RESEARCH ARTICLE Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism Thaher Pelaseyed1,2, Raghuvir Viswanatha1,2,3, Ce´ cile Sauvanet1,2, Joshua J Filter2, Michael L Goldberg2, Anthony Bretscher1,2* 1Weill Institute for Molecular and Cell Biology, Cornell University, Ithaca, United States; 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States; 3Department of Genetics, Harvard Medical School, Boston, United States Abstract How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK. DOI: 10.7554/eLife.22759.001 *For correspondence: apb5@ cornell.edu Competing interests: The authors declare that no competing interests exist. Funding: See page 16 Received: 28 October 2016 Accepted: 27 March 2017 Published: 21 April 2017 Reviewing editor: Pekka Lappalainen, University of Helsinki, Finland Copyright Pelaseyed et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Introduction All nucleated cells can polarize to generate morphologically and biochemically distinct regions at the cell surface. For example, the apical and basolateral domains of epithelial cells have distinct protein and lipid compositions, and microvilli are restricted to the apical domain. How cells maintain morphologically distinct regions of their cell surface is not clear. We have been addressing this issue by examining how microvilli are assembled and specifically localized to the apical surface of epithelial cells (Sauvanet et al., 2015). A critical component of epithelial microvilli is ezrin, the founding member of the closely-related ezrin/radixin/moesin (ERM) protein family, that serves as a regulated membrane-F-actin linking protein (Bretscher, 1983, 1989; Fehon et al., 2010). Genetic knockout of ezrin in the mouse results in enterocytes with shorter and disorganized microvilli. In the fruit fly, loss of the single ERM protein is lethal, but when selectively knocked out in photoreceptor cells, microvilli are lost (Karagiosis and Ready, 2004; Saotome et al., 2004; Speck et al., 2003). Thus, ERM proteins provide a critical function in polarized morphogenesis. ERM proteins are regulated by a reversible head-to-tail interaction (Figure 1A). Like all ERMs, ezrin contains an N-terminal FERM domain that binds the plasma membrane and a C-terminal F-actin-binding domain (ezrin-CTD) that can attach to the underlying actin filaments that make up the core of microvilli (Gould et al., 1989; Turunen et al., 1994). In the closed inactive state, the FERM domain is tightly associated with the ~80 residues of ezrin-CTD, masking the membrane association and F-actin-binding sites (Gary and Bretscher, 1995; Pearson et al., 2000; Reczek and Bretscher, 1998). Linking these two regions is a ~150 residue a-helical region that folds into an anti- Pelaseyed et al. eLife 2017;6:e22759. DOI: 10.7554/eLife.22759 1 of 18 Research article Biochemistry Cell Biology A Closed/ inactive FERM Open/ active PM ezrin LOK 1 1 LOK-N LOK-C 318 474 585 FERM CTD Kinase 348 568 PKK1 Kinase 499 PKK1 895 968 PKK2 968 PKK2 pT567/ezrin-CTD P pT567/ezrin F-actin PIP2 IP3 PI PI(3)P PI(4)P PIP3 B ezrin-CTD ATP LOK LOK pT567 Coomassie 1.0 0.8 0.6 0.4 0.2 0.0 + ++ + -+ - ++ C 250 130 55 ezrin ATP PIP2 LOK LOK + ++ + +++ - -++ - +++ D ezrin LOK 250 130 ezrin/pT567 ++ -+ ++ +++ + ++ +++ + 70 35 ezrin/pT567 55 ezrin 70 70 35 **** ezrin 70 pT567 1.0 0.8 0.6 0.4 0.2 0.0 **** **** 70 pT567/ezrin pT567 1.0 0.8 0.6 0.4 0.2 0.0 **** **** 70 E ezrin LOK pT567 Coomassie + ++ ++ + + ++ ++ + F ezrin-CTD ATP LOK 70 LOK-N LOK-N (3X) 70 ezrin/pT567 ezrin pT567 ++ ++ + G ezrin +- +++ -++- - PIP2 LOK --- +- LOK-N (3X) --- -+ 55 ezrin/pT567 35 55 ezrin 35 55 pT567 ++ ++++ - - - +++ -+- -+- - +- - + 70 70 70 PIP2 DOPC DOPC:PIP2 DOPC:DOPS DOPC:DOPS:PIP2 pT567/ezrin-CTD pT567/ezrin 35 ns 1.0 **** **** **** *** 1.0 0.8 0.6 0.4 0.8 0.6 0.4 0.2 0.2 0.0 0.0 Figure 1. 