Figure 3.

Entry and exit of Bright from lipid rafts requires nucleocytoplasmic shuttling, alternative post-translational modifications, but not association with Btk. (A) Schematic of Bright indicating domains and positions of substitution mutations. (B) Cytoplasmic-nuclear shuttling is a requirement for Bright's inclusion into lipid rafts. NIH/3T3 fibroblasts were transfected with constructs encoding wild type and nuclear export signal-defective (NES, G532A) and nuclear localisation signal-defective (NLS, K466A) Bright. Fractions from discontinuous (5–40%) sucrose gradient purification of lipid rafts were analysed by western for Bright. (C) Palmitoylation of Bright requires cysteine 342. Bright constructs (indicated at the top) were transfected into Cos-7 cells, and after 48 h, were incubated as indicated with ¹⁴C palmitic acid. Whole cell lysates were subjected to immunoprecipitation using antibodies against Bright and VSV as indicated (left). SDS–PAGE separated proteins were transferred onto nitrocellulose, and the metabolic incorporation of ¹⁴C palmitic acid was determined by autoradiography. Solvent control is indicated by −. (D) Specification of Bright to lipid rafts requires cysteine 342. NIH/3T3 fibroblasts were transfected with constructs indicated in the figure. Crude plasma membrane and lipid rafts, prepared as described in previous legends, were analysed by anti-Bright western blotting. (E) Sumo-I modification of Bright is lost after mutation of ⁴⁰¹KIKK. Cos-7 cells were transfected with GFP-SUMO-I and GFP-Bright expression constructs, as indicated. Whole cell lysates were prepared using RIPA buffer and analysed by western using α-Bright anti-serum. An arrow points to a GFP-Sumo-I conjugated species of GFP-Bright. (F) Bright forms a transient, stimulation-specific complex with Sumo-I-conjugation enzymes PIAS1 and Ubc9 in lipid rafts. Indicated B cells (∼10⁸) were stimulated mildly (30 s; 100 ng α-μ). Lipid rafts were collected on gradients, subjected to immunoprecipitation with anti-Bright antiserum, and then analysed by western using the antibodies indicated.