Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association

Abstract

Muskelin is an intracellular protein with a C-terminal kelch-repeat domain that was initially characterized as having functional involvement in cell spreading on the extracellular matrix glycoprotein thrombospondin-1. As one approach to understanding the functional properties of muskelin, we have combined bioinformatic and biochemical studies. Through analysis of a new dataset of eight animal muskelins, we showed that the N-terminal region of the polypeptide corresponds to a predicted discoidin-like domain. This domain architecture is conserved in fungal muskelins and reveals a structural parallel between the muskelins and certain extracellular fungal galactose oxidases, although the phylogeny of the two groups appears distinct. In view of the fact that a number of kelch-repeat proteins have been shown to self-associate, co-immunoprecipitation, protein pull-down assays and studies of cellular localization were carried out with wild-type, deletion mutant and point mutant muskelins to investigate the roles of the discoidin-like and kelch-repeat domains. We obtained evidence for cis- and trans-interactions between the two domains. These studies provide evidence that muskelin self-associates through a head-to-tail mechanism involving the discoidin-like domain.

RanBPM, Muskelin, p48EMLP, p44CTLH, and the armadillo-repeat proteins ARMC8α and ARMC8β are components of the CTLH complex

“…Muskelin associated with p48EMLP, Twa1, p44CTLH, and ARMC8α. Self-association of Muskelin, which was characterized in a previous report (Prag et al, 2004), was also confirmed in this assay (Fig. 3A, lane 3).…”

“…We identified these proteins by searches of the NCBI's non-redundant protein sequence database. The 78-kDa band was identified as Muskelin (Adams et al, 1998;Prag et al, 2004), which was previously shown to associate with RanBPM in a yeast two-hybrid system (Umeda et al, 2003). The 62-kDa band was a novel protein, ARMC8α.…”

A Specific Interaction between Muskelin and the Cyclin-dependent Kinase 5 Activator p39 Promotes Peripheral Localization of Muskelin

“…8A). This localization was seen in all cells examined (26 of 26 cells imaged) and resembles the reported localization of muskelin in other cell types (23,24). Cells that were transfected with p39 alone showed p39 accumulation in the nucleus if expression levels were high.…”

“…Kelch domains are structural repeats first observed in the Drosophila actin cross-linking protein Kelch that permit proteins to fold into a cylindrical, "␤-propeller structure" (22). Although several kelch domain proteins are also actin-binding proteins (22), muskelin does not bind directly to either actin or tubulin (23). It is, however, associated with the actin cytoskeleton and has multiple effects on cell adhesion and cytoskeletal structure in adherent cells (24).…”

Adipocyte Enhancer-binding Protein-1 Promotes Macrophage Inflammatory Responsiveness by Up-Regulating NF-κB via IκBα Negative Regulation

“…Our findings suggest that AEBP1 physically interacts with IB␣ via its DLD, which is known be a protein-protein interaction mediator (Johnson et al, 1993;Prag et al, 2004). Indeed, DLD of AEBP1 was found to be required for protein-protein interaction between AEBP1 and MAPK .…”