shown in bold. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF326353. (B) Alignment of human SLAP-2 and SLAP. Identical amino acids are boxed and highlighted. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. The overall amino acid similarity between SLAP-2 and SLAP is 36%. In the SH2 and SH3 domains alone the similarity between SLAP-2 and SLAP is 48% and between SLAP-2 and the Src family kinase Hck is 45%. (C) Northern blot analysis of SLAP-2 expression. Left: human tissues and right: tumor cell lines including promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogeneous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. Blots were stripped and reprobed with an actin probe (bottom panel). (D) RT-PCR of SLAP-2 from purified human primary cells. Specific SLAP-2 primers or control GAPDH primers were used in a standard PCR reaction with cDNA templates obtained from resting (rest.) and activated (act.) CD4+ T cells (stimulated with PHA), CD19+ B cells (stimulated with pokeweed mitogen), or CD14+ monocytes (human blood fractions MTC panel [CLONTECH Laboratories, Inc.]).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195979&req=5

fig2: Cloning of a novel inhibitory adaptor, SLAP-2. (A) Human SLAP-2 cDNA sequence. The putative myristoylation site is shown in bold. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF326353. (B) Alignment of human SLAP-2 and SLAP. Identical amino acids are boxed and highlighted. Open and solid triangles indicate the boundaries of SH3 and SH2 domains, respectively. The overall amino acid similarity between SLAP-2 and SLAP is 36%. In the SH2 and SH3 domains alone the similarity between SLAP-2 and SLAP is 48% and between SLAP-2 and the Src family kinase Hck is 45%. (C) Northern blot analysis of SLAP-2 expression. Left: human tissues and right: tumor cell lines including promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogeneous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. Blots were stripped and reprobed with an actin probe (bottom panel). (D) RT-PCR of SLAP-2 from purified human primary cells. Specific SLAP-2 primers or control GAPDH primers were used in a standard PCR reaction with cDNA templates obtained from resting (rest.) and activated (act.) CD4+ T cells (stimulated with PHA), CD19+ B cells (stimulated with pokeweed mitogen), or CD14+ monocytes (human blood fractions MTC panel [CLONTECH Laboratories, Inc.]).
Mentions: Sequencing of retroviral library cDNA inserts recovered from clone 780 identified the Src-like adaptor protein, SLAP (21). One function previously attributed to SLAP is that of a negative regulator of TCR signaling, although SLAP is also expressed in B cells (22). The isolation of the SLAP cDNA from our screen not only validated the efficacy of the functional selection used, but also established its role in the BCR signaling pathway. Clone 584 contained a cDNA encoding a novel protein of 261 a.a. (Fig. 2 A). Sequence analysis revealed that it shares structural homology with both SLAP and Src-family kinases, and contains an NH2-terminal myristoylation consensus sequence (MGX1–4 T/S), one SH2- and one SH3 domain, and a unique COOH terminus, but lacks an enzymatic domain (Fig. 2 B). We therefore named it SLAP-2 for Src-like adapter protein-2. Subsequent analyses of the remaining positive clones identified seven other SLAP-2–containing clones and two more SLAP-containing clones. All of the SLAP-2 and SLAP inserts contained the entire open reading frame and were transcribed in the sense orientation, suggesting that SLAP-2 is a negative regulator.

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