Elsevier

Experimental Cell Research

Volume 313, Issue 16, 1 October 2007, Pages 3579-3591
Experimental Cell Research

Research Article
Cytolinker cross-talk: Periplakin N-terminus interacts with plectin to regulate keratin organisation and epithelial migration

https://doi.org/10.1016/j.yexcr.2007.07.005Get rights and content

Abstract

Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin–periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.

Introduction

Plakin proteins play an instrumental role in cytoskeletal dynamics and tissue integrity. The members of the plakin family connect intermediate filaments (IFs) to desmosomes and hemidesmosomes. In addition, plakin proteins can interact with all three cytoskeletal networks and link IFs, actin microfilaments and microtubules to each other [1], [2]. The family comprises seven genes, plectin desmoplakin, BPAG-1, envoplakin, periplakin, epiplakin and MACF-1, but alternative splicing increases the variety of cytolinker proteins especially by generating several functionally distinct isoforms of plectin, BPAG-1 and MACF-1 [2], [3]. Analysis of gene targeted mouse models and human inherited diseases underlines the role of plakins in the maintenance of tissue integrity. Desmoplakin is indispensable during embryonic development as desmoplakin null mouse embryos fail to develop beyond day E6.5 [4] Plectin mutations cause another syndrome with both skin and muscle manifestation, epidermolysis bullosa simplex with muscular dystrophy [5], [6]. The epithelial isoform of BPAG-1 is a target for autoantibodies in bullous pemphigoid, an epidermal blistering disease. In addition, autoantibodies against several plakin proteins, including envoplakin and periplakin, are found in patients with paraneoplastic pemphigus [7], [8].

Notably, plakins do not merely act as passive connectors between IFs and desmosomes but, as shown by knock-out and knock-down experiments, actively regulate the organisation of IF cytoskeleton. Epiplakin, a unique family member comprising solely from globular plectin repeat domains and intervening linkers, has recently been implicated in organisation of intermediate filament networks. Knock-down of epiplakin in simple epithelial cells results in a cell-specific disruption of keratin and vimentin networks [9]. We have demonstrated that periplakin participates in the organisation of keratin filaments to thick cables parallel to scratch wound edges of MCF-7 simple epithelial cells [10]. Moreover, periplakin and desmoplakin dynamics differ at wound edge cells [10]. Keratin organisation and dynamics are also compromised in plectin-null keratinocytes [11]. Intriguingly, ablation of periplakin slowed down MCF-7 epithelial migration [10] whereas the migration of keratinocyte sheets was accelerated in the absence of plectin [11].

An important molecular interaction of plakins is binding to intermediate filaments mediated by the C-terminal plectin repeat domain of plakins. Elucidation of the crystal structure of desmoplakin IF binding domain defined the plectin repeat as a 38-residue-long α-hairpin that is followed by two antiparallel α-helices [12]. The plectin repeats fold to globular domains separated by linker sequence with 4.5 or 5 repeats per domain [12], [13]. Periplakin C-terminus has no globular domains but a conserved linker domain with similarity to two plectin repeats that form the linker next to the fifth globular domain in plectin C-terminus [14]. This linker sequence alone is sufficient for periplakin binding to intermediate filaments [15], [16], [17].

The structures of plakin N-terminal domains, apart from the calponin-homology actin-binding domains, are less well characterised. A recent study of the crystal structure of a fragment of BPAG1 N-terminus has revealed a structure consisting of two pairs of spectrin repeats that flank a putative SH3 domain [18]. Interactions between desmoplakin N-terminal and desmosomal proteins such as plakophilins, plakoglobin and desmosomal cadherins have been characterised in detail (see e.g. [19] for a review). Likewise, interaction between plectin and α4 integrin in hemidesmosomes is well understood [20]. However, much less is known about interacting partners and targeting of periplakin and envoplakin. Presently, only one N-terminal periplakin-interacting partner, kazrin, has been identified. Kazrin is a previously uncharacterised protein that is targeted to keratinocyte plasma membranes [21]. Thus, the mechanisms that regulate periplakin localisation and function are not yet fully resolved.

In order to characterise the molecular interactions of periplakin N-terminus we performed co-immunoprecipitation experiments on a stably transfected cell line expressing HA-tagged periplakin N-terminus. In this report, we identify plectin as a periplakin-interacting protein and show that ablation of plectin compromises subcellular distribution of periplakin in HaCaT keratinocytes. These data suggest a mechanism for plakin function via interactions between different family members.

Section snippets

Cell culture and transfections

Human mammary adenocarcinoma cells (MCF-7) and HaCaT keratinocytes were maintained in Dulbecco's minimal essential media (DMEM Sigma, UK) supplemented with 10% foetal calf serum and 5% GPS (l-glutamine, glucose, penicillin, streptomycin). Generation of stably transfected cell lines expressing the pP1/2N construct [15] and scratch wound assays to monitor collective epithelial migration were carried out as described before [10]. siRNA transfections with Oligofectamine reagent were carried out as

Generation of stably transfected MCF-7 cell clone expressing periplakin N-terminus

We generated a stably transfected subclone of MCF-7 breast adenocarcinoma cell line, MCF-C6-PPL, expressing HA-tagged periplakin N-terminus, p1/2N construct [15] comprising the subdomains NN, Z, Y and X [14], to identify proteins that regulate targeting of periplakin to different membrane and cytoplasmic locations. This domain was chosen because earlier immunofluorescence studies had demonstrated that periplakin N-terminus shows the same subcellular distribution as the whole protein in cultured

Discussion

We have shown in this paper that the N-terminal ‘plakin-box’ domain of periplakin is found in protein complexes containing plectin, another member of plakin family of cytoskeletal linker proteins. We identified plectin as an interaction partner for periplakin in a proteomic screen of protein complexes co-immunoprecipitated from MCF-7 cells expressing HA-tagged periplakin N-terminus. The interaction was confirmed by co-immunoprecipitation of full-length endogenous proteins and by co-localisation

Acknowledgments

We would like to thank Ms Joanne Robson and Dr. William Simon for helping in MALDI-TOF analysis. This study was supported by British Skin Foundation, BBSRC and Cancer Research UK.

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