PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2015 May 1; 290(18): 11384–11392.
Published online 2015 March 19. doi:  10.1074/jbc.M115.643791
PMCID: PMC4416843

Structural Basis for the Phosphorylation-regulated Interaction between the Cytoplasmic Tail of Cell Polarity Protein Crumbs and the Actin-binding Protein Moesin*

Zhiyi Wei,§¶,1 Youjun Li,‡,1 Fei Ye,§ and Mingjie Zhang§,2

Abstract

The type I transmembrane protein crumbs (Crb) plays critical roles in the establishment and maintenance of cell polarities in diverse tissues. As such, mutations of Crb can cause different forms of cancers. The cell intrinsic role of Crb in cell polarity is governed by its conserved, 37-residue cytoplasmic tail (Crb-CT) via binding to moesin and protein associated with Lin7–1 (PALS1). However, the detailed mechanism governing the Crb·moesin interaction and the balance of Crb in binding to moesin and PALS1 are not well understood. Here we report the 1.5 Å resolution crystal structure of the moesin protein 4.1/ezrin/radixin/moesin (FERM)·Crb-CT complex, revealing that both the canonical FERM binding motif and the postsynaptic density protein-95/Disc large-1/Zonula occludens-1 (PDZ) binding motif of Crb contribute to the Crb·moesin interaction. We further demonstrate that phosphorylation of Crb-CT by atypical protein kinase C (aPKC) disrupts the Crb·moesin association but has no impact on the Crb·PALS1 interaction. The above results indicate that, upon the establishment of the apical-basal polarity in epithelia, apical-localized aPKC can actively prevent the Crb·moesin complex formation and thereby shift Crb to form complex with PALS1 at apical junctions. Therefore, Crb may serve as an aPKC-mediated sensor in coordinating contact-dependent cell growth inhibition in epithelial tissues.

Keywords: Cell Growth, Cell Polarity, Protein Structure, Scaffold Protein, X-ray Crystallography, Crumbs, FERM Domain, Moesin, aPKC

Introduction

The establishment of epithelial cell polarity is a complicated and dynamic process that involves cell-cell adhesions, assembly of junction complexes, reorganization of cytoskeleton, directional transportation of vesicles, and specific localization of proteins and lipids (1,4). Over the last two decades, genetic and cell biology studies have identified three tripartite protein complexes, namely the crumbs (Crb)3 complex composed of Crb·PALS1·PALS1-associated tight junction (PATJ), the partition-defective (PAR) complex composed of PAR3/PAR6/aPKC, and the Disc large (DLG) complex composed of DLG/lethal giant larvae (LGL)/Scribble, as the principle cell polarity regulators (5,10). Among these regulators, only Crb is a transmembrane protein and necessary for both the apical-basal cell polarity and the assembly of the zonula adherens in Drosophila epithelia (11,13). In early Drosophila embryo development, dysfunction mutations of Crb lead to the loss of the apical-membrane identity, and overexpression of Crb causes expansion of the apical-membrane size at the expense of the basolateral membranes (14, 15).

As a type I transmembrane protein, Drosophila Crb is composed of an extracellular region, a transmembrane domain, and a 37-residue cytoplasmic tail (Crb-CT) that contains a FERM binding motif (FBM), a PDZ binding motif (PBM), and several potential aPKC phosphorylation sites (16,19) (Fig. 1A). Expression of transmembrane domain-tethered Crb-CT alone can rescue most of the embryonic polarity defects in crb mutant (15, 16), indicating that the cytoplasmic tail plays a crucial role for the functions of Crb. Mechanistically, the PBM of Crb-CT is known to specifically bind to the PDZ-Src homology 3 (SH3)-guanylate kinase supramodule of PALS1 and stabilizes the apical Crb complex (20), whereas the FBM is considered not to be directly engaged in the polarity complex formation but instead to be involved in the Hippo signaling pathway and the apical membrane-cytoskeleton regulations (21,25). Coincidently, the aPKC phosphorylation sites in Crb-CT are located near or within the Crb-FBM (Fig. 1A), implying that the interaction(s) between Crb-FBM and its target(s) may be regulated by aPKC-mediated phosphorylation of Crb-CT.

FIGURE 1.
Crb-CT specifically binds to moesin-FERM. A, schematic diagram showing the domain organization of moesin and Crb and the amino acid sequence alignment of the cytoplasmic tails of different Crb proteins (hs for human, mm for mouse, xt for Xenopus tropicalis ...

