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

Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan.
Biological & Pharmaceutical Bulletin (Impact Factor: 1.83). 05/2003; 26(4):409-16. DOI: 10.1248/bpb.26.409
Source: PubMed


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.

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Available from: Teruaki Oku, Oct 07, 2015
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    • "Previous studies indicated that full-length CRN2 possesses binding sites for F-actin in the conserved WD40-repeat domain forming the seven-bladed β-propeller659 and in the conserved part of the C-terminal linker region34596061. More specifically, a conserved arginine residue, R30 in CRN1 and R28 in CRN2, which is surface exposed and located within the seventh β-propeller blade3, has turned out to be essential for F-actin binding of both coronin proteins1262. "
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    ABSTRACT: CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration.
    Scientific Reports 01/2012; 2:241. DOI:10.1038/srep00241 · 5.58 Impact Factor
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    • "The extended multiple sequence alignment presented here together with the recently determined crystal structure of murine coronin-1A [17] now allows a reevaluation of previous mutagenesis studies. Truncation studies have shown that the coronin domain, including the β-propeller and its C-terminal extension, is necessary for F-actin binding [30,39]. Mapping the sequence conservation within 13 short coronin members onto the surface of the crystal structure revealed two regions, one formed by blades 1, 6, and 7 and one formed by blades 6 and 7 and a portion of the C-terminal extension, to represent possible actin binding sites [17]. "
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    ABSTRACT: Coronins belong to the superfamily of the eukaryotic-specific WD40-repeat proteins and play a role in several actin-dependent processes like cytokinesis, cell motility, phagocytosis, and vesicular trafficking. Two major types of coronins are known: First, the short coronins consisting of an N-terminal coronin domain, a unique region and a short coiled-coil region, and secondly the tandem coronins comprising two coronin domains. 723 coronin proteins from 358 species have been identified by analyzing the whole-genome assemblies of all available sequenced eukaryotes (March 2011). The organisms analyzed represent most eukaryotic kingdoms but also cover every taxon several times to provide a better statistical sampling. The phylogenetic tree of the coronin domains based on the Bayesian method is in accordance with the most recent grouping of the major kingdoms of the eukaryotes and also with the grouping of more recently separated branches. Based on this "holistic" approach the coronins group into four classes: class-1 (Type I) and class-2 (Type II) are metazoan/choanoflagellate specific classes, class-3 contains the tandem-coronins (Type III), and the new class-4 represents the coronins fused to villin (Type IV). Short coronins from non-metazoans are equally related to class-1 and class-2 coronins and thus remain unclassified. The coronin class distribution suggests that the last common eukaryotic ancestor possessed a single and a tandem-coronin, and most probably a class-4 coronin of which homologs have been identified in Excavata and Opisthokonts although most of these species subsequently lost the class-4 homolog. The most ancient short coronin already contained the trimerization motif in the coiled-coil domain.
    BMC Evolutionary Biology 09/2011; 11(1):268. DOI:10.1186/1471-2148-11-268 · 3.37 Impact Factor
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    • "ms ( as seen in Figure 4a ) . In Figure 4b we show a panel of four modeled Arp2 / 3 proteins in Leishmania where all of them possess the WDR , but no other known binding sites . WDR domains dominantly interfere with coronin function , acting as multi - molecular scaffolding domains by bringing together inter - acting proteins on a single surface ( Oku et al . , 2003 ; Yan et al . , 2005 ) . They are likely to interfere with normal cell function by scavenging interacting proteins away from en - dogenous , full - length coronin . If Arp2 / 3 complex is , in fact , one of those proteins that interact with coronin ( Humphries et al . , 2002 ; Yan et al . , 2005 ) , other portions ( except WDR ) of the "
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    ABSTRACT: Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented.
    Genetics and Molecular Biology 07/2009; 32(3):652-65. DOI:10.1590/S1415-47572009000300033 · 1.20 Impact Factor
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