Fig 1.
Strategy for protein-analysis and vector validation
(A) Schematic representation of the pEBB vector containing biotinylation target-domain (BTD) and (B) Simultaneous two-color target analysis of ANKRD54 wt and Δ3 mutant in Cos7 cells. The first three lanes (from left side of the blot) represent immunoprecipitation (IP) with streptavidin (SA) beads and the last three lanes show whole cell lysate (WCL). Anti-ANKRD54 (green) primary antibody recognizes the C-terminus of the protein and anti-IRDye 680-Streptavidin (red) recognizes the BTD domain.
Fig 2.
Characterization of the high-affinity ANKRD54/BTK interaction.
(A) Transfection by electroporation of Namalwa cells and (B) transiently-transfected Cos7 cell lysate mixed with Namalwa (Nam) lysate, followed by co-immunoprecipitation (IP) of endogenous BTK. The missing expression of BTK from the whole cell lysate (WCL) of untransfected Cos7 cells (lane 1) shows that these cells do not express BTK.
Fig 3.
Nuclear exclusion of BTK-NLS fusion protein by ANKRD54.
Subcellular localization of pEGFP-BTK-NLS alone (from left to right) and in the presence of wild-type ANKRD54, Δ3-mutant and mock. The white arrowheads in the second upper panel indicate nuclei devoid of BTK-NLS and the lower panels indicate merged images.
Table 1.
SH3-domains identified by bio-panning of recombinant ANKRD54 against a human SH3-domain library.
Fig 4.
ANKRD54 selectively binds to BTK.
Cos7 cells transiently-transfected with ANKRD54-wt or ANKRD54-Δ3 mutants followed by immunoprecipitation with streptavidin magnetic-beads. The IP complexes were incubated with whole cell lysates obtained from wt and Btk-KO primary spleen cells. The absence of BTK (second lane from left to right) did not enhance the enrichment to LYN. β-Actin used as quantity-index of protein, indicating equal-loading control of WCL.
Fig 5.
Schematic visualization of the SH3-domain and sequence-alignment of the C-terminal SH3-domains of the ANK54RD-binders identified in the screen.
Secondary structural elements of BTK-SH3-domain and homology alignment of the last 20-amino-acids of the human SH3-domains, according to Kärkkäinen [25] database. The NCF1, BZRAP1 and SH3KBP1 proteins contain more than one SH3-domain and the sequence corresponds to the SH3-domain binding to ANKRD54. SNY-amino-acids create a right-handed 310 helix [27]. *The biotinylated synthetic BTK-peptide (22-aa) reported in [9] was used as bait for the interaction with endogenous ANKRD54. **The TXK/RLK-SH3 was not identified in this screening, although ANKRD54 influenced the nucleocytoplasmic shuttling of full-length TXK [9].
Fig 6.
Residues in the 310 helix important for the interaction of ANKRD54 to BTK.
Cos7 cells were separately transfected with ANKRD54-wt and the following BTK-SH3 mutants: P265A, Y268A and P265A/Y268A. The obtained lysates were mixed and processed for immunoprecipitation.