The B cell-specific major raft protein, Raftlin, is necessary for the integrity of lipid raft and BCR signal transduction

Abstract

Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identification of proteins that co-purified with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.

Figures

Fig. 1. Raft purification from Raji B cells. (A) Raji cells were lysed and fractionated using sucrose gradient ultracentrifugation. Each fraction from 6 × 105 cells was loaded onto SDS 12% polyacrylamide gels, and visualized by silver staining or blotted using CTB and anti-Lyn MAb. The numbers indicate the fractions from the top. (B) The raft fraction was loaded on SDS–PAGE (10% gel) and visualized by Coomassie Brilliant Blue staining (1.5 × 108 cells) or silver staining (1.5 × 107 cells). The main bands (a–o) were excised, and the proteins included in the gel were determined using LC-ESI-MS/MS. (C) As in (A), but the fractions were immunoblotted using anti-human Raftlin Ab.

Fig. 2. Deduced amino acid sequences of Raftlins. (A) A comparison of the deduced amino acid sequences of human Raftlin and chicken Raftlin. (B) A comparison of the deduced amino acid sequences of human Raftlin and human Raftlin-2. (C) Northern blot analysis of mouse Raftlin expression in mouse tissues. Mouse G3PDH was a control for the amount of RNAs. (D) Western blot analysis of human Raftlin expression in several human cell lines. Lysates from 1 × 105 cells were immunoblotted with anti-human Raftlin Ab. Similar levels of protein loading were confirmed by western blotting with anti-STAT5b Ab.

Fig. 3. The effect of fatty acylation on the cellular localization of Raftlin. (A) Comparison of the N-terminal amino acid sequences of human Lck and human Raftlin. S-palmitoylation and N-myristoylation sites of human Lck are shown as Pal and Myr. (B and C) Cellular localization of human Raftlin expressed in HEK293T cells. Wild-type and mutated Raftlins (WT, G2A, C3S and G2A C3S) expressed as GFP fusion proteins were observed by fluorescence microscopy (B), and the fractions using sucrose gradient ultracentrifugation were blotted with anti-GFP Ab and CTB (C). (D and E) Effect of fatty acylation inhibitors on the localization of human Raftlin in HEK293T cells. Wild-type Raftlin fused GFP was expressed in the absence or presence of 2-hydroxymyristic acid or 2-bromopalmitic acid, and observed by fluorescence microscopy (D). After sucrose gradient ultracentrifugation, combined raft fractions (R; fractions 1–5) and combined soluble fractions (S; fractions 9–13) were blotted with anti-GFP Ab (E). Relative intensity (R/S) was plotted.

Fig. 4. Localization of Raftlin in Daudi cells. (A) Co-localization of BCR and Raftlin in Daudi cells. Cells were stimulated with anti-human IgM Ab for indicated periods and stained with a combination of anti-human Raftlin Ab and anti-human IgM Ab (a), or CTB and anti-human IgM Ab (b), or CTB and anti-human Raftlin Ab (c). Antiserum passed through a recombinant Raftlin protein column (antigen-depleted anti-Raftlin) was used as a negative control. (B) Immunoprecipitation of BCR and Raftlin using Daudi cells. Cells (6 × 107) stimulated with or without anti-human IgM (20 µg/ml) for 5 min at 37°C were lysed with digitonin-lysis buffer and immunoprecipitated with anti-human IgM Ab and protein-G–Sepharose 4FF (Amersham Bioscience) or anti-human Raftlin IgG-immobilized Sepharose. Immunoprecipitants were divided into three aliquots and blotted with anti-human IgM, anti-human Raftlin and anti-phosphotyrosine Abs (4G10).

