Journal of Cell Science 113, 2471-2483 (2000) 2471 Printed in Great Britain © The Company of Biologists Limited 2000 JCS4693 Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis Ilse Hofmann1,*, Claudia Mertens1, Monika Brettel1, Volker Nimmrich1,‡, Martina Schnölzer2 and Harald Herrmann1 1Division of Cell Biology/A0100 and 2Protein Analysis Facility/R0800, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany *Author for correspondence (e-mail: [email protected]) ‡Present address: Suny, Downstate Medical Center, New York, USA Accepted 19 April; published on WWW 14 June 2000 SUMMARY Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm- proteins is saturable at an approximately equimolar ratio. repeat protein family. They are both constitutively In extracts from HaCaT cells, distinct soluble complexes expressed in most vertebrate cells, in two splice forms containing PKP1a and desmoplakin I (DPI) have been named a and b, and display a remarkable dual location: identified by co-immunoprecipitation and sucrose density they occur in the nuclei of cells and, in epithelial cells, at fractionation. The significance of these interactions of the plasma membrane within the desmosomal plaques. We PKP1a with IF proteins on the one hand and desmoplakin have shown by solid phase-binding assays that both PKP1a on the other is discussed in relation to the fact that PKP1a and PKP2a bind to intermediate filament (IF) proteins, in is not bound – and does not bind – to extended IFs in vivo. particular to cytokeratins (CKs) from epidermal as well as We postulate that (1) effective cellular regulatory simple epithelial cells and, to some extent, to vimentin. In mechanisms exist that prevent plakophilins from line with this we show that recombinant PKP1a binds unscheduled IF-binding, and (2) specific desmoplakin strongly to IFs assembled in vitro from CKs 8/18, 5/14, interactions with either PKP1, PKP2 or PKP3, or vimentin or desmin and integrates them into thick (up to combinations thereof, are involved in the selective 120 nm in diameter) IF bundles extending for several µm. recruitment of plakophilins to the desmosomal plaques. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation Key words: Plakophilin, Desmoplakin, Cytokeratin, Intermediate experiments. In particular, the binding of PKP1a to IF filament, Desmosome, Junction INTRODUCTION 1994; Schmidt et al., 1997; PKP2: Mertens et al., 1996, 1999; PKP3: Bonné et al., 1999; Schmidt et al., 1999; for a review Among the diverse forms of the plaque-bearing adhering see Hatzfeld, 1999). In addition, some other less-well-studied junctions (for a review, see Schmidt et al., 1994) the proteins are found within the desmosomal plaque (e.g. Tsukita desmosomes are characterized by their molecular composition and Tsukita, 1985; Schwarz et al., 1990; Hatzfeld and and by their specific anchorage of bundles of intermediate Nachtsheim, 1996; Kowalczyk et al., 1999a). filaments (IFs; Schwarz et al., 1990; Kowalczyk et al., 1999a). The basic protein PKP1a (‘band 6 protein’, Kapprell et al., They represent clusters of isoforms of two types of 1988), known to bind cytokeratins (CKs) in vitro (Kapprell et transmembrane glycoproteins, the desmogleins (Dsg1-3) and al., 1988; Hatzfeld et al., 1994; Smith and Fuchs, 1998), has desmocollins (Dsc1-3), both members of the larger family of been identified on the basis of its amino acid (aa) sequence, cadherins. In their carboxy-terminal, cytoplasmic domains, together with its splice variant PKP1b, as a member of a large these desmosomal cadherins assemble the common plaque family of proteins characterized by variable numbers of so- protein, plakoglobin (Cowin et al., 1986; Franke et al., 1987a,b, called arm-repeats comprising a motif of a mean number of 42 1989; Fouquet et al., 1992), and a distinct set of desmosomal aa (Schäfer et al., 1993; Hatzfeld et al., 1994; Heid et al., 1994; plaque proteins. These include general desmosomal proteins Schmidt et al., 1994). This arm-repeat motif, first identified in such as desmoplakin I (DPI) and cell type-specific proteins the developmentally defined gene armadillo of Drosophila such as desmoplakin II (DPII; Franke et al., 1982; Mueller and (Peifer and Wieschaus, 1990; Peifer et al., 1994), has been Franke, 1983; Cowin et al., 1985) as well as members of the found in more than a dozen other junctional plaque and nuclear plakophilin (PKP) subfamily of arm-repeat proteins (PKP1: proteins, including plakoglobin (Franke et al., 1989) and β- Kapprell et al., 1988, 1990; Hatzfeld et al., 1994; Heid et al., catenin (McCrea et al., 1991). 