Fig. 1: Plasma membrane recruitment of the iSH2 domain induces clathrin and dynamin dependent endocytosis.
a Crystal structure of PI3K (PDB 2Y3A) and the AP2 binding motifs of mouse p85β iSH2 domain. b AF2-predicted structures of the 16 aa iSH2 peptide binding to AP2. Left: rank 2 structure showing the iSH2 YxxΦ motif binding to µ subunit. Right: rank 5 structure showing the iSH2 di-Leucine motif binding to the σ subunit. Gray: AF2-predicted µ subunit, Orange: AF2-predicted σ subunit, Magenta: AF2-predicted 16 aa iSH2 peptide, Cyan and Green (left): µ subunit and TGN38 peptide from PDB 2XA7. Tan and Green (right): σ subunit and CD4 peptide from PDB 2JKR. Motif amino acids are represented in ball-stick model. c Western blot of the AP2 α subunit for pulldown assay using GST-iSH2 and AP2 core. Similar results were obtained from 3 independent experiments. d Confocal images of endocytic vesicles produced by plasma membrane targeting of the iSH2 domain. HeLa cells were transiently transfected with Lyn-ECFP-FRB, mCherry-PH(Akt), and EYFP-FKBP or EYFP-FKBP-iSH2. Images show before and after 100 nM rapamycin addition. Similar results were obtained from more than 3 independent experiments. e Confocal images of iSH2-induced vesicles colocalized with endocytosis marker molecules: mCherry-Rab5 (early endosome) and LAMP1-mRFP (lysosome). mCherry (cytosol) and mCherry-KDEL (ER) were used as negative controls. The graph shows Pearson’s correlation between iSH2 and marker molecules. mCherry, n = 40 cells. Rab5, n = 39 cells. LAMP1, n = 41 cells. KDEL, n = 41 cells. P values: mCherry–Rab5, <1.0 × 10–12. mCherry–LAMP1, <1.0 × 10–12. Rab5–KDEL, <1.0 × 10–12. LAMP–KEDL, 2.4 × 10–12. f Quantified iSH2-mediated endocytosis indices (see Methods) of wild type iSH2 and mutants in AP2 binding motifs. YF-iSH2, n = 29 cells. motifGS, n = 24 cells. ∆motif, n = 12 cells. D597A, n = 24 cells. Y599A, n = 24 cells. L601A, n = 21 cells. YF, n = 31 cells. YF-iSH2, n = 32 cells. EDEDA-GSAGG, n = 33 cells. YF, n = 31 cells. P values: (left) motifGS, 1.1 × 10–7. ∆motif, 1.0 × 10–4. D597A, 5.4 × 10–7. Y599A, 1.0. L601A, 3.1 × 10–7. YF, 1.7 × 10–9. (right) YFiSH2–EDEDA-GSAGG, 9.5×10–6. EDEDA-GSAGG–YF, 3.3 × 10–10. g TIRF images of iSH2 vesicles colocalized with AP2. Similar results were obtained from more than 3 independent experiments. h, i Confocal images of iSH2 vesicles showing dynamin and clathrin dependency. Vesicle formation was suppressed in the presence of the dominant negative form of dynamin (K44A) or AP180C. Dyn WT, n = 37 cells. Dyn K44A, n = 37 cells. mCherry, n = 30 cells. AP180C-mCherry, n = 40 cells. P values: (h) 5.3 × 10–12, (i) 1.2 × 10–7. Box whisker plots represent median, 1st, 3rd quartiles and 1.5×inter-quartile range. P values: *: <0.05, **: <0.01, ***: <0.001, ****: <0.0001. n.s.: not significant. e, f Steel-Dwass test (two sided). In the left panel of (f), only p values against YF-iSH2 are shown. h, i Wilcoxon rank sum test (two sided).