The ezrin-radixin-moesin (ERM) proteins are a family of widely distributed membrane-associated proteins that provide a structural linkage between plasma membranes and cortical cytoskeletons (26,30). These three proteins share similar domain organizations and high sequence identities, all having an N-terminal FERM domain and a C-terminal domain that can fold back to bind to the FERM domain forming an autoinhibited conformation (Fig. 1A) (31,33). The autoinhibited ERMs can be activated by phosphorylation on a conserved Thr (Thr-567 in ezrin, Thr-564 in radixin, Thr-558 in moesin) at their respective inhibitory C-terminal domain and/or via binding to phosphatidylinositol 4,5-bisphosphate (PIP2) (34,37). The activated ERMs then bind to membranes via their FERM domains (directly to membrane lipids and/or transmembrane proteins) and to actin filaments via their C-terminal actin binding motifs. In Drosophila epithelia, Crb exhibits close co-localization with moesin and stabilizes the apical membrane-cytoskeleton, leading to reinforcement of the zonula adherens and effective coupling between epithelial morphogenesis and cell polarity (25). However, the detailed mechanism governing the Crb·moesin interaction and Crb-mediated regulation between apical-basal cell polarity and apical cytoskeleton reorganization are not well understood.

Here we biochemically and structurally characterized the interaction between Crb-CT and moesin FERM domain. The 1.5 Å resolution crystal structure of the moesin-FERM·Crb-CT complex reveals a typical FERM/FBM binding mode in which the FBM of Crb-CT forms a short β-strand and fits into the canonical F3 lobe target binding site. To our surprise, the PBM of Crb-CT also contributes to the binding to moesin-FERM by occupying the PIP2 binding site at the F1/F3 cleft, implying that Crb-CT may mimic the role of PIP2 in the activation of the ERM family proteins. We further show by NMR spectroscopy that phosphorylation of Crb-CT by aPKC disrupts the Crb·moesin association, likely by preventing the formation of the β-strand conformation of the Crb-FBM. The biological implications of these findings are discussed.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

The mouse moesin FERM domain (residues 1–297) and fly Crb-CT (residues 2110–2146) were amplified by PCR using their respective full-length cDNA as the templates. Each PCR product was individually cloned into a modified pET-32 M vector. Various mutants were created using standard two-step PCR-based methods and confirmed by DNA sequencing. Recombinant proteins each with an N-terminal Trx-His6 tag were expressed in Escherichia coli BL21(DE3) cells at 16 °C. The expressed proteins were purified by a nickel-nitrilotriacetic acid-agarose affinity chromatography followed by a size-exclusion chromatography.

Isothermal Titration Calorimetry (ITC) Assay

ITC was carried out on a MicroCal VP-ITC at 25 °C. All proteins were dissolved in a buffer containing 50 mm Tris, pH 7.5, 400 mm NaCl, 1 mm EDTA, and 1 mm DTT. The titration processes were performed by injecting 5–10-μl aliquots of protein samples in a syringe (concentration of 200 μm) into protein samples in cell (concentration of 20 μm) at time intervals of 120 s to ensure that the titration peak returned to the baseline. The relatively high salt concentration is used to ensure that the FERM domains used in the binding studies behave well in solution. The data were analyzed using the Origin 7.0 program and fitted by the one-site binding model.

Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR samples containing 1.5 mm Crb-FBM (2111NKRATRGTYSPSAQE2125) or Crb-FBM_Thr(P)-2118 peptide were dissolved in 50 mm potassium phosphate, pH 6.5, with 90% H2O, 10% D2O. NMR spectra were acquired at 283 K on a Varian Inova 750-MHz spectrometer. Mixing times of 300 and 75 ms were used for the nuclear Overhauser effect spectroscopy (NOESY) and total correlated spectroscopy (TOCSY) experiments, respectively. Spectra were processed using NMRPipe (38) and analyzed with Sparky software.

Crystallization

For crystallization, the tagged proteins were treated with a small amount of human rhinovirus 3C protease at 4 °C overnight to cleave the fusion tags and further purified by a step of size-exclusion chromatography. Crystals of the Moesin-FERM·Crb-CT complex were obtained by the hanging drop vapor diffusion method at 16 °C within 2 days. To set up a hanging drop, 1 μl of concentrated protein mixture at a 1:1 stoichiometric ratio was mixed with 1 μl of crystallization solution with 20% PEG3350 and 0.2 m ammonium iodine. Before diffraction experiments, crystals were soaked in the crystallization solution containing an additional 30% glycerol for cryoprotection. The diffraction data were collected at the Shanghai Synchrotron Radiation Facility and were processed and scaled using HKL2000 (39).

