Figure 6.

Unregulated LOK causes ezrin depolarization. (A) Schematic of truncation constructs and chimeras used. ATH, AT1-46 homology. (B) LOK-RFP and truncations were localized relative to F-actin. LOK and the LOK C-terminal domain localize to microvilli, whereas the N-terminal kinase domain does not. For clarity, only a small region of the apical domain is shown, and the full cell is shown in the vertical cross section. (C) Cells were cotransfected with indicated RFP-tagged LOK constructs along with ezrin-iFlag. The cells were lysed, ezrin-iFlag was immunoprecipitated (Flag IP), and phosphorylation of ezrin-iFlag was measured by quantitative Western blotting. The fold increase in ezrin phosphorylation as a result of each LOK truncation was normalized to the overexpression of full-length LOK. (D) Cells expressing the RFP-tagged LOK N-terminal kinase domain alone (LOK-1–348-RFP) were stained for ezrin and F-actin and presented as either an apical, basolateral, or side view, and cells transfected with full-length LOK-RFP (bottom) are presented in the side view. Cells expressing the LOK kinase domain alone exhibit delocalized ezrin staining, whereas cells expressing full-length LOK do not. (E) Cells expressing indicated LOK-RFP fusion proteins were stained as in D, and the ezrin depolarization phenotypes were quantified by confocal microscopy. Only when the LOK kinase domain is functional but unregulated does it cause ezrin to mislocalize. Note that although cells expressing LOK-K65R do not have mislocalized ezrin, fewer have microvilli, as reported in Fig. 8 C. (F) Cells overexpressing a fusion of the LOK kinase domain to the integrin-binding region (IBR) of talin were processed as in D. Arrows point to one F-actin–containing basal membrane–associated structure. When the kinase domain alone is intentionally mislocalized, ezrin is preferentially located around these structures. (G) Cell expressing full-length LOK fused to the IBR and stained for ezrin and F-actin. Even when intentionally mislocalized basally, full-length LOK does not cause ezrin to mislocalize, indicating that it is only active in the apical domain. (H) Cells expressing LOK-RFP and derivatives were stained with ezrin and F-actin, and the presence of microvilli was scored as in Figs. 3 B and 4 D. Active, mislocalized LOK constructs cause cells to lose microvilli. Data in B, E, and H are means ± SD of three or four independent experiments. Cross sections were expanded fivefold vertically for clarity, and yellow asterisks mark transfected cells. WB, Western blot. Bars: (B, En face images) 1 µm; (B [main images], D, F, and G) 10 µm.