Skip to main content
. 2013 Mar 26;8(3):e59842. doi: 10.1371/journal.pone.0059842

Figure 6. Mutations of specific tyrosine residues and proline rich domains of BANK1 abrogated the association with PLCg2.

Figure 6

(A) Schematic representation of the motifs and the structure of the human BANK1 splicing variants and analysis of the motifs affecting the BANK1-PLCg2 binding. The Dof/BCAP/BANK (DBB) motif (amino acids 199–327), the double ankyrin repeat-like (ANK) motifs (amino acids 339–402) and the presumptive coiled coils (CC) region (amino acids 677–705) are indicated. Tyrosine residues susceptible to be phosphorylated are shown by Y. Residues Y125, Y146, Y161, Y416, Y484 and Y488 that predict the putative SH2 binding sites are indicated as bold Y (http://scansite.mit.edu/motifscan_seq.phtml). The positions of the mutated amino acids are indicated above the BANK1 drawing. (B) Alignment of the BANK1 amino acid motifs in different species indicating the mutated residues corresponding to putative SH2 and SH3 binding domains, alignment was done using data downloaded from www.ensembl.org. (C) Phosphorylation and immunoprecipitation analysis of the wild type and mutated forms of BANK1 co-expressed with the constitutively active form of BLK (BLK-YF) and PLCg2. Relative expression of the constructs was monitored by western blot (IB) of an aliquot (1/10) of the transfected HEK293T cell extract. The rest of the lysates were used for immunoprecipitation (IP) using anti-PLCg2 (Abcam) and it is shown in the lower row. Lane 0 represents immunoprecipitation without the IP antibody. (D) Quantification of BANK1 phosphorylation and immunoprecipitation using anti-PLCg2 antibody. Bands of the western blots were quantified using ImageJ program. Values of expression of BANK1 in the lysates (IB:Anti-BANK1) were taken to normalize the results of phosphorylation and immunoprecipitation of BANK1. The relative expression of BANK1 was: 1, 1.03, 0.93, 0.85,0.69, 0,81, and 0.73 from lane 1 to 7 in the lysates interrogated with anti-BANK1. The Y484-8F mutation significantly reduced both the tyrosine phosphorylation of BANK1 and the binding to PLCg2. The PP513LL mutation does not affect the tyrosine phosphorylation of BANK1, however it leads to a decrease in binding to PLCg2. (E) Co-expression in HEK293T cells of the two isoforms of BANK1 renders equivalent recovery of both isoforms in the anti-PLCg2 immunoprecipitate, indicating that exon 2 did not participate in the binding between BANK1 and PLCg2.