Regular articlePACE-1, a novel protein that interacts with the C-terminal domain of ezrin
Introduction
Adhesion receptors play an essential role in regulating cell shape, cell adhesion, cell motility, and responses to the extracellular environment. Accumulating evidence points to the importance in these events of four related proteins: ezrin, radixin, moesin, and their more distant relative, merlin, which together comprise the ERM subclass of the FERM (band 4.1, ezrin-radixin-moesin) superfamily [1], [2]. All four proteins play a role in linking transmembrane glycoproteins to the cytoskeleton, and may provide this function directly, such as the complex among CD44, ezrin, and actin [3], [4] or indirectly, as exemplified for the cystic fibrosis transmembrane conductance regulator (CFTR), which interacts with ezrin through a PDZ-containing protein ezrin/moesin/radixin-binding phosphoprotein 50 (EBP50) [5]. However, such interactions not only provide a structural link but can also serve as a point of regulation to mediate cellular changes in response to both extracellular and intracellular cues [reviewed in [2], [6], [7]].
Structurally, ERM proteins are composed of three conserved protein domains (Fig. 1A): (i) the N-terminal or FERM homology domain, which contains binding sites for transmembrane receptors, scaffolding and signalling molecules; (ii) the central α-helical domain, which in ezrin, radixin, and merlin is followed by a short polyproline stretch; and (iii) the C-terminal region, which in ezrin, radixin, and moesin terminates in 34 amino acids that function as the major F-actin binding site. The N-terminal and C-terminal regions in ERMs, also known as the N-ERMAD and C-ERMAD (association domains) [8], mediate self-association and this interaction masks many of the binding sites, including those for transmembrane proteins and actin, resulting in an inactive conformation. In support of this conformational model of ERM regulation, the cytoplasmic pool of ezrin has been shown to exist mainly as closed monomers [9], [10] with activation requiring dissociation of the ERMAD domains. At least in part, this activation is mediated by phosphorylation of a conserved threonine residue in the C-terminal region of ERMs (T567, T558, and T564 in ezrin, radixin, and moesin, respectively), which suppresses the interaction between ERMAD domains [11]. In addition, the involvement of lipids has also been invoked by in vitro assays of ERM activation [12] and also for the in vivo functions of ERMs [13]. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has been shown to bind to the FERM domain of ezrin [14] and mutation of the PtdIns(4,5)P2 binding site in the ezrin FERM domain results in an altered cellular distribution of the protein [15]. However, recent findings also point to the existence of both phosphorylation-dependent and -independent mechanisms of ERM activation [13], thereby highlighting the complexity of these regulatory events.
A major challenge in the ERM field is to understand how the proposed structural functions of ERMs integrate with signalling pathways, and in turn, how these interactions may coordinate the assembly of complex cellular structures such as microvilli and lamellipodia [2]. A better understanding of the composition of molecular complexes formed with ERM proteins may greatly facilitate knowledge in this area. To identify new components of ERM–molecular complexes we undertook a yeast 2-hybrid library screen using human ezrin as bait and report here on the identification of a novel ezrin-interacting protein, PACE-1.
Section snippets
Yeast 2-hybrid analysis
Human ezrin cDNA in the pGEM-4Z vector [16] was excised with NcoI, end-filled with Klenow polymerase, and inserted into the SmaI site of the yeast 2-hybrid bait vector pGBT9 (Clontech, Oxford, UK). The resulting construct generates a fusion protein of the GAL4 DNA binding domain in frame with human ezrin lacking the first 10 amino acids, pGBT9/ezrin(11–585). This construct was used in conjunction with a human kidney Matchmaker cDNA library in the pGAD10 vector (Clontech) to screen for ezrin
Screening for novel proteins associating with ezrin
In a yeast 2-hybrid strategy to identify novel ezrin-associating proteins, the pGBT9/ezrin(11–585) construct (Fig. 1B) was used as bait to screen a pGAD10 human kidney cDNA library. All His-positive/β-galactosidase-positive clones identified were subjected to second round screening without the bait construct and in a different yeast strain to eliminate auto-activating inserts and false positive interactions, respectively. After second round screening 46 positive clones were N-terminal sequenced
Pace-1 is a novel ezrin binding molecule
Numerous interacting proteins have been described for ezrin and other members of the ERM family. These proteins can be broadly categorised into transmembrane receptors including adhesion molecules, signalling molecules, scaffolding molecules, actin and related proteins, and other ERM proteins. However, apart from the interaction with actin and spectrin (and the intra- and inter-molecular association between the C-ERMAD with the N-ERMAD domains of ERMs) most of the ERM–protein interactions
Acknowledgements
The authors thank our colleagues who have contributed reagents essential to this study. We also thank Tony Hunter (The Salk Institute, San Diego) and Axel Knebel (University of Dundee) for valuable advice concerning the kinase domain of PACE-1. This work was supported by funding from the BBSRC, Breakthrough Breast Cancer Research, and Cancer Research UK.
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