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 mM ezrin-CTD. Data are presented as mean ± SE, n = 3, two-way Figure 1 continued on next page Pelaseyed et al. eLife 2017;6:e22759. DOI: 10.7554/eLife.22759 2 of 18 Research article Biochemistry Cell Biology Figure 1 continued 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 mM 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 mM ezrin is specifically primed by 90 mM of PIP2 micelles and not by IP3 or other phospholipids at 90 mM 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 mM 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 mM ezrin-CTD but not full-length ezrin in presence of 90 mM 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: 10.7554/eLife.22759.002 The following source data and figure supplement are available for figure 1: Source data 1. Experimental replicates for Figure 1B,C, D, F and G. DOI: 10.7554/eLife.22759.003 Figure supplement 1. Phosphorylation of full-length ezrin by full-length LOK requires PIP2 micelles or PIP2-containing liposomes. DOI: 10.7554/eLife.22759.004 parallel coiled coil hairpin in the closed structure (Li et al., 2007). In the open structure, the unmasked FERM domain binds the plasma membrane, the unmasked C-terminal domain binds F-actin, and the a-helical region presumably unravels. The unmasked FERM domain can bind many proteins, including the NHERF family adaptor proteins EBP50 and E3KARP, and the trans-membrane proteins CD44, ICAMs, and b-dystroglycan (Mori et al., 2008; Reczek et al., 1997). The transition between closed and open ERMs requires phosphorylation of a specific threonine residue (T567 in ezrin; T564 in radixin and T558 in moesin) (Nakamura et al., 1995; Simons et al., 1998). Additionally, in vivo phosphorylation was found to require binding of the FERM domain to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) (Fievet et al., 2004; Hao et al., 2009). Structural analysis of the radixin FERM domain in the presence or absence of IP3 led to the suggestion that PIP2 binding to FERM directly changes the conformation so as to partially release the C-terminal domain to make the phosphorylation site accessible (Hamada et al., 2000). Consistent with this model, in the closed configuration, the T567 that becomes phosphorylated is buried within the interface between the FERM and the ezrin-CTD (Li et al., 2007; Pearson et al., 2000). Despite the findings above, how ezrin activation is restricted to the apical membrane was not resolved. At steady state, about one half of the ezrin in an epithelial cell is phosphorylated. Moreover, in vivo phosphorylation of ezrin turns over on the time-scale of minutes, similar to the lifetime of microvilli. These observations strongly suggest a morphogenetic principle: a dynamic system of local ezrin activation by phosphorylation coupled with delocalized deactivation by dephosphorylation. Lymphocyte-oriented-kinase (LOK) and its close paralog Ste20-like-kinase (SLK) were then found to be the major ezrin kinases in epithelial cells. Remarkably, LOK and SLK are currently the only kinases known to selectively localize to the apical membrane of epithelial cells (Viswanatha et al., 2012). LOK had previously been identified as the kinase that phosphorylates ezrin in lymphocytes (Belkina et al., 2009). LOK and SLK belong to the germinal center-like kinase (GCK) -V subfamily of kinases. They consist of a conserved N-terminal kinase domain, a less conserved intermediate region, and a moderately conserved C-terminal domain (Figure 1A). A single ortholog exists in Drosophila melanogaster (Slik) and Caenorhabditis elegans (GCK4). In the fly, Slik has been identified as the sole kinase that phosphorylates the single ERM ortholog, moesin (Carreno et al., 2008; Hipfner et al., 2004; Kunda et al., 2008). It is notable that LOK is highly selective for members of the ERM family, as the kinase’s target sequences requires a conserved tyrosine at position À2 relative to the substrate threonine (Belkina et al., 2009). Here, we describe an in vitro system to reveal why ezrin has to bind PIP2 to become a substrate for phosphorylation by LOK. As far as we are aware, this is the first example of a protein that has to bind a phosphoinositide lipid to serve as a kinase substrate. Our results indicate that PIP2 binding to the ezrin FERM domain transmits a conformational change through the a-helical region to weaken the FERM/ezrin-CTD association. PIP2-priming of ezrin permits the LOK C-terminal domain to act as Pelaseyed et al. eLife 2017;6:e22759. DOI: 10.7554/eLife.22759 3 of 18 Research article Biochemistry Cell Biology a wedge between the FERM and ezrin-CTD to allow the kinase domain to gain access and phosphorylate T567. In