Structure Determination

The initial phase was determined by molecular replacement using the apo form of moesin-FERM (PDB code 1EF1) as the searching model. The model was refined in Phenix (40) against the 1.5 Å dataset. The Crb-CT peptide was built subsequently in COOT (41). In the final stage, an additional TLS refinement was performed in Phenix. The final model was further validated by using MolProbity (42). The refinement statistics are listed in Table 1. All structure figures were prepared using PyMOL. The sequence alignments were prepared and presented using ClustalW (43).

TABLE 1
Statistics of data collection and model refinement

RESULTS

Crb-CT Specifically Binds to Moesin FERM Domain

Unlike its extracellular region, the Crb-CT, especially the PBM, FBM, and aPKC phosphorylation sites, is highly conserved across different species and isoforms (Fig. 1A). Specifically, the fly Crb-CT is essentially the same as mammalian Crb2-CT, and therefore, we continued to use the fly Crb-CT after our recent study of the Crb-CT/PALS1 interaction (20). To investigate the Crb·moesin interaction, we used highly purified recombinant Crb-CT and moesin-FERM proteins. Quantitative binding assays showed that Crb-CT directly binds to moesin-FERM with a disassociation constant (Kd) of ~5 μm in the presence of 400 mm NaCl in the assayed buffer (Fig. 1B). Interestingly, although sharing a high amino acid sequence identity with moesin-FERM, the FERM domain of merlin had no detectable binding to Crb-CT (Fig. 1C), indicating that moesin-FERM encodes its intrinsic target binding specificity. A moesin-FERM chimera (termed as moesin-FERMF3/Merlin), in which the F3 lobe was replaced by the corresponding F3 lobe of merlin, failed to bind to Crb-CT (Fig. 1D), indicating that the F3 lobe is chiefly responsible for the binding of moesin-FERM to Crb-CT.

Overall Structure of the Moesin-FERM·Crb-CT Complex

To understand the molecular basis governing the moesin·Crb interaction, we determined the structure of moesin-FERM in complex with Crb-CT by x-ray crystallography (Table 1). The moesin-FERM·Crb-CT complex crystals were diffracted to up to 1.5 Å resolution. In the crystals the moesin-FERM·Crb-CT complex forms a heterodimer with one complex per asymmetric unit. The final structural model contains most of residues from the complex, except for a flexible loop (residues 2127–2135) of Crb-CT, which connects its FBM and PBM (Fig. 1A and Fig. 2). Moesin-FERM adopts a typical cloverleaf architecture composed of three lobes (F1, F2, and F3). The overall fold of the FERM domain in the moesin·Crb complex structure is essentially identical to the apo moesin-FERM structure (the overall RMSD of 1.6 Å with the 285 aligned residues). The two highly conserved motifs in Crb-CT, FBM and PBM, bind to moesin-FERM at the F3 lobe and a cleft between the F1 and F3 lobes, respectively (Fig. 2).

FIGURE 2.
Overall structure of the moesin-FERM·Crb-CT complex. A, the ribbon diagram structure of moesin-FERM·Crb-CT complex with the three lobes of moesin-FERM, F1 (green), F2 (cyan), and F3 (blue), Crb-FBM (gold), and Crb-PBM (purple) drawn in ...

The Crb-FBM Binding Site in the F3 Lobe of Moesin-FERM

In FERM-containing proteins, F3 lobes act as the major target binding site. A groove (known as the αβ-groove) mainly formed by β5F3 and α1F3 is a well characterized target binding region in FERM domains including those of moesin (44), radixin (45,48), talin (49, 50), Merlin (51), and myosin-X (52, 53). Most of the previously characterized targets, especially those interacting with ERM proteins, bind to the αβ-groove in a β-strand or β-strand-like structures (Fig. 3B). In the moesin-FERM·Crb-CT complex, the FBM of Crb also adopts a β-strand structure and binds to the αβ-groove in the F3 lobe of moesin-FERM (Fig. 3A), extending the anti-parallel β-sheet formed by β5F3, β6F3, and β7F3. The FBM/F3 interaction is mainly mediated by hydrogen bonds. Some hydrophobic interactions (e.g. Pro-2121Crb inserts its aliphatic side chain into a hydrophobic cleft formed by Ile-245F3 and Ile-248F3) also contribute to the FBM/F3 interaction. The three consecutive and strictly conserved residues in the FBM, Gly-2117, Thr-2118, and Tyr-2119 (termed as the GTY motif and known as the iconic sequence of FBMs), are intimately involved in the FBM/F3 interaction (Fig. 3A). The very tight packing between Gly-2117 and the ring of Phe-250F3 is afforded by the lack of side chain of Gly-2117. The side chain of Thr-2118 forms a pair of hydrogen bonds with Asp-247F3. Tyr-2119 forms a strong hydrogen bond with His-288F3 (bond length of 2.7 Å) in addition to interacting with Met-285F3 through their hydrophobic side chains. Consistent with our structural analysis, the substitution of Tyr-2119 with Ala abolished Crb-CT binding to moesin-FERM (Fig. 3C).

