Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region

Abstract

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.

The identification of a new actin-binding region in p57

“…The results from western blotting revealed that cross-linking of p57 by DSS led to the appearance of a dimer band (46 kDa, the monomer is 23 kDa) (Figure 4A), which was consistent with the report of Oku et al [8] since no trimer or other higher-molecular-weight band was observed. However, in the p57 297-461/Mut sample, no dimer band was detected ( Figure 4A).…”

“…The leucine zipper domain is closely related to the dimerization (or oligomerization) of p57 molecule [8,9]. Therefore, we suppose that the dimerization (or oligomerization) of the p57 297-461 monomers may lead to the conformational change in the region surrounding the actin-binding site, which results in the presence of the actin-binding activity.…”

“…Since p57 297-461 has the actin-binding activity and is a homodimer through the leucine zipper domain [8], its activity to cross-link actin filaments was further investigated. p57 297-461 without GST tag was used to cross-link pre-assembled actin filaments and a low speed co-sedimentation assay was utilized to test the effect of cross-linking [23].…”

“…The Nterminal extension of p57 is composed of approximately 70 amino acid residues conserved in most mammalian coronin-like proteins. The C-terminal extension contains a leucine zipper domain, which evolved from a coiled coil of the coronin-like protein family and was reported to mediate the homotypic dimerization or trimerization of p57 [7][8][9].…”

Actin-binding Proteins Coronin-1a and IBA-1 are Effective Microglial Markers for Immunohistochemistry

“…Recently, gene knockout of coronin-1a in murine T cells leads to reduced ability to respond to chemotactic gradients and also increased susceptibility to apoptotic stimuli (Foger et al 2006). Although the relationship between the actin cytoskeleton and coronin-1a is incompletely understood, these properties have lead researchers to believe that coronin-1a is involved in modulating rearrangement of the actin cytoskeleton during immune-specific functions (Oku et al 2005).…”