Hansen, Soren Prag; (2003) Structural and functional analysis of muskelin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Muskelin is an intracellular, kelch-repeat protein that is functionally involved in cell spreading on thrombospondin-1. The aim of this thesis project was to investigate the role of muskelin domains in the subcellular distribution and the regulation of the protein using cellular, biochemical, and bioinformatic approaches. Using bioinformatics to compare muskelin orthologues from Mus musculus, Homo sapiens, Rattus norvegicus, Danio rerio, Drosophila melanogaster, and Anopheles gambiae, highly conserved regions within muskelin were identified, with the combination of a discoidin domain, a LisH motif, a C-terminal to LisH motif, and six kelch repeats. A further bioinformatic analysis of the kelch repeat proteins in whole genomes from Homo sapiens, Drosophila melanogaster, and Anopheles gambiae, demonstrated that muskelin has a unique molecular architecture amongst the large family of kelch repeat proteins. EGFP-tagged forms of muskelin were prepared and used to study its cellular localisation and to explore possible regulatory mechanisms. EGFP-MK had a cytoplasmic distribution and also formed mobile, granular inclusion bodies in 25% of the transfected cells. Formation of the inclusion bodies was a specific property of EGFP-wild-type muskelin, because EGFP alone or EGFP-muskelin mutated at key residues within kelch repeat 4 (Y488A/V495A or G474S/G475S) or mutations in the discoidin domain (K132A/I133A/V134A) were equivalently expressed yet had a uniformly diffuse distribution. Because of the complex domain structure of muskelin, a rational view of domain boundaries has proven essential for successful expression of truncated forms. Truncated forms composed of the discoidin or the kelch repeats alone did not form inclusion bodies. In biochemical pull-down assays, muskelin was found to self-associate by a mechanism dependent on both the discoidin domain and the fourth kelch repeat. Mutations in kelch repeat 4 altered the sensitivity to limited proteolysis by proteinase K, suggesting that the protein conformation is altered. To begin to explore physiological factors that regulate the formation of inclusion bodies, the possible roles of five putative protein kinase C (PKC) phosphorylation sites that are conserved between muskelin species orthologues were evaluated in the cellular and biochemical assays. Activation of PKC decreased the formation of the inclusion bodies and resulted in phosphorylation of muskelin. Using a site-directed mutagenesis approach, two PKC phosphorylation sites critical for this activity were identified. Both of these sites are predicted to lie on the same face of the [beta]-propeller structure composed by the six kelch-repeats. Together, these studies have developed new evidence that muskelin is a multidomain protein with multiple protein-protein interaction sites.

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