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

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.

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Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

Author(s):

Christine L. Humphries ◽

Heath I. Balcer ◽

Jessica L. D'Agostino ◽

Barbara Winsor ◽

David G. Drubin ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Complex Activity ◽

Actin Binding Protein ◽

Coiled Coil Domain ◽

Allele Specific ◽

And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

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Molecular cloning of a novel actin-binding protein, p57, with a WD repeat and a leucine zipper motif

FEBS Letters ◽

10.1016/0014-5793(95)00393-n ◽

1995 ◽

Vol 364 (3) ◽

pp. 283-288 ◽

Cited By ~ 72

Author(s):

Kensuke Suzuki ◽

Jun Nishihata ◽

Yuko Arai ◽

Nakayuki Honma ◽

Kazuo Yamamoto ◽

...

Keyword(s):

Molecular Cloning ◽

Leucine Zipper ◽

Binding Protein ◽

Actin Binding ◽

Leucine Zipper Motif ◽

Actin Binding Protein ◽

Wd Repeat

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A Novel Human Actin-Binding Protein Homologue That Binds to Platelet Glycoprotein Ib

Blood ◽

10.1182/blood.v92.4.1268.416k12_1268_1276 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1268-1276 ◽

Cited By ~ 1

Author(s):

Wen-feng Xu ◽

Zhi-wei Xie ◽

Dominic W. Chung ◽

Earl W. Davie

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Protein ◽

Full Length ◽

Actin Binding ◽

Intracellular Domain ◽

Platelet Surface ◽

Full Length Cdna ◽

Actin Binding Protein ◽

Amino Terminal

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIb. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other. © 1998 by The American Society of Hematology.

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IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites

Journal of General Virology ◽

10.1099/vir.0.81270-0 ◽

2005 ◽

Vol 86 (11) ◽

pp. 3031-3038 ◽

Cited By ~ 13

Author(s):

WonKyung Kang ◽

Noriko Imai ◽

Yu Kawasaki ◽

Toshihiro Nagamine ◽

Shogo Matsumoto

Keyword(s):

Bombyx Mori ◽

Fluorescent Protein ◽

Coiled Coil ◽

Terminal Region ◽

Mutant Protein ◽

Yeast Two Hybrid ◽

Focus Formation ◽

Coiled Coil Domain ◽

Nuclear Foci ◽

Two Hybrid

The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1–110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1–78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.

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The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200106089 ◽

2001 ◽

Vol 154 (6) ◽

pp. 1209-1224 ◽

Cited By ~ 178

Author(s):

Åsa E.Y. Engqvist-Goldstein ◽

Robin A. Warren ◽

Michael M. Kessels ◽

James H. Keen ◽

John Heuser ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Live Cells ◽

Cell Cortex ◽

Coated Pits ◽

Actin Binding Protein ◽

Real Time Analysis

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

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A novel Amoeba proteus 120 kDa actin-binding protein with only 1 filamin repeat and a coiled-coil region

Biochemistry and Cell Biology ◽

10.1139/o06-177 ◽

2007 ◽

Vol 85 (1) ◽

pp. 22-31 ◽

Cited By ~ 2

Author(s):

Magdalena Sobczak ◽

Elżbieta Kocik ◽

Maria Jolanta Rędowicz

Keyword(s):

High Probability ◽

Binding Protein ◽

Coiled Coil ◽

Amoeba Proteus ◽

Actin Binding ◽

Protein Dimerization ◽

Reading Frame ◽

Actin Binding Protein ◽

Coiled Coil Region ◽

Very High

A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear–nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ∼95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1–104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209–324), and a central region (residues 344–577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.

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Redox-dependent oligomerization through a leucine zipper motif is essential for MG53-mediated cell membrane repair

AJP Cell Physiology ◽

10.1152/ajpcell.00382.2010 ◽

2011 ◽

Vol 301 (1) ◽

pp. C106-C114 ◽

Cited By ~ 39

Author(s):

Moonsun Hwang ◽

Jae-kyun Ko ◽

Noah Weisleder ◽

Hiroshi Takeshima ◽

Jianjie Ma

Keyword(s):

Cell Membrane ◽

Leucine Zipper ◽

Fluorescent Protein ◽

Coiled Coil ◽

Mechanical Damage ◽

Thiol Group ◽

Membrane Repair ◽

Coiled Coil Domain ◽

Green Fluorescent

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.

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