Characterization of gelsolin truncates that inhibit actin depolymerization by severing activity of gelsolin and cofilin.
Gelsolin is a calcium-activated actin-binding protein with six subdomains. The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A Ca2+-sensitive truncate containing G2-6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2-3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains.
Fujita
H
H
Division of Gene Regulation, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan. h_fujita@med.hokudai.ac.jp
Allen
P G
PG
Janmey
P A
PA
Azuma
T
T
Kwiatkowski
D J
DJ
Stossel
T P
TP
Furu-uchi
K
K
Kuzumaki
N
N
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D018260:Gelsolin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics
D051339:Actin Depolymerizing Factors; D000199:Actins; D018260:Gelsolin; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D002118:Calcium