Regular article
PACE-1, a novel protein that interacts with the C-terminal domain of ezrin

https://doi.org/10.1016/S0014-4827(02)00054-XGet rights and content

Abstract

The ERM proteins (ezrin, radixin, moesin) together with merlin comprise a subgroup of the band 4.1 superfamily. These proteins act as membrane cytoskeletal linker proteins mediating interactions between the cytoplasmic domains of transmembrane proteins and actin. To better understand how the ERM proteins function to regulate these junctional complexes, a yeast 2-hybrid screen was undertaken using ezrin as a bait. We describe here the identification and cloning of a novel protein, PACE-1, which binds to the C-terminal domain of ezrin. Characterization of PACE-1 in human breast cancer cell lines demonstrates it to have two distinct intracellular localizations. A proportion of the protein is associated with the cytoplasmic face of the Golgi apparatus. This distribution is dependent upon the presence of the PACE-1 N-terminal myristoylation consensus sequence but is not dependent on an association with ezrin. In contrast, PACE-1 colocalises with ezrin in the lamellipodia, where ezrin has a role in cell spreading and motility. A notable feature of PACE-1 is the presence of a putative N-terminal kinase domain; however, in biochemical assays PACE-1 was shown to have associated rather than intrinsic kinase activity. Together these data suggest that PACE-1 may play a role in regulating cell adhesion/migration complexes in migrating cells.

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.

References (53)

  • M.D. Resh

    Fatty acylation of proteinsnew insights into membrane targeting of myristoylated and palmitoylated proteins

    Biochim. Biophys. Acta.

    (1999)
  • J.F. Hancock et al.

    A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane

    Cell.

    (1990)
  • S. McLaughlin et al.

    The myristoyl-electrostatic switcha modulator of reversible protein-membrane interactions

    Trends Biochem. Sci.

    (1995)
  • G.G. Borisy et al.

    Actin machinerypushing the envelope

    Curr. Opin. Cell Biol.

    (2000)
  • R.D. Mullins

    How WASP-family proteins and the Arp2/3 complex convert intracellular signals into cytoskeletal structures

    Curr. Opin. Cell Biol.

    (2000)
  • C.D. Nobes et al.

    Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia

    Cell

    (1995)
  • K. Takahashi et al.

    Direct interaction of the Rho GDP dissociationinhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein

    J. Biol Chem.

    (1997)
  • S.F. Pietromonaco et al.

    Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence

    J. Biol. Chem.

    (1998)
  • R.F. Lamb et al.

    Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts

    Curr. Biol.

    (1997)
  • A. Bretscher et al.

    ERM proteins and merlinintegrators at the cell cortex

    Nat. Rev. Mol. Cell Biol.

    (2002)
  • S. Yonemura et al.

    Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2

    J. Cell Biol.

    (1998)
  • R. Gary et al.

    Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site

    Mol. Biol. Cell.

    (1995)
  • M. Berryman et al.

    Ezrin oligomers are major cytoskeletal components of placental microvillia proposal for their involvement in cortical morphogenesis

    J. Cell Biol.

    (1995)
  • A. Bretscher et al.

    Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains

    Biochemistry

    (1995)
  • T. Matsui et al.

    Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association

    J. Cell Biol.

    (1998)
  • S. Yonemura et al.

    Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteinsan essential role for polyphosphoinositides in vivo

    J. Cell Sci.

    (2002)
  • Cited by (17)

    • SCYL3, as a novel binding partner and regulator of ROCK2, promotes hepatocellular carcinoma progression

      2023, JHEP Reports
      Citation Excerpt :

      SCY1-like pseudokinase 3 (SCYL3) (accession number BC014662) is located on chromosome 1q24.2 and encodes a protein of 110 kDa that has a kinase domain and four HEAT repeats. SCYL3 is also known as protein-associating with the carboxyl-terminal domain of ezrin (PACE-1) because it physically interacts with the C-terminal domain of ezrin.5 Currently, only two reports have discussed the possible functional role of SCYL3.

    • A comprehensive screening method for investigating the potential binding targets of doxorubicin based on protein microarray

      2021, European Journal of Pharmacology
      Citation Excerpt :

      The 401 candidate proteins include adhesion proteins (CDH6(Gugnoni et al., 2017)) and integrin proteins (ITGBL1(Li et al., 2015)) related to cell adhesion. Ubiquitination-related protein GGA3(Parachoniak et al., 2011), cell adhesion regulator protein SCYL3(Sullivan et al., 2003), and protein phosphorylase PPM1F(Luo et al., 2015) are all related to cell migration and are doxorubicin-binding proteins with SNR values greater than 10. Cell migration-related proteins may be potential targets of doxorubicin worthy of further study.

    • Co-translational processing of glycoprotein 3 from equine arteritis virus: N-Glycosylation adjacent to the signal peptide prevents cleavage

      2013, Journal of Biological Chemistry
      Citation Excerpt :

      It was proposed recently that signal anchors insert head first into the translocon and subsequently but still at the translocon rotate 180° to expose the N terminus to the cytoplasm (33). To facilitate this inversion, the N terminus should contain a net charge of +3 to fulfill the “positive inside rule” (34). However, the signal peptide of Gp3 has only one positively charged amino acid, which is only even present in around one-third of all Gp3 sequences.

    View all citing articles on Scopus
    View full text