FIGURE 3.
The moesin-FERM/Crb-FBM interaction. A, a stereo view of the molecular details of the moesin-FERM/Crb-FBM interaction. Notably, Ser-2120 at the Crb-CT adopts a dual-conformation. Hydrogen bonds are indicated by dashed lines. B, two examples showing that ...

The PBM of Crb Binds to the F1/F3 Cleft of Moesin-FERM

An unexpected finding in the moesin·Crb complex structure is that the Crb-PBM directly contacts the moesin FERM domain (Fig. 2A), a mode that has not been observed in any other FERM domains characterized biochemically and structurally. The last seven residues of Crb-CT, which contains its PBM, fit snuggly into the cleft formed by highly conserved residues from the F1 and F3 lobes (Fig. 4A). The C-terminal tail carboxyl group of Crb-PBM forms a salt bridge with Lys-278F3. Leu-2145Crb at the −1 position of PBM inserts into a hydrophobic pocket formed by Leu-281F3, Phe-250F3, and the aliphatic part of Lys-278F3. Glu-2143Crb at the −3 position of PBM forms two salt bridges with Lys-60F1 and Lys-83F1. The residues immediately N-terminal to Crb-PBM also play a role in binding to the F1/F3 cleft (Fig. 4A). Glu-2142Crb makes two hydrogen bonds with the main chains of Leu-61 and Asn-62 in the β4/β5 loop of the F1 lobe. The two proline residues, Pro-2140Crb and Pro-2141Crb, are involved in hydrophobic interactions with residues from the F1 and F3 lobes. Consistent with the above structural analysis, removal of the last four residues from Crb-CT weakened its binding to moesin-FERM by ~5-fold assayed in high salt buffers (Fig. 4B). We expect that the contribution of the PBM to the Crb-CT/moesin-FERM is larger at physiological salt concentrations, given the heavy involvement of the charged residues in the interaction. It is important to note that the residues involved in the moesin-FERM·Crb-CT interaction are highly conserved in ERM proteins as well as Crb homologs across species (Fig. 1A), suggesting that this interaction is conserved throughout metazoan.

FIGURE 4.
The moesin-FERM/Crb-PBM interaction. A, a stereo view of the molecular details of the moesin-FERM/Crb-PBM interaction. B, ITC-based measurement displaying the interaction between Crb-CT-ΔPBM and moesin-FERM. C and D, comparison of the bindings ...

ERM proteins are believed to associate with membranes either by binding to transmembrane proteins or directly to phospholipids. The association of ERMs with PIP2 potentiates the activation of ERM proteins (34, 37). Interestingly, the Crb-PBM binding site in F1/F3 cleft overlaps with the PIP2 head group binding site on moesin-FERM (54) and the Crb-PBM mimics PIP2 binding to moesin by engaging positively charged residues in the F1/F3 cleft (Fig. 4, C and D), indicating that the bindings of Crb and PIP2 to moesin-FERM are mutually exclusive. Therefore, it is hypothesized that the association of Crb with ERMs may potentiate the activation as well as membrane localization of ERMs better than phospholipids alone.

