Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping.
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The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.Keywords:
Gelsolin
Actin-binding protein
Actinin
Actina
MDia1
Actin remodeling
The oligomeric molecular chaperone CCT is essential for the folding of the highly abundant protein actin, which in its native state forms actin filaments that generate the traction forces required for cell motility. In addition to folding proteins, CCT can provide a platform for protein complex assembly and binds actin filaments assembled in vitro. Some individual subunits of CCT, when monomeric, have been shown to be functionally active, and in particular, the CCTepsilon subunit is involved in the serum response factor pathway that controls actin transcription. Thus, there is a complex interplay between CCT and actin that extends beyond actin folding. CCT has recently been shown to bind gelsolin, an actin filament severing protein that increases actin dynamics by generating filament ends for further actin polymerization. However, the biological significance of the CCT:gelsolin interaction is unknown. Here, using a co-immunoprecipitation assay, we show that CCT binds directly to gelsolin in its calcium-activated, actin-severing conformation. Furthermore, using actin filaments retained from fixed and permeabilized cells, we demonstrate that CCT can inhibit the actin filament severing activity of gelsolin. As our work and that of others shows gelsolin is not folded by CCT, the CCT:gelsolin interaction represents a novel mode of binding where CCT may modulate protein activity. The data presented here reveal an additional level of interplay between CCT and actin mediated via gelsolin, suggesting that CCT may influence processes depending on gelsolin activity, such as cell motility.
Gelsolin
Actin-binding protein
MDia1
Actin remodeling
Chaperone (clinical)
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Gelsolin
MDia1
Actina
Actin-binding protein
Actinin
Actin remodeling
Cite
Citations (0)
Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.
Gelsolin
Actin-binding protein
MDia1
Actin remodeling
Actina
Treadmilling
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Gelsolin is an actin filament-severing and -capping protein which is inhibited by polyphosphoinositides (PPI).Severing requires gelsolin binding to the side of the filaments through a site in segments 2 and 3 (52-3) to position another site in segment 1 (Sl) to sever filaments.In this paper, we report that 52-3, like S1, caps actin filaments.Since neither S1 and 52-3 caps as well as gelsolin, and neither severs actin filament, S2-3 may actively contribute to severing by capping filaments cooperatively with S1.We used deletional mutagenesis to 10cate the S2-3 sequence required for actin filament side binding, capping, and PPI binding and found that these sites are located close to the NH, terminus of 52 (residues 161-172).S3, a segment which has no known function up to now and does not by itself bind actin, contributes to stable capping and may contain an additional PPI-binding site.Actin filament severing provides a rapid mechanism for remodeling the actin cytoskeleton in cells and is a unique biochemical reaction which is not yet completely understood.Gelsolin and some of its homologs are exquisitely efficient actinsevering proteins which also cap actin filaments
Gelsolin
MDia1
Actin-binding protein
Actin remodeling
Actina
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The effect of nonmuscle actin ADP‐ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP‐ribosylation. ADP‐ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends. ADP‐ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin. The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments. The results suggest that nonmuscle actin ADP‐ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.
Gelsolin
MDia1
ADP-ribosylation
Actin-binding protein
Actin remodeling
Treadmilling
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Citations (58)
Gelsolin
Actin-binding protein
MDia1
Actin remodeling
Conformational change
Actina
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Citations (44)
Gelsolin
MDia1
Actin-binding protein
ADP-ribosylation
Actin remodeling
Cite
Citations (15)
Gelsolin
MDia1
Actin remodeling
Actin-binding protein
Actina
Cite
Citations (21)
The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.
Gelsolin
Actin-binding protein
Actinin
Actina
MDia1
Actin remodeling
Cite
Citations (156)