Fig 2: LOK kinase domain recognizes a docking site distal to T567 phosphorylation site.(A) In vitro kinase assay showing that 10 nM LOK and 30 nM LOK-N phosphorylate GST-ezrin-CTD, and the truncations GST-ezrin-490–585 and GST-ezrin-520–585. Phosphorylation decreases in the truncations GST-ezrin-530–585, GST-ezrin-555–585 and GST-ezrin-490(Δ520–530)−585. All substrate concentrations were 18 µM. Schematic illustration of GST-ezrin-CTD and truncations shows the distal docking site in orange letters and box. LOK kinase consensus sequence is shown in black, in which the T567 phosphorylation site is in red letters and the conserved Y565 is in blue letters. Data are represented as mean ± SE, n = 3, two-way ANOVA (See also Figure 6—source data 1), **p<0.0021, ****p<0.0001. (B) A working model depicting the proposed coincidence detection mechanism for ezrin phosphorylation. (1) Ezrin is recruited to PIP2 and primed for phosphorylation. (2) Autoinhibition of LOK is relieved as LOK-C wedges in between FERM and ezrin-CTD. (3) LOK-N binds to distal docking site on ezrin-CTD and, (4) phosphorylates T567. (5) LOK remains tethered to activated ezrin, allowing phosphorylation of microvillar ezrin in a positive feedback loop.DOI: http://dx.doi.org/10.7554/eLife.22759.01610.7554/eLife.22759.017Figure 6—source data 1.Experimental replicates for Figure 6A.DOI: http://dx.doi.org/10.7554/eLife.22759.017

Fig 3: In vitro phosphorylation of full-length ezrin requires PIP2 and LOK C-terminal domain.(A) Left panel: A cartoon illustration of cytoplasmic closed/inactive ezrin versus membrane-tethered open/active ezrin acting as crosslinker between the plasma membrane (PM) and the cytoskeletal F-actin. Right panel: The domain structure of ezrin and LOK constructs used in this study. The numbers indicate amino acids residues at protein domain boundaries. (B) In vitro kinase assay showing that 10 nM LOK phosphorylates 18 µM ezrin-CTD. Data are presented as mean ± SE, n = 3, two-way ANOVA (See also Figure 1—source data 1), ****p<0.0001. (C) In vitro kinase assay showing LOK-mediated phosphorylation of full-length ezrin in presence of 90 µM of PIP2 micelles. Data are presented as mean ± SE, n = 3, two-way ANOVA (See also Figure 1—figure supplement 1 and Figure 1—source data 1), ****p<0.0001. (D) In vitro kinase assay showing that 18 µM ezrin is specifically primed by 90 µM of PIP2 micelles and not by IP3 or other phospholipids at 90 µM concentrations. Blots are derived from same membrane. Data are presented as mean ± SE, n = 3, two-way ANOVA (See also Figure 1—source data 1), ****p<0.0001. (E) In vitro kinase assay showing that unilamellar liposomes DOPC:PIP2 (90 mol% DOPC, 10% PIP2) or DOPC:DOPS:PIP2 (80 mol% DOPC, 10 mol% DOPS, 10 mol% PIP2) promote phosphorylation of 18 µM ezrin by 10 nM LOK, whereas DOPC (100 mol% DOPC) or DOPC:DOPS (70 mol% DOPC, 30 mol% DOPS) fail to promote LOK-mediated ezrin phosphorylation. (F–G) 10 nM LOK-N phosphorylates 18 µM ezrin-CTD but not full-length ezrin in presence of 90 µM PIP2. Data are presented as mean ± SE, n = 3, two-way ANOVA (See also Figure 1—source data 1), ***p<0.0002, ****p<0.0001. Total ezrin is shown in red and phosphorylation of T567 in green in dual color Western blots.DOI: http://dx.doi.org/10.7554/eLife.22759.00210.7554/eLife.22759.003Figure 1—source data 1.Experimental replicates for Figure 1B,C, D, F and G.DOI: http://dx.doi.org/10.7554/eLife.22759.003

Fig 4: LOK C-terminus is a regulatory domain that inhibits the LOK kinase domain.(A) In vitro kinase assay showing that LOK-C (0–65 µM) inhibits phosphorylation of 18 µM ezrin phosphorylation by 10 nM LOK (red panel) and 10 nM LOK-N-EBP50t (yellow panel) in presence of 90 µM PIP2. (B) In vitro kinase assay showing that LOK-C (0–65 µM) inhibits LOK-mediated phosphorylation of 18 µM ezrin-CTD (blue panel), and LOK-N-mediated phosphorylation of 18 µM ezrin-CTD (purple panel). Total ezrin is shown in red and phosphorylation of T567 in green in dual color western blots. (C) Inhibition curves of LOK/ezrin+PIP2 (red), LOK-N-EBP50t/ezrin+PIP2 (yellow), LOK/ezrin-CTD (blue), and LOK-N/ezrin-CTD (purple), with their corresponding IC50-values. Data are represented as mean ± SE, n = 2 (see also Figure 5—source data 1).DOI: http://dx.doi.org/10.7554/eLife.22759.01410.7554/eLife.22759.015Figure 5—source data 1.Data summary and analysis for Figure 5C, based on experimental replicates represented in Figure 5A–B.DOI: http://dx.doi.org/10.7554/eLife.22759.015

Fig 5: Full length LOK can phosphorylate ezrin, ezrin ∆α1 and ezrin ∆α7 in a PIP2-dependent manner.Full length LOK (10nM) was incubated with 18µM ezrin or ezrin variant and ATP for 10 min at 37°. Samples were resolved by SDS-PAGE and blotted for ezrin (red) and pT567 (green).DOI: http://dx.doi.org/10.7554/eLife.22759.018