aPKC Phosphorylates Crb and in Turn Abolishes the Moesin·Crb Interaction

Although aPKC-dependent phosphorylation of Crb is essential for epithelial apical-basal cell polarity in Drosophila (19), the molecular basis underlying this phosphorylation-dependent cell polarity establishment and maintenance is not clear. The aPKC phosphorylation sites (Thr-2115 and Thr-2118) are located near/in Crb-FBM (Fig. 3A). We demonstrated earlier on that Crb-FBM is not involved in the binding of Crb-CT to PALS1 (20). Therefore, we focused our investigation on the potential phosphorylation-regulated interaction between Crb-CT and moesin-FERM. Perhaps it is not too surprising that a phosphorylation-mimic mutation of Crb-CT (Crb-CT_T2118E) only moderately weakened its binding to moesin-FERM (Fig. 5A), as the side chain of Thr-2118 is solvent-exposed and makes relatively minor contribution to the binding by forming hydrogen bond with Asp-247F3. Surprisingly, a synthetic phosphor-peptide of Crb-CT, in which Thr-2118 of the FBM (i.e. GT2118Y) was specifically phosphorylated, displayed no detectable binding to moesin-FERM (Fig. 5B), indicating that aPKC-mediated phosphorylation can completely disrupt the Crb·moesin interaction. Based on the structure shown in Fig. 3A, the large difference between the phosphor-mimetic mutation of Crb-CT (Crb-CT_T2118E) and the corresponding phosphor-Crb-CT (Crb-CT_Thr(P)-2118) in their bindings to moesin-FERM cannot be explained solely by their charge differences. Instead, the phosphorylation of Thr-2118 might alter the conformation of Crb-CT, and such phosphorylation-induced conformational alterations cannot be fully mimicked by the T2118E mutation. To test this hypothesis, we compared the structural changes of Crb-CT induced by Thr-2118 phosphorylation by NMR spectroscopy. The NOE pattern of WT Crb-FBM peptide derived from the 1H homonuclear NOESY spectrum indicates that the peptide adopts a largely extended structure (Fig. 5D) and thus can easily form the observed β-strand structure upon forming complex with moesin-FERM. In contrast, a stretch of (i, i+2) NOEs surrounding Thr(P)-2118 were detected for the Crb-FBM_Thr(P)-2118 peptide (Fig. 5, E and F), indicating that phosphorylation of Thr-2118 induces the formation of a turn-like structure between Arg-2116 and Ser-2120, and this turn-like structure is likely stabilized by the interaction of the side chains between Arg-2116 and Thr(P)-2118 (Fig. 5, E and F). As a consequence, the formation of the turn-like structure of the phosphor-Crb-FBM likely prevents Crb-CT from binding to moesin-FERM.

FIGURE 5.
Phosphorylation of Thr-2118 abolishes the moesin·Crb interaction. A–C, ITC-based measurements showing the binding profiles between Crb-CT_T2118E (A) or Crb-CT_Thr(P)-2118 (B) and moesin-FERM and between Crb-CT_Thr(P)-2118 and PALS1 PDZ-SH3-GK ...

DISCUSSION

The interaction between Crb-CT and moesin characterized here is ~50-fold weaker than the Crb-CT/PALS1 interaction that we demonstrated earlier on (20). Would the crumbs·moesin interaction even occur if PALS1 is present? We believe that both interactions can exist in cells, but the two interactions likely occur in different regions/time points/growth conditions in living cells. There are several possible scenarios that can occur in cells. Although the binding between crumbs and moesin is not very strong, the enrichment of moesin by actin filaments (via the C-terminal actin binding domain of moesin) can increase the binding avidity between crumbs and moesin. In polarized epithelial cells, PALS1 is normally enriched in the apical cell cortex together with aPKC; the PALS1/crumbs interaction would dominate under such condition. On the other hand, the moesin/crumbs complex may have the advantage of forming in regions with enriched moesin/actin filaments (e.g. at leading edges of non-polarized migrating cells).

Actin cytoskeleton reorganization is a major step during the establishment of apical-basal cell polarity. A series of recent studies has shown that the actin cytoskeleton organization is closely linked to the Hippo signaling pathway, as numerous Hippo-mediated cellular processes (e.g. cell growth and differentiation, cell-cell, and cell-matrix contact-induced tissue morphogenesis/homeostasis, cell migrations, etc.) involve changes in the F-actin structures (55,60). We demonstrate in this study that aPKC-mediated phosphorylation of Crb-CT disrupts its binding to moesin and thus can result in weakening of the moesin-tethered coupling of Crb-harboring plasma membranes to cortical actin cytoskeleton and consequent reduction of cortical tensions in polarized epithelia. This cortical tension change may then trigger the activation of the Hippo signaling pathway and prevent cells from further growth. Because aPKC-mediated phosphorylation of Crb-CT does not affect the interaction between Crb-CT and PALS1 (Fig. 5C), aPKC phosphorylation-mediated release of Crb from moesin is likely to be accompanied by increased formation of the Crb·PALS1 complex at the tight junctions, a process favorable for cell polarity establishment and stabilization. As such, a plausible picture of the role of Crb in connecting apical-basal cell polarity establishment and contact-induced cell growth inhibition emerges (Fig. 6). In this picture, formation of the apical-basal polarity leads to apical enrichment and activation of aPKC and subsequent phosphorylation of Crb-CT. The phosphorylated Crb dissociates from moesin and in turn promotes Crb to form complex with PALS1, thereby stabilizing apical-basal polarity with concomitant cell growth inhibition possibly via activation of the Hippo signaling pathway. In addition to its role in the regulation of apical-basal cell polarity and apical membrane/cytoskeleton interactions, Crb-CT has also been reported to directly function in the Hippo signaling pathway by binding to the FERM domain-containing protein Expanded (22, 23). Both Crb/Expanded and Crb·moesin interactions involve the conserved FBM with aPKC phosphorylation sites. Understanding the relations of Crb-CT to its diverse targets may provide further insights for the regulatory roles of Crb in distinct cellular processes. Our study here indicates that aPKC plays a vital regulatory role in determining the functions of Crb in cell growth or cell polarity establishment.

