SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation

Abstract

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.

Figure 1

The SLP-65 gene encodes a protein with several regions of similarity to SLP-76 and is transcribed in lymphoid tissues. (A) Deduced amino acid sequence of murine SLP-65. Dots, Potential tyrosine-phosphorylation sites are marked. Box, The COOH-terminal SH2 domain. Underline, Peptide sequences obtained from affinity-purified SLP-65. (B) SLP-65 transcripts of ∼2 kb were detected by Northern blot analysis of RNA from the indicated tissues and cell lines. The SLP-65 probe used was derived from the ORF, and a reference signal for ribosomal 28S RNA was used as a loading control. (C) Anti–SLP-65 precipitates were prepared from postnuclear supernatants of pervanadate/H₂O₂-stimulated J558Lμm3 cells, transiently transfected either with the parental vector (lane 1) or an expression vector encoding SLP-65 with an NH₂-terminal HA tag (lane 2). Precipitated proteins were analyzed by antiphosphotyrosine (anti-pY) immunoblotting.

Figure 2

SLP-65 is constitutively associated with Vav and Grb-2. J558Lμm3 cells were left unstimulated (lanes 1 and 2) or stimulated for 2 min with 50 μM pervanadate/ H₂O₂ (lanes 3 and 4). Cleared cellular lysates were subjected to immunoprecipitation with preimmune serum (pre-i., lanes 1 and 3) or anti–SLP-65 antibodies (lanes 2 and 4). Tyrosine-phosphorylated SLP-65 was detected by antiphosphotyrosine immunoblotting (anti-pY, top). The Vav and Grb-2 proteins were detected by probing the same filter with anti-Vav (middle) and anti–Grb-2 antibodies (bottom).

Figure 3

The NH₂-terminal SH3 domain of Grb-2 is sufficient to bind SLP-65. Grb2-binding proteins were purified from lysates of unstimulated (lanes 1, 3, 5, and 7) and pervanadate/H₂O₂-stimulated J558Lμm3 cells (lanes 2, 4, 6, and 8) using GST fusion proteins containing different domains of Grb-2: NH₂-terminal SH3 domain (lanes 1 and 2), NH₂-terminal SH3 plus SH2 domain (lanes 3 and 4), SH2 domain (lanes 5 and 6), and complete Grb-2 (lanes 7 and 8). GST was used as a control (lanes 9 and 10). Proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting (top and bottom, respectively).

Figure 4

Tyrosine phosphorylation of SLP-65 is dependent on BCR expression. BCR-negative J558L cells (lanes 1–3) and BCR-positive J558Lμm3 cells (lanes 4–6) were unstimulated (lanes 1 and 4) or stimulated with 25 μM pervanadate/H₂O₂ either for 10 s (lanes 2 and 5) or 2 min (lanes 3 and 6). Proteins complexed with GST–Grb-2[SH3-SH2] fusion proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting (top and bottom, respectively).