Fig. 5.

BIN1 phosphorylation at T348 regulates BIN1–Tau interaction by modulating open/closed conformation of BIN1. a Alignment of Amphiphysin 1 and BIN1iso1; domains not to scale. The underlined sequence indicates the BIN1 PRD sequence interacting with the BIN1 SH3 domain. b Lambda protein phosphatase (λ-PP) treatment dephosphorylates BIN1; 2 lanes per condition. c.In vitro phosphorylation assays with recombinant proteins show that Cdk2 and Cdk5 phosphorylate BIN1 at T348. Also see Fig. S4. d, e Immunoblots and quantification showing the effects of U0126 and CsA (10 μM; 2.5 h) on BIN1 and Tau phosphorylation. Inset shows the effect of 10 nM CsA on BIN1 phosphorylation. Mean ± SD from 3 independent experiments. One-way ANOVA and paired t test; * p < 0.05; **p < 0.01. f Behavior of BIN1-SH3 domain in the whole BIN1 isoform 1 protein as a function of phosphorylation by Cdk2 or of a mutation at threonine (T) 348 to glutamate (E) as monitored by ¹H-¹⁵N HSQC spectra of BIN1iso1 CLAP T348E protein (in blue), Cdk2-phospho-BIN1iso1 (superimposed in red), and BIN1iso1 protein (superimposed in green). Also see Fig. S22. g Titration of BIN1-SH3 domain with concentration of CLAP (334-355) or phospho-T348 CLAP (334-355) peptides. Normalized saturation curves (shown for residue 559), built from the gradual chemical shift changes (normalized; 1 denotes the largest change), are shown as pink stars for CLAP (334-355) and red stars for phospho-CLAP (334-355). Saturation curves are in cyan and green for CLAP (334-355) and phospho-CLAP (334-355), respectively. Also see Fig. S23. h Representative images of PNC overexpressing BIN1iso1 and the BIN1iso1 T348E, its systematically open form, showing PLA signals and Tau and BIN1 immunolabeling. i Boxed areas in h are 2.4× magnified. j PLA density after normalization with respective BIN1 immunofluorescence in PNC overexpressing BIN1iso1 and BIN1iso1 T348E (for clarity, datasets were further normalized with the mean of BIN1iso1). N = 3 independent experiments. Red bars and black squares indicate sample median and mean, respectively. Wilcoxon rank-sum test; ***p < 0.001