Identification of critical functional and regulatory domains in gelsolin.
Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11-amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin-severing proteins.
Kwiatkowski
D J
DJ
Hematology-Oncology Unit, Massachusetts General Hospital, Boston.
Janmey
P A
PA
Yin
H L
HL
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D018260:Gelsolin; D005796:Genes; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D021382:Protein Sorting Signals; D004247:DNA; D002118:Calcium