FIGURE 6.
A model showing aPKC-induced shift of Crb from the moesin complex to the PALS1 complex. In this model the establishment of the apical-basal polarity leads to apical enrichment and activation of aPKC and subsequent phosphorylation of Crb-FBM. The phosphorylation ...

Acknowledgment

We thank the Shanghai Synchrotron Radiation Facility (SSRF) BL17U for x-ray beam time.

*This work was supported by Research Grants Council of Hong Kong Grants 663811, 663812, 664113, AoE/M09/12, and T13–607/12R and by the Asia Fund for Cancer Research (to M. Z.).

The atomic coordinates and structure factors (code 4YL8) have been deposited in the Protein Data Bank (http://wwpdb.org/).

3The abbreviations used are:

Crb
crumbs
FERM
protein 4.1/ezrin/radixin/moesin
aPKC
atypical protein kinase C
PALS1
protein associated with Lin7–1
PDZ
postsynaptic density protein-95/Disc large-1/Zonula occludens-1
CT
cytoplasmic tail
FBM
FERM binding motif
PBM
PDZ-binding motif
ERM
ezrin/radixin/moesin
ITC
isothermal titration calorimetry
PIP2
phosphatidylinositol 4,5-bisphosphate.

REFERENCES

1. Martin-Belmonte F., Perez-Moreno M. (2012) Epithelial cell polarity, stem cells, and cancer. Nat. Rev. Cancer 12, 23–38 [PubMed]
2. Rodriguez-Boulan E., Macara I. G. (2014) Organization and execution of the epithelial polarity programme. Nat. Rev. Mol. Cell Biol. 15, 225–242 [PMC free article] [PubMed]
3. Tepass U. (2012) The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival. Annu. Rev. Cell Dev. Biol. 28, 655–685 [PubMed]
4. Margolis B., Borg J. P. (2005) Apicobasal polarity complexes. J. Cell Sci. 118, 5157–5159 [PubMed]
5. Bulgakova N. A., Knust E. (2009) The Crumbs complex: from epithelial-cell polarity to retinal degeneration. J. Cell Sci. 122, 2587–2596 [PubMed]
6. Knust E., Bossinger O. (2002) Composition and formation of intercellular junctions in epithelial cells. Science 298, 1955–1959 [PubMed]
7. Nance J., Zallen J. A. (2011) Elaborating polarity: PAR proteins and the cytoskeleton. Development 138, 799–809 [PubMed]
8. Suzuki A., Ohno S. (2006) The PAR-aPKC system: lessons in polarity. J. Cell Sci. 119, 979–987 [PubMed]
9. Tepass U., Tanentzapf G., Ward R., Fehon R. (2001) Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35, 747–784 [PubMed]
10. Bilder D. (2004) Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors. Genes Dev. 18, 1909–1925 [PubMed]
11. Pocha S. M., Knust E. (2013) Complexities of Crumbs function and regulation in tissue morphogenesis. Curr. Biol. 23, R289–R293 [PubMed]
12. Tepass U. (1996) Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia of Drosophila. Dev. Biol. 177, 217–225 [PubMed]
13. Tepass U., Theres C., Knust E. (1990) Crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61, 787–799 [PubMed]
14. Wodarz A., Grawe F., Knust E. (1993) CRUMBS is involved in the control of apical protein targeting during Drosophila epithelial development. Mech. Dev. 44, 175–187 [PubMed]
15. Wodarz A., Hinz U., Engelbert M., Knust E. (1995) Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67–76 [PubMed]
16. Klebes A., Knust E. (2000) A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Curr. Biol. 10, 76–85 [PubMed]
17. Klose S., Flores-Benitez D., Riedel F., Knust E. (2013) Fosmid-based structure-function analysis reveals functionally distinct domains in the cytoplasmic domain of Drosophila crumbs. G3 3, 153–165 [PMC free article] [PubMed]
18. Laprise P., Beronja S., Silva-Gagliardi N. F., Pellikka M., Jensen A. M., McGlade C. J., Tepass U. (2006) The FERM protein Yurt is a negative regulatory component of the crumbs complex that controls epithelial polarity and apical membrane size. Dev. Cell 11, 363–374 [PMC free article] [PubMed]
19. Sotillos S., Díaz-Meco M. T., Caminero E., Moscat J., Campuzano S. (2004) DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila. J. Cell Biol. 166, 549–557 [PMC free article] [PubMed]
