Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner

Abstract

Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation.

Fig. 1.

SLAP and c-Cbl deficiency lead to alterations in B cell development. (A) Analysis of cells from bone marrow from C57BL/6 (B6), c-Cbl-deficient (c-Cbl^−/−), SLAP-deficient (SLAP^−/−), or SLAP and c-Cbl doubly deficient mice (DKO). (B) Analysis of splenic B cells (B220⁺) from B6, c-Cbl^−/−, SLAP^−/−, or DKO mice. (C) Analysis of peritoneal B cells (CD19⁺) from B6, c-Cbl^−/−, SLAP^−/−, or DKO mice. (A–C) The 2D contour plots are representative of results obtained from six mice per genotype. (D) Average percentage of B1 B cells in the peritoneal lavage fluid from B6 (light gray bar), SLAP^−/− (black bar), c-Cbl^−/− (white bar), or DKO (dark gray bar) mice. (E) Average percentage of B2 B cells in the peritoneal lavage fluid from B6 (light gray bar), SLAP^−/− (black bar), c-Cbl^−/− (white bar), or DKO (dark gray bar) mice. These data are the average percentage of cells determined from six mice per genotype. The asterisks denote statistically significant differences as compared with the B6 controls.

Fig. 2.

SLAP associates with the BCR upon stimulation and internalization. (A) Immunoblot of whole-cell lysate (WCL) from BAL-17 cells 0, 5, 10, and 20 min after BCR stimulation. GST pull-downs were performed by using either GST SLAP or GST as a control. Proteins retained during the GST pull-down were detected with an antibody specific for phosphotyrosine. Immunoblots are representative of at least three independent experiments with similar results. (B and C) Immunoblot of WCL from BAL-17 cells stably expressing either GFP or SLAP-GFP 0 and 5 min after BCR stimulation. Coimmunoprecipitations were performed on WCL lysates by using an anti-GFP antibody. Proteins retained during coimmunoprecipitation were detected with an antibody specific for phosphotyrosine (B) or Igα (C). Immunoblots are representative of at least three independent experiments with similar results. (D) Deconvolution microscopy was performed on BAL-17 cells stably expressing either GFP or SLAP-GFP (green). The cells were then fixed 0, 5, 15, and 30 min after BCR stimulation, and IgM (red) and GFP/SLAP-GFP (green) were visualized. Images are representative of at least five independent transfections and at least 20 independent cells captured per transfection.

Fig. 3.

Expression of SLAP and c-Cbl down-regulates surface and total BCR levels in BAL-17 cells, and both the SH2 and C-terminal domains of SLAP are required for BCR down-modulation. (A) Percentage of surface IgM expression (±SEM) on GFP⁺ BAL-17 cells transiently transfected with GFP alone, c-Cbl plus GFP, GFP-SLAP, or c-Cbl plus GFP-SLAP. IgM levels were compared with that expressed on GFP-only-transfected cells. The data are the average of at least three independent experiments. (B) Percentage of total IgM expression (±SEM) on GFP⁺ BAL-17 cells transiently transfected with GFP alone, c-Cbl plus GFP, SLAP-GFP, or c-Cbl plus SLAP-GFP. Total IgM levels were compared with levels expressed on GFP-only-transfected cells. The data are the average of at least three independent experiments. (C) Expression and localization of GFP fluorescence (green), c-Cbl (red), and IgM (blue) in Bal-17 cells that were transiently transfected with GFP or SLAP-GFP with or without c-Cbl and analyzed by deconvolution microscopy. For each experiment >10 cells per transfection condition were analyzed from three independent transfections. (D) SLAP-GFP constructs: GFP only (V), SLAP-GFP (WT), SH3 point mutant (SH3*), SH2 point mutant (SH2*), and deletion of C terminus (DC). (E) Percentage of surface IgM expression (±SEM) on GFP⁺ BAL-17 cells transiently transfected with GFP-SLAP either alone or with c-Cbl expression constructs. IgM expression was compared with IgM expressed on GFP-only-transfected cells. The data are the average of at least three independent experiments.

Fig. 4.

The SH2 domain of SLAP is required for its colocalization with IgM in BAL-17. Expression and localization of GFP fluorescence (green), c-Cbl (blue), and IgM (red) in BAL-17 cells transiently transfected with c-Cbl and GFP-SLAP expression constructs and analyzed by deconvolution microscopy. For each experiment >10 cells per transfection condition were analyzed from three independent transfections.

Fig. 5.

Altered BCR recycling upon SLAP and c-Cbl expression in BAL-17. (A) Ligand-independent recycling of internalized IgM. BAL-17 cells were transiently transfected with GFP and c-Cbl, SLAP-GFP and c-Cbl, SLAP-GFP and Cbl-b, or SLAP-GFP and a E3 ubiquitin ligase-deficient mutant of c-Cbl(C3AHN). Four hours after transfection, surface IgM was labeled with a biotinylated F(ab) specific for IgM. After washing, the cells were incubated at 37°C for 0, 1, 3, or 4 h and immediately fixed in 1.6% paraformaldehyde, and the remaining surface IgM was detected by using streptavidin-Cy 5. The data are expressed as the average of three independent transfections (±SEM). (B) BAL-17 cells were transiently transfected with GFP-SLAP alone, GFP-SLAP(SH2*) and c-Cbl, GFP-SLAP(SH3*) and c-Cbl, or GFP-SLAP(DC) and c-Cbl. The experiment was performed as described in A. The data are expressed as the average of three independent transfections (±SEM). (C) BAL-17 cells were transiently transfected with GFP-SLAP and c-Cbl, and the experiment was performed as described in A, except that at the 1-h time point, the cells were split and the Src family kinase inhibitor PP2 was added to one-half of the cells. The data are expressed as the average of three independent transfections (±SEM). (D) Model of how SLAP as an adaptor of c-Cbl may lead to (1) increased internalization, (2) decreased recycling, and (3) ubiquitination of activated components of the BCR complex targeting them for degradation in the lysozome or proteosome.