Figure 4. The constitutive-active form of BLK enhances the binding between BANK1 and PLCG2.

(A) Schematic representation of the constructs used to study the association between BANK1 and PLCg2 in transfected HEK293 cells. The constructs coding for wild-type forms of BLK, LYN, GFP and PLCg2 or the indicated mutated forms were fused to the epitope V5 at the C-termini. BANK1 was targeted with the Flag epitope at the N- terminus. The catalytic domains of BLK and PLCg2 are shown in red. The kinase dead form (BLK-KL-v5) has a substitution K (lysine) to L (leucine) at position 269 and the constitutively active form (BLK-YF-v5) has a Y501F substitution that prevents the phosphorylation of the inhibitory tyrosine. The lipidation in the amino terminal of BLK is indicated as back line. The myristoylation site was deleted by G2V substitution (glycine to valine) and the addition of an extra by palmitoylated site by L3C substitution (leucine to cysteine). The Src homology 3 domains (SH3) that bind to proline-rich motifs are drawn in orange and the SH2 domains in yellow. The Pleckstrin homology domain (PH) that binds to phosphatidylinositol lipids is shown in blue. In BANK1 are shown the Dof/BCAP/BANK (DBB) motif (amino acids 199–327), the double ankyrin repeat-like (ANK) motifs (amino acids 339-402) and the putative coiled coil (CC) region (amino acids 677–705). (B) HEK293 cells were transiently co-transfected with plasmids coding for the wild-type form of BLK, its functionally mutated forms (KL and YF), LYN or GFP in addition to plasmids expressing BANK1 and PLCg2. The lysates were immunoprecipitated using anti-PLCg2 antibody (above) and immunoblotted sequencially with anti-BANK1 antibody, anti-V5 to detect PLCg2, Srcs kinases and GFP and anti-phosphotyrosine antibody. (C). Mutation of lipidation sites of the kinases influence the formation of the BANK1-PLCg2 complex and the overall tyrosine phosphorylation on PLCg2. The blots were interrogated as in B.