Fig 1: The N-terminal SH3 domain of CRKL binds to a specific PXXPXK motif of SASH1. (A) Shown are several SASH1 deletion constructs tagged with an N-terminal GFP- or C-terminal V5-epitope, which were used to map the CRKL binding site. Length of SASH1 constructs as well as the CRKL domain architecture also are indicated. (B) Endogenous CRKL was precipitated with an anti-CRKL antibody in HEK293 cells expressing GFP or different SASH1 deletion constructs with N-terminal GFP tags. Full-length GFP-SASH1 (aa1–1247) and deletion constructs containing the C-terminal part of SASH1 (aa1–887 and 709–1247) co-precipitated with CRKL. (C) Reciprocal immunoprecipitations were performed with SASH1 constructs carrying a C-terminal V5-epitope tag. In accordance to GFP-SASH1 constructs, precipitation with anti-V5 and anti-CRKL antibodies showed that endogenous CRKL binds to the C-terminal part of SASH1 (aa590–1247). (D) SASH1 contains 3 distinct PXXPXK motifs (P1–3) in the C-terminal region. Mutation of the second PXXPXK motif (P2, aa984–989) of GFP-SASH1 is sufficient to disrupt co-precipitation with endogenous CRKL (left panel). Anti-RFP immunoprecipitations show that the interaction of CRKL (fused to RFP at the C-terminus) with C3G and SASH1, but not GAB1, is abolished by the W160R mutation that inactivates the N-terminal SH3 domain of CRKL (right panel). (E) Recombinant expression of SASH1 with a C-terminal V5 tag in HEK293 cells abrogates co-precipitation of C3G by endogenous CRKL (unpaired t test; n = 3 independent experiments; P = .0310), while the interaction with GAB1 is not disturbed. (F) The affinity between SASH1 and CRKL was determined by dynamic mass redistribution (DMR) experiments using a peptide containing the second PXXPXK motif of SASH1 (aa982–991) and the N-terminal SH3 domain of CRKL purified from E coli. SASH1 binds CRKL with an apparent dissociation constant of dissociation constant (±SD) of 7.378 ± 1.637 µmol/L. Ctrl, control; IP, immunoprecipitation; Mr (K), molecular range (kilodalton); P1–3, PXXPXK motifs; Pro-rich, proline-rich region; rel, relative; RFP, red fluorescent protein; SAM, sterile a motif; WB, Western blot.