INTRODUCTION Among the diverse forms of the plaque-bearing adhering junctions (for a review, see Schmidt et al., 1994) the desmosomes are characterized by their molecular composition and by their specific anchorage of bundles of intermediate filaments (IFs; Schwarz et al., 1990; Kowalczyk et al., 1999a). They represent clusters of isoforms of two types of transmembrane glycoproteins, the desmogleins (Dsg1-3) and desmocollins (Dsc1-3), both members of the larger family of cadherins. In their carboxy-terminal, cytoplasmic domains, these desmosomal cadherins assemble the common plaque protein, plakoglobin (Cowin et al., 1986; Franke et al., 1987a,b, 1989; Fouquet et al., 1992), and a distinct set of desmosomal plaque proteins. These include general desmosomal proteins such as desmoplakin I (DPI) and cell type-specific proteins such as desmoplakin II (DPII; Franke et al., 1982; Mueller and Franke, 1983; Cowin et al., 1985) as well as members of the plakophilin (PKP) subfamily of arm-repeat proteins (PKP1: Kapprell et al., 1988, 1990; Hatzfeld et al., 1994; Heid et al., 1994; Schmidt et al., 1997; PKP2: Mertens et al., 1996, 1999; PKP3: Bonné et al., 1999; Schmidt et al., 1999; for a review see Hatzfeld, 1999). In addition, some other less-well-studied proteins are found within the desmosomal plaque (e.g. Tsukita and Tsukita, 1985; Schwarz et al., 1990; Hatzfeld and Nachtsheim, 1996; Kowalczyk et al., 1999a). The basic protein PKP1a (‘band 6 protein’, Kapprell et al., 1988), known to bind cytokeratins (CKs) in vitro (Kapprell et al., 1988; Hatzfeld et al., 1994; Smith and Fuchs, 1998), has been identified on the basis of its amino acid (aa) sequence, together with its splice variant PKP1b, as a member of a large family of proteins characterized by variable numbers of so- called arm-repeats comprising a motif of a mean number of 42 aa (Schäfer et al., 1993; Hatzfeld et al., 1994; Heid et al., 1994; Schmidt et al., 1994). This arm-repeat motif, first identified in the developmentally defined gene armadillo of Drosophila (Peifer and Wieschaus, 1990; Peifer et al., 1994), has been found in more than a dozen other junctional plaque and nuclear proteins, including plakoglobin (Franke et al., 1989) and β- catenin (McCrea et al., 1991). 2471 Journal of Cell Science 113, 2471-2483 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS4693 Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm- repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several μm. 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 experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound – and does not bind – to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques. Key words: Plakophilin, Desmoplakin, Cytokeratin, Intermediate filament, Desmosome, Junction SUMMARY Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis Ilse Hofmann 1, *, Claudia Mertens 1 , Monika Brettel 1 , Volker Nimmrich 1,‡ , Martina Schnölzer 2 and Harald Herrmann 1 1 Division of Cell Biology/A0100 and 2 Protein Analysis Facility/R0800, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany *Author for correspondence (e-mail: i.hofmann@dkfz-heidelberg.de) ‡ Present address: Suny, Downstate Medical Center, New York, USA Accepted 19 April; published on WWW 14 June 2000