Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP-2-mediated endocytosis

Abstract

Class IA phosphoinositide 3-kinase (PI3K) galvanizes fundamental cellular processes such as migration, proliferation, and differentiation. To enable multifaceted roles, the catalytic subunit p110 utilizes a multi-domain, regulatory subunit p85 through its inter SH2 domain (iSH2). In cell migration, their product PI(3,4,5)P₃ generates locomotive activity. While non-catalytic roles are also implicated, underlying mechanisms and its relationship to PI(3,4,5)P₃ signaling remain elusive. Here, we report that a disordered region of iSH2 contains previously uncharacterized AP-2 binding motifs which can trigger clathrin and dynamin-mediated endocytosis independent of PI3K catalytic activity. The AP-2 binding motif mutants of p85 aberrantly accumulate at focal adhesions and upregulate both velocity and persistency in fibroblast migration. We thus propose the dual functionality of PI3K in the control of cell motility, catalytic and non-catalytic, arising distinctly from juxtaposed regions within iSH2.

Figure 1. Plasma membrane recruitment of iSH2 domain induces clathrin and dynamin dependent endocytosis.

(a) Crystal structure of PI3K (PDB 2y3a) and AP-2 binding motifs of mouse p85β iSH2 domain. (b) Confocal images of endocytic vesicles produced by plasma membrane targeting of 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. (c) 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. (d) Quantified iSH2-mediated endocytosis indices (see method) of wild type and mutants in di-leucine motif and acidic cluster, but not YxxΦ motif. (e) TIRF images of iSH2 vesicles colocalized with AP-2. (f, g) Confocal images of iSH2 vesicles showing dynamin and clathrin dependency. Vesicle formation was suppressed in the presence of dominant negative form of dynamin (K44A) or AP180C. 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. (c, d) Steel-Dwass test. In the right panel of (d), p-values against YF-iSH2 were only shown. (f, g) Wilcoxon rank sum test.

Figure 2. iSH2-mediated endocytosis is independent of PI3K catalytic activity and C-terminal 46 aa region is necessary and sufficient.

(a) Confocal images of PI(3,4,5)P3 sensor PH(Akt) and iSH2 vesicles. Quantifications are shown on the right. LY294002: PI3K inhibitor, iSH2(DN): deletion mutant lacking p110 binding site. (b) Top: Amino acid sequence alignment of AP-2 binding motif region of human and mouse p85α, p85β, p55γ isoforms. Bottom: Quantification of iSH2 vesicles produced by each isoform. (c) Secondary structure of mouse p85β iSH2 domain and quantification of PH(Akt) translocation and iSH2 vesicles. 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.

Figure 3. Mutation in AP-2 binding motifs of p85β increases focal adhesion localization.

(a) Schematic of receptor tyrosine kinase-dependent and focal adhesion-dependent PI3K pathways. (b) Western blot of total- and phospho-Akt (T308) and its quantification. Cells were treated with 50 ng/mL PDGF for indicated time. pAkt/Akt level was normalized to DKO/p85β-wt 5 min. Error bars represent standard deviations. (c) Doubling time of DKO and p85 rescued MEF cells. (d) Confocal images of p85β-wt and p85β-motifGS cells and their quantification. Yellow: EYFP-p85β, Magenta: immunofluorescence against vinculin. (e) Western blot of total- and phospho-FAK (Y397) and its quantification. (f) FAK activity dependency of p85 focal adhesion localization. Cells were treated with DMSO or 10 μM PF-573228 (FAK inhibitor; FAKi) for 5 min and EYFP-p85β intensity were divided by the values of time=0. Box whisker plots represent median, 1st, 3rd quartiles and 1.5×inter-quartile range. P-value:****: < 0.0001. (d) Wilcoxon rank sum test.

Figure 4. Mutation in AP-2 binding motifs of p85β enhances cell motility in random and chemotactic migration.

(a) Representative tracks of 2D random migration on fibronectin coated plates. Cells were allowed to migrate at 37°C with 5% CO₂ and 10% FBS. 0.25 mg/mL Hoechst 33342 was used for tracking cells. (b, c, d) Quantification of migration parameters. Error bars in (c) and (d) represent 2×SEM (95% CI). (e) Representative tracks of chemotaxis in μ-Slide chemotaxis chamber (ibidi). Cells were allowed to migrate at 37°C with 5% CO₂ in the presence of 1–20% FBS gradient. 0.25 mg/mL Hoechst 33342 was used for tracking cells. (f, g, i and j) Quantification of migration parameters. Error bars in (g and i) represent 2×SEM (95% CI). (h) Schematic of displacement: d, distance: D, and forward displacement: y. Persistence ratio was defined as d/D, while Forward migration index was defined as y/D. Box whisker plots represent median, 1st, 3rd quartiles and 1.5×inter-quartile range. (b, f, and j) Steel-Dwass test was performed and p-values against DKO/p85β-wt were indicated. P-values: ****: < 0.0001. n.s.: not significant. In (j), p-values of Steel-Dwass test were < 0.001 for wt-DKO, <0.05 for wt-DKO/p85α-wt, a <0.001 for wt-DKO/p85β-motifGS, respectively, while the other pairs were not significant.