Mechanism of Actin Filament Bundling by Fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin cons...

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Bibliographic Details
Published in:The Journal of biological chemistry 2011-08, Vol.286 (34), p.30087-30096
Main Authors: Jansen, Silvia, Collins, Agnieszka, Yang, Changsong, Rebowski, Grzegorz, Svitkina, Tatyana, Dominguez, Roberto
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
ACTIN
Actin Bundling
Actin Cytoskeleton - chemistry
Actin Cytoskeleton - genetics
Actin Cytoskeleton - metabolism
Animals
Anticancer Drug
BASIC BIOLOGICAL SCIENCES
Binding Sites
Carrier Proteins - chemistry
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Line, Tumor
Cell Motility
CRYSTAL STRUCTURE
CRYSTALLOGRAPHY
Crystallography, X-Ray
DESIGN
ELECTRON MICROSCOPY
Fascin
Filopodia
GLYCEROL
Humans
IN VITRO
Mice
Microfilament Proteins - chemistry
Microfilament Proteins - genetics
Microfilament Proteins - metabolism
Molecular Biophysics
MUTAGENESIS
Mutagenesis Mechanisms
Mutation
MUTATIONS
NEOPLASMS
PHOSPHORYLATION
Phosphorylation - genetics
PHOSPHOTRANSFERASES
POLYETHYLENE GLYCOLS
Protein Kinase C - genetics
Protein Kinase C - metabolism
Protein Structure, Tertiary
PROTEINS
Pseudopodia - chemistry
Pseudopodia - genetics
Pseudopodia - metabolism
Pseudopodia - ultrastructure
RESIDUES
RESOLUTION
Structure-Activity Relationship
SYMMETRY
TARGETS
X-ray Crystallography
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Summary:Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M111.251439