20. Li Y., Wei Z., Yan Y., Wan Q., Du Q., Zhang M. (2014) Structure of Crumbs tail in complex with the PALS1 PDZ-SH3-GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex. Proc. Natl. Acad. Sci. U.S.A. 111, 17444–17449 [PubMed]
21. Chen C. L., Gajewski K. M., Hamaratoglu F., Bossuyt W., Sansores-Garcia L., Tao C., Halder G. (2010) The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 107, 15810–15815 [PubMed]
22. Ling C., Zheng Y., Yin F., Yu J., Huang J., Hong Y., Wu S., Pan D. (2010) The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded. Proc. Natl. Acad. Sci. U.S.A. 107, 10532–10537 [PubMed]
23. Robinson B. S., Huang J., Hong Y., Moberg K. H. (2010) Crumbs regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain protein Expanded. Curr. Biol. 20, 582–590 [PMC free article] [PubMed]
24. Varelas X., Samavarchi-Tehrani P., Narimatsu M., Weiss A., Cockburn K., Larsen B. G., Rossant J., Wrana J. L. (2010) The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-β-SMAD pathway. Dev. Cell 19, 831–844 [PubMed]
25. Médina E., Williams J., Klipfell E., Zarnescu D., Thomas G., Le Bivic A. (2002) Crumbs interacts with moesin and β(Heavy)-spectrin in the apical membrane skeleton of Drosophila. J. Cell Biol. 158, 941–951 [PMC free article] [PubMed]
26. Sato N., Funayama N., Nagafuchi A., Yonemura S., Tsukita S., Tsukita S. (1992) A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J. Cell Sci. 103, 131–143 [PubMed]
27. Bretscher A., Edwards K., Fehon R. G. (2002) ERM proteins and merlin: integrators at the cell cortex. Nat. Rev. Mol. Cell Biol. 3, 586–599 [PubMed]
28. Fehon R. G., McClatchey A. I., Bretscher A. (2010) Organizing the cell cortex: the role of ERM proteins. Nat. Rev. Mol. Cell Biol. 11, 276–287 [PMC free article] [PubMed]
29. Fiévet B., Louvard D., Arpin M. (2007) ERM proteins in epithelial cell organization and functions. Biochim. Biophys. Acta 1773, 653–660 [PubMed]
30. Bretscher A., Chambers D., Nguyen R., Reczek D. (2000) ERM-Merlin and EBP50 protein families in plasma membrane organization and function. Annu. Rev. Cell Dev. Biol. 16, 113–143 [PubMed]
31. Gould K. L., Bretscher A., Esch F. S., Hunter T. (1989) cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1. EMBO J. 8, 4133–4142 [PubMed]
32. Lankes W. T., Furthmayr H. (1991) Moesin: a member of the protein 4.1-talin-ezrin family of proteins. Proc. Natl. Acad. Sci. U.S.A. 88, 8297–8301 [PubMed]
33. Funayama N., Nagafuchi A., Sato N., Tsukita S., Tsukita S. (1991) Radixin is a novel member of the band 4.1 family. J. Cell Biol. 115, 1039–1048 [PMC free article] [PubMed]
34. Hirao M., Sato N., Kondo T., Yonemura S., Monden M., Sasaki T., Takai Y., Tsukita S., Tsukita S. (1996) Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol. 135, 37–51 [PMC free article] [PubMed]
35. Matsui T., Maeda M., Doi Y., Yonemura S., Amano M., Kaibuchi K., Tsukita S., Tsukita S. (1998) Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140, 647–657 [PMC free article] [PubMed]
36. Simons P. C., Pietromonaco S. F., Reczek D., Bretscher A., Elias L. (1998) C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton. Biochem. Biophys. Res. Commun. 253, 561–565 [PubMed]
37. Nakamura F., Huang L., Pestonjamasp K., Luna E. J., Furthmayr H. (1999) Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides. Mol. Biol. Cell 10, 2669–2685 [PMC free article] [PubMed]
38. Delaglio F., Grzesiek S., Vuister G. W., Zhu G., Pfeifer J., Bax A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 [PubMed]
39. Otwinowski Z., Minor W. (1997) Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326
40. Adams P. D., Grosse-Kunstleve R. W., Hung L. W., Ioerger T. R., McCoy A. J., Moriarty N. W., Read R. J., Sacchettini J. C., Sauter N. K., Terwilliger T. C. (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 [PubMed]
