Two Regions Responsible for the Actin Binding of p57, a Mammalian Coronin Family Actin-Binding Protein.

Abstract

The actin-binding protein p57, a member of the coronin protein family, is expressed in a variety of immune cells. It has five WD repeats and a coiled-coil motif containing a leucine zipper, both of which are known to mediate protein-protein interactions. In order to identify the precise actin-binding regions in p57, and to assess the contribution of these structural motifs, we prepared various truncated p57 as fusion proteins with glutathione S-transferase (GST) and examined their actin-binding activity. A co-sedimentation assay demonstrated that p57(1-371) (C-terminal truncated p57) had the ability to bind F-actin, but p57(372-461) (a fragment containing the coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p57(1-34)) was found to bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that p57(1-34) was co-localized with F-actin in COS-1 cells after the transfection with the p57(1-34) construct. Deletion of (10)KFRHVF(15), a sequence conserved among coronin-related proteins, from p57(1-34) abolished its actin-binding activity, suggesting that this sequence with basic and hydrophobic amino acids is crucial for p57 to bind to F-actin. However, the N-terminal deletion mutant p57(63-461) retained the binding ability to F-actin. This result suggests the presence of a second actin-binding region. Further deletion analysis revealed that p57(111-204), which includes the second and third WD repeats, also exhibited weak actin-binding activity in the co-sedimentation assay. Taken together, these data strongly suggest that at least two regions within Met-1 to Asp-34 and Ile-111 to Glu-204 of p57 are responsible for its binding to the actin cytoskeleton.

F-actin Binding Region of SPIN90 C-terminus Is Essential for Actin Polymerization and Lamellipodia Formation

“…Other F-actin binding proteins, such as N-WASP, Btk, and p57 have been shown to possess basic and hydrophobic amino acid-enriched regions that interact with corresponding acidic amino acid clusters on actin (19,22,27). Consistent with this, we found that SPIN90 contains a highly hydrophobic and basic region at the end of its C-term (582-722).…”

“…Our results revealed that GST alone and GST-C-term (269-656) failed to co-sediment with Factin, whereas GST-C-terminus (269-722) and GST-C-term (657-722) both bound F-actin ( Figure 2B). These results suggest that the region responsible for SPIN90 binding to F-actin is located at the SPIN90-C-term (657-722), implicating that the basic and hydrophobic amino acids are essential for F-actin binding of SPIN90 (11,19,22,27). When we estimated the dissociation constant (K d ) for a SPIN90-full, -Cterminus (269-722) and -C-term (657-722) with respect to F-actin, we found that the association of the GST-SPIN90-full, -C-terminus (269-722), and -Cterm (657-722) with F-actin became saturated as the F-actin concentration increased.…”

“…From there, we focused on SPIN90-C-terminus (269-722), which contains an Arp2/3 complex binding domain (amino acids 336-407), a G-actin binding domain (amino acids 527-549), and additional sequences of unknown functions. Similar F-actin co-sedimentation assays were performed to avoid overlapping of the band representing GST-SPIN90-C-term (582-722; ∼45kDa) with that of actin (43 kDa), we generated a GSTconstruct including amino acids 657-722 containing a highly basic and hydrophobic region (19,22,27). Our results revealed that GST alone and GST-C-term (269-656) failed to co-sediment with Factin, whereas GST-C-terminus (269-722) and GST-C-term (657-722) both bound F-actin ( Figure 2B).…”

In Vivo and in Vitro Characterization of Novel Neuronal Plasticity Factors Identified following Spinal Cord Injury

“…Coronin 1b is an actin-binding protein, belonging to the Coronin family, and may bind also to tubulin (27). It is important in cytoskeleton remodeling, lamellipodia extensions and mitosis; moreover, Coronin 1b is localized to the cortical cytoskeleton, where it is co-localized with F-actin (36,37). It is known that cytoskeletal organization and remodeling are essential cellular modifications during sprouting and axonal elongation (for review, see Ref.…”

The identification of a new actin-binding region in p57

“…A previous study suggested that two regions within N-terminal and central part of p57 (Met-1 to Asp-34 and Ile-111 to Glu-204) are responsible for actin binding [12]. However, it has been reported that the deletion of the C-terminal 65 amino acids from Xcoronin, a Xenopus homologue of coronin, significantly reduces its actin-binding activity [13].…”