2472 I. Hofmann and others The plakophilins (PKP1-PKP3) are remarkable as they occur restriction sites and appropriate oligonucleotide pairs to introduce stop constitutively in the nucleoplasm of cells normally forming codons or start codons, respectively. desmosomes, cells induced to form desmosomes and cells cDNAs coding for the complete CK5 and CK14, respectively, were devoid of desmosomes (e.g. Mertens et al., 1996; Schmidt isolated from a human epidermis cDNA library (Clontech, Palo Alto, ′ 32 et al., 1997; Bonné et al., 1999). In certain states of CA, USA; 5 -Stretch Plus, HL 1112b) using P-labelled partial differentiation, plakophilins are recruited to the plasma clones of human CK5 and 14 (provided by L. Langbein; Langbein et al., 1993) employing standard procedures (Sambrook et al., 1989). membrane in a cell type-specific manner, targeted to very Both clones were mutagenized to include their start codons as part of specific ensembles of plaque proteins where specific IF unique NdeI sites, and were subsequently cloned into the prokaryotic proteins are inserted. PKP1a, originally isolated from bovine expression vector pET-21b (Novagen, Madison, WI, USA). muzzle epithelium as ‘band-6-protein’ under harsh extractive conditions, has been found primarily in desmosomes of Protein purification stratified and complex epithelia (Franke et al., 1983; Kapprell Total human vimentin (Herrmann et al., 1993), the human vimentin et al., 1988, 1990; Hatzfeld et al., 1994; Heid et al., 1994; rod domain (Rogers et al., 1995), mouse desmin (Rogers et al., 1995), Schmidt et al., 1997). By contrast, PKP2, also present in total CK8 and CK18 (Hofmann and Franke, 1997), the CK8 rod and two splice forms designated a and b, is characteristic of the CK18 rod (Bader et al., 1991), all subcloned into the pDS5 desmosomes of one-layered (‘simple’) epithelia and certain plasmid, were introduced into E. coli, strain TG1. Human vimentin, mouse desmin (Li et al., 1994) and the mouse desmin rod domain non-epithelial, desmosome-possessing tissues such as were purified according to Hofmann et al. (1991), and the human myocardium, but has also been localized to certain complex vimentin rod domain according to Herrmann et al. (1996). CK5, and stratified epithelia where it colocalizes with PKP1a and/or CK14, CK8, CK18, CK8 rod and CK18 rod were prepared as PKP3 (e.g. Mertens et al., 1996, 1999). PKP3 has been found described (Coulombe and Fuchs, 1990; Hofmann and Franke, 1997). in desmosomes of both simple and stratified epithelia but not E. coli strain BL21 was transformed with plasmids containing the in hepatocytes and in myocardium (Bonné et al., 1999; cDNA of human PKP1a, various subdomains derived from PKP1a, Schmidt et al., 1999). neurofilament protein NF-L (Heins et al., 1993) and lamin LIII (Stick, Desmoplakins DPI and DPII, the latter being a splice 1988), subcloned into the pET-vector system. For the generation of variant of DPI (Green et al., 1990; Virata et al., 1992), together recombinant proteins, transformed bacteria were grown overnight in with envoplakin, periplakin, plectin and bullous pemphigoid 400 ml TB medium at 37°C under rigorous shaking. PKP1a and truncated versions of PKP1a were enriched in inclusion body fractions antigen 1 (BPAG1), have been grouped into a distinct protein and purified as described (Hofmann et al., 1991). The inclusion body family, the plakins (Ruhrberg and Watt, 1997). All these fraction was dissolved in 40 ml of column buffer I (8 M urea, 5 mM proteins have been localized, in one or the other cell type, to Tris-HCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, pH 7.5) and certain IFs and their plasma membrane anchorage sites and centrifuged for 90 minutes in a Beckman Ti 50 rotor at 35,000 rpm share a common domain structure, i.e. a central rod domain (Beckman Instruments, München, Germany). The supernatant was flanked by globular amino- and carboxy-terminal domains. directly applied to a 30 ml DEAE-sepharose column in column buffer Given their expression pattern, abundance, and the absence of I. The proteins PKP1a, PKP1a-N268, PKP1a-N353 and PKP1a- a homologous protein in actin-anchoring cell junctions, DPI N331his were recovered in the flow-through fraction and were directly and DPII have always been prime candidates for a role in the applied to a 15 ml CM-sepharose column equilibrated in column specific connections between desmosomes and IFs. buffer I. Bound protein was eluted in a 50 ml gradient of NaCl (0-0.3 M) with column buffer I. Peak fractions were monitored by SDS- Correspondingly, transient transfection studies using cDNA PAGE and purified protein was pooled and stored at −80°C until use. constructs encoding truncated DPI have suggested that the NF-L bound to the DEAE-sepharose column was eluted in a 50 ml carboxy-terminal desmoplakin domain interacts with IFs gradient of NaCl (0-0.3 M) in column buffer I. It did not, however, (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; bind to CM-sepharose in column buffer I. Therefore fractions eluted Bornslaeger et al., 1996). This concept has been confirmed by from the DEAE-sepharose column were dialzyed into column buffer in vitro studies using CKs and the recombinant carboxy- II (8 M urea, 30 mM sodium formate, 1 mM EDTA, 1 mM EGTA, 1 terminal desmoplakin domain (Kouklis et al., 1994), as well mM DTT, pH 4.0) and subsequently applied to a CM-sepharose as by observations made in yeast two-hybrid experiments column equilibrated in column buffer II.