41. Emsley P., Cowtan K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 [PubMed]
42. Davis I. W., Leaver-Fay A., Chen V. B., Block J. N., Kapral G. J., Wang X., Murray L. W., Arendall W. B., 3rd, Snoeyink J., Richardson J. S., Richardson D. C. (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 [PMC free article] [PubMed]
43. Thompson J. D., Higgins D. G., Gibson T. J. (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 [PMC free article] [PubMed]
44. Pearson M. A., Reczek D., Bretscher A., Karplus P. A. (2000) Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell 101, 259–270 [PubMed]
45. Hamada K., Shimizu T., Yonemura S., Tsukita S., Tsukita S., Hakoshima T. (2003) Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex. EMBO J. 22, 502–514 [PubMed]
46. Terawaki S., Kitano K., Hakoshima T. (2007) Structural basis for type II membrane protein binding by ERM proteins revealed by the radixin-neutral endopeptidase 24.11 (NEP) complex. J. Biol. Chem. 282, 19854–19862 [PubMed]
47. Takai Y., Kitano K., Terawaki S., Maesaki R., Hakoshima T. (2008) Structural basis of the cytoplasmic tail of adhesion molecule CD43 and its binding to ERM proteins. J. Mol. Biol. 381, 634–644 [PubMed]
48. Mori T., Kitano K., Terawaki S., Maesaki R., Fukami Y., Hakoshima T. (2008) Structural basis for CD44 recognition by ERM proteins. J. Biol. Chem. 283, 29602–29612 [PMC free article] [PubMed]
49. García-Alvarez B., de Pereda J. M., Calderwood D. A., Ulmer T. S., Critchley D., Campbell I. D., Ginsberg M. H., Liddington R. C. (2003) Structural determinants of integrin recognition by talin. Mol. Cell 11, 49–58 [PubMed]
50. Wegener K. L., Partridge A. W., Han J., Pickford A. R., Liddington R. C., Ginsberg M. H., Campbell I. D. (2007) Structural basis of integrin activation by talin. Cell 128, 171–182 [PubMed]
51. Li Y., Wei Z., Zhang J., Yang Z., Zhang M. (2014) Structural basis of the binding of Merlin FERM domain to the E3 ubiquitin ligase substrate adaptor DCAF1. J. Biol. Chem. 289, 14674–14681 [PMC free article] [PubMed]
52. Hirano Y., Hatano T., Takahashi A., Toriyama M., Inagaki N., Hakoshima T. (2011) Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain. EMBO J. 30, 2734–2747 [PMC free article] [PubMed]
53. Wei Z., Yan J., Lu Q., Pan L., Zhang M. (2011) Cargo recognition mechanism of myosin X revealed by the structure of its tail MyTH4-FERM tandem in complex with the DCC P3 domain. Proc. Natl. Acad. Sci. U.S.A. 108, 3572–3577 [PubMed]
54. Hamada K., Shimizu T., Matsui T., Tsukita S., Hakoshima T. (2000) Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J. 19, 4449–4462 [PubMed]
55. Aragona M., Panciera T., Manfrin A., Giulitti S., Michielin F., Elvassore N., Dupont S., Piccolo S. (2013) A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 154, 1047–1059 [PubMed]
56. Dupont S., Morsut L., Aragona M., Enzo E., Giulitti S., Cordenonsi M., Zanconato F., Le Digabel J., Forcato M., Bicciato S., Elvassore N., Piccolo S. (2011) Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 [PubMed]
57. Sansores-Garcia L., Bossuyt W., Wada K., Yonemura S., Tao C., Sasaki H., Halder G. (2011) Modulating F-actin organization induces organ growth by affecting the Hippo pathway. EMBO J. 30, 2325–2335 [PubMed]
58. Zhao B., Li L., Wang L., Wang C. Y., Yu J., Guan K. L. (2012) Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev. 26, 54–68 [PubMed]
59. Piccolo S., Dupont S., Cordenonsi M. (2014) The biology of YAP/TAZ: hippo signaling and beyond. Physiol. Rev. 94, 1287–1312 [PubMed]
60. Low B. C., Pan C. Q., Shivashankar G. V., Bershadsky A., Sudol M., Sheetz M. (2014) YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth. FEBS Lett. 588, 2663–2670 [PubMed]

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology