Role of plectin as a cytolinker and docking site of signaling complexes. Molecular and structural analyses

Research project P 14520 Structural and functional analysis of plectin Gerhard WICHE 26.6.2000 Plectin is a widely expressed protein of very large size that has all the attributes of a multifunctional crosslinking and organizing element of the cytoskeleton. It displays a multi-domain structure, versatile binding activities, and subcellular localizations that enable it to strengthen cells against mechanical stress forces. In fact, hereditary gene defects in plectin cause epiderm-olysis bullosa simplex (EBS)-MD, a severe human skin blistering disease with muscular dys-trophy and symptoms of neurodegeneration. Recent studies indicated an unprecedented variety of tissue-specific plectin isoforms with structurally different amino-termini generated by differential splicing. The major objectives of the proposed project are: (i) Molecular characterization of plectin isoform-mediated interactions at the cytoskeleton-plasma membrane interface, including known plectin-binding proteins, such as cytoskeletal IF and microfilament proteins (vimentin, actin), and transmembrane, subplasma membrane skeleton, and junctional complex-associated proteins (integrins alpha.7 and beta4, beta-dystroglycan, spectrin, utrophin, alpha-actinin). (ii) Biochemical assessment of the role of plectin as scaffolding and docking site for proteins involved in cellular signaling, such as the ubiquitous cytoplasmic tyrosine kinases Fer and RACKI. (iii) Three-dimensional analysis of functionally important plectin subdomains, including plectin`s multifunctional amino-terminal actin-binding domain and its carboxy-terminal repeat domains. The experimental approach is based primarily on the expression of recombinant proteins, corresponding to various subdomains of plectin isoforms and of its interacting partners, in bacteria (or in baculovirus-infected insect cells), and the structure-function analysis of these proteins and their complexes, using a combination of biochemical, biophysical, crystallographic, and cell biological techniques. In addition, the yeast-two-hybrid system will be used as a primary tool to identify new interacting proteins. This should enable a comprehensive biochemical analysis of plectin`s proposed role as multi-functional cytolinker and scaffolding protein and provide new insights into the structure and molecular mechanics of the cytoskeleton- membrane interface.

Plectin is the prototype of an emerging family of versatile cytoskeletal linker proteins which are widespread and generally of very large size (>500 kDa). In both humans and mice, plectin has been shown to play an important role in stabilizing cells against mechanical stress, and its deficiency leads to severe skin blistering, muscular dystrophy, and neurodegeneration. In this project we studied molecular properties and functions of plectin on the biochemical level. We focused first on a versatile protein docking site of plectin which forms a bridge to intermediate filaments, the most robust cytoskeletal filament system existing in cells. We found that structural elements known as cysteine-bridges are stabilizing this domain and using computer modeling we predicted their positions within a 3-dimensional molecular model. We then expressed and purified plectin`s actin binding domain (ABD), an equally important part of the molecule, crystallized it, and deduced its atomic structure by X-ray crystallography. In the course of this work we made the discovery that alternative, very short sequences preceding this domain (generated by differential splicing of the gene) determine fate and function of the rest of the molecule even within the same cell. This opened interesting new perspectives on how cells manage to diversify and fine-tune varied functions of cytolinker proteins, a subject that will be further explored and followed up in future studies. Based on another set of data from this project we developed and strengthened a new hypothesis regarding cytolinker functions that go beyond their widely accepted role as mechanical stabilizers of cells. We found that due to its large surface area, multi-domain structure, and versatile binding activities, plectin serves as a scaffolding platform and docking site for signaling complexes. We demonstrated this for the cytoplasmic tyrosine kinase Fer, the PKC-activating protein RACK1-protein kinase C complex, and muscle AMP-kinase, the key regulatory enzyme of energy homeostasis. Furthermore, one of the variants of plectin was found to bind to an ubiquitine ligase involved in protein degradation. Not only serves plectin as a docking site for these enzymes, but as shown in 3 cases, it affects also their signaling activities. Thus cytolinkers such as plectin may be a missing link, long looked for, in cytoplasmic signaling pathways. We call this concept the platform hypothesis and it should be interesting and a challenging task to assess its validity in future research.