Inhibition of p21-activated kinase rescues symptoms of fragile X syndrome in mice

PAK1 interacts with FMRP. (A) Immunoprecipitation followed by Western blot analysis. Brain extract was subjected to immunoprecipitation with either rabbit serum (negative control), PAK1 antibody (α-PAK1), or α-PAK1 plus a blocking peptide. Western blots were probed for either FMRP or PAK1. For Input, 2% of the extract used for a single immunoprecipitation was loaded on the gel. (B) Schematic structure of FMRP, highlighting various functional domains including three RNA-binding motifs (RGG, KH1, and KH2) and the phosphorylation site (S499, represented by a gray asterisk). The constructs used for in vitro binding included full-length (WT), truncated (ΔRGG, ΔS499, and ΔKH), or mutated (I304N) FMRP. ΔRGG refers to the FMRP variant with a deletion of the RGG box at amino acids 526–555. The deleted area in ΔS499 spans amino acids 443–527 and includes the phosphorylation site, S499, as well as putative phosphorylation sites. The isoleucine to asparagine missense mutation in the KH2 domain mimics that previously reported in a human FXS patient (I304N, represented by a black asterisk). The ΔKH deletion mutant lacks both KH domains in tandem corresponding to amino acids 207–425. The numbers refer to the amino acid positions designated by the SwissProt Q06787 entry. Adapted from ref. 38. (C) Characterization of the interaction between PAK1 and various FMRP variants in vitro. In vitro-translated FMRP variants were incubated with GST or GST-PAK1 and glutathione Sepharose beads. The complexes isolated by this method were subjected to SDS/PAGE and Western blotted for FMRP. For Input, 10% of in vitro-translated FMRP sample before the binding reaction was carried out was loaded on the gel.