Fig. 5. Disruption of the Raftlin gene in chicken DT40 B cells. (A) The intron and exon structure of the human Raftlin gene. The first methionine is indicated as Met, and the numbered boxes represent exons. (B) Targeting constructs of chicken Raftlin. Hygro, hisD, and Neo denote the hygromycin-, histidinol- and neomycin-resistance genes, respectively. The small arrows indicate the primers for screening of homologous recombinants. (C) Genomic PCR of wild-type (WT) and Raftlin-deficient DT40 cells (Raftlin–). Wild-type allele (WT) and mutated alleles containing hygromycin-, histidinol- and neomycin-resistance genes (Hygro, HisD and Neo, respectively) were amplified by PCR. (D) Raftlin expression in wild-type and mutated cells. RT–PCR (upper panel) and western blotting (lower panel) of chicken Raftlin were performed using wild-type (+/+/+), single-recombinated (+/+/–), double-recombinated (+/–/–) and knock out (–/–/–) cells. The primers for RT–PCR were designed on the bases of the sequences of the first and third exons. (E) Western blotting of wild-type (WT), Raftlin-deficient cells (Raftlin–), and transformants of chicken Raftlin cDNA (Raftlin–/cRaftlin) and human Raftlin cDNA (Raftlin–/hRaftlin) into Raftlin-deficient cells using anti-chicken Raftlin Ab and anti-human Raftlin Ab.

Fig. 6. Proliferation assay of Raftlin-deficient DT40 cells. (A) Growth curves of DT40 cells. Cells were suspended at 5 × 104 cells per ml in the medium. They were cultured for the indicated time and the cell numbers were counted. (B) Cell cycle analysis. Various mutant DT40 cells were treated in hypotonic solution containing PI and subjected to DNA content analysis by FACScan.

Fig. 7. Raftlin is necessary for the integrity of lipid rafts. (A) Western blot analysis of proteins in raft and soluble fractions. DT40 cells unstimulated (–) or stimulated with 5 µg/ml M4 for 3 min (+) were lysed and fractionated in a sucrose gradient. Combined raft fractions (fractions 1–4) from 1.5 × 106 cells and combined soluble fractions (fractions 8–13) from 1.5 × 105 cells were loaded onto SDS–PAGE (10%) and visualized by silver staining (Silver Staining). The asterisk indicates chicken and human Raftlin, and the amount of protein in each fraction is shown at the bottom of the panel. For western blotting, raft and soluble fractions from 1 × 106 cells were resolved by SDS–PAGE and stained with anti-Raftlin Ab, anti-Lyn Ab, anti-BLNK Ab and CTB. (B) BCR and GM1 expression on the DT40 cell surface. (Left column) Various type DT40 cells were stained using Alexa488- conjugated CTB with (thin continuous lines) or without (thick continuous lines) 10-fold excess of non-labeled CTB. Unstained cells were used as a negative control (dotted lines). (Right column) To detect surface IgM levels, various genotypes of DT40 cells were stained with M4 antibody followed by Alexa488-conjugated anti-mouse IgM (continuous lines). The cells stained with only Alexa488-conjugated anti-mouse IgM were used as a negative control (dotted lines). The numbers indicate the average of Alexa488 intensity. (C) The effect of cholesterol depletion on the raft proteins and GM1. Wild-type (lanes 1, 5, 9, 13), Raftlin– (lanes 2, 6, 10, 14), Raftlin–/cRaftlin, (lanes 3, 7, 11, 15) and Raftlin–/hRaftlin cells (lanes 4, 8, 12, 16) were treated with or without MβCD, lysed, and fractionated in a sucrose gradient. Combined raft (fractions 1–4; lanes 1–4, 9–12) and combined soluble fractions (fractions 8–13; lanes 5–8, 13–16) from 1 × 106 cells were resolved by SDS–PAGE and blotted with anti-Raftlin, anti-Lyn and anti-BLNK Abs, and CTB–HRP.

Fig. 8. Loss of Raftlin expression reduced BCR-signal transduction. (A) BCR-induced tyrosine phosphorylation of cellular proteins. Various mutant DT40 cells were stimulated with anti-IgM antibody (M4) (5 µg/ml) for the indicated time. The whole-cell lysates prepared from 106 cells were loaded onto SDS–PAGE (10%) and analyzed by western blotting with anti-phosphotyrosine antibody (4G10). (B) BCR-induced tyrosine-phosphorylation of Lyn and BLNK. Indicated DT40 variants (8 × 106 cells for Lyn and 5 × 106 cells for BLNK) were stimulated with (+) or without (–) M4 (5 µg/ml) for 2 min. The cells were lysed with NP40 lysis buffer and immunoprecipitated using anti-Lyn or anti-BLNK Abs. The precipitants were divided into two parts and blotted with the indicated antibodies. (C) BCR-induced calcium mobilization. Various mutant DT40 cells were stimulated with rabbit anti-mouse IgM followed by M4. Arrows indicate the time points of M4 addition.