support of this model, we design a chimeric protein and show that it can recapitulate the activity of LOK in vitro and ex vivo. Further, we define a distal site in ezrin about 40 residues from T567 that is also necessary for phosphorylation by the kinase domain. In addition to being required for ezrin phosphorylation, the LOK C-terminal domain negatively regulates the kinase activity of LOK. Thus, the PIP2-dependent mechanism of ezrin phosphorylation by LOK involves several distinct steps, thereby ensuring the specificity of the reaction. Results In vitro phosphorylation of ezrin by LOK requires PIP2 and the LOK C-terminal domain We first established an in vitro phosphorylation assay for purified LOK using the isolated ezrin-CTD as substrate. LOK phosphorylated the ezrin-CTD as detected using pT567 antibody (Figure 1B), but failed to phosphorylate full-length ezrin (Figure 1C). We therefore explored the possible role of phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) as previous reports have suggested a regulatory role for this phospholipid (Fievet et al., 2004; Hao et al., 2009). In the presence of PIP2 micelles, LOK was readily able to phosphorylate ezrin (Figure 1C), but PIP2 did not influence phosphorylation of the ezrin-CTD by LOK (Figure 1—figure supplement 1A). The requirement for PIP2 was specific, as other phosphoinositides, or soluble IP3, failed to substitute for PIP2 in phosphorylation of ezrin (Figure 1D). The requirement of PIP2 for ezrin phosphorylation was not simply a negative charge effect as PIP2-dependency was reproduced in 100-nm-sized synthetic unilamellar liposomes of DOPC or DOPC:DOPS containing 10 mol% PIP2, which represent a more physiological system (Figure 1E). We next explored which domains of LOK are required for ezrin phosphorylation. LOK consists of an N-terminal kinase domain (LOK-N) and a C-terminal region (LOK-C) containing putative polo kinase kinase (PKK) domains of unknown function (Figure 1A). In vitro, both full-length LOK and LOK-N can phosphorylate ezrin-CTD, although the isolated kinase domain was less active (Figure 1F). Whereas intact LOK phosphorylated ezrin in a PIP2-dependent manner, LOK-N was unable to phosphorylate ezrin, even in the presence of PIP2 micelles and unilamellar DOPC:DOPS liposomes containing 10 mol% PIP2 (Figure 1G and Figure 1—figure supplement 1B). We conclude that in vitro phosphorylation of full-length ezrin requires both PIP2 and the C-terminal domain of LOK. In vitro phosphorylation requires active participation by the central ahelical domain of ezrin The FERM domain of ezrin is linked to the ezrin-CTD by a ~150 residue a-helical coiled coil hairpin, as represented in the structure of the close homolog Sfmoesin (Figure 2A) (Li et al., 2007). This region consists of three a-helices that connect the FERM domain to the ezrin-CTD via an antiparallel coiled coil (Li et al., 2007). In the absence of the a-helical region, the individual FERM and ezrinCTD domains bind avidly together (Pearson et al., 2000). To explore whether binding of PIP2 to the FERM domain simply weakens the association with the ezrin-CTD as proposed (Hamada et al., 2000), we asked whether a complex of isolated GST-ezrin-CTD and FERM could be phosphorylated by LOK or LOK-N in the presence of PIP2. Remarkably, while GST-ezrin-CTD was phosphorylated by LOK and LOK-N, we found that the isolated GST-ezrin-CTD+FERM complex could not be phosphorylated by LOK or LOK-N in the presence of PIP2 (Figure 2B). Furthermore, LOK failed to phosphorylate GST-ezrin-CTD in complex with FERM in presence of DOPC:DOPS:PIP2 unilamellar liposomes (Figure 2—figure supplement 1). We conclude that the central a-helical region is required for translating PIP2-binding to the FERM domain into a weakened association between FERM and ezrin-CTD domains, thus allowing in vitro phosphorylation of ezrin by LOK. PIP2 primes ezrin to serve as an efficient substrate for phosphorylation by LOK Having established the broad features of the PIP2-dependent phosphorylation of ezrin by LOK, we explored the kinetic parameters of the phosphorylation reactions in order to gain more insight into Pelaseyed et al. eLife 2017;6:e22759. DOI: 10.7554/eLife.22759 4 of 18 Research article Biochemistry Cell Biology A Coiled coil _