Open AccessFunctions of [His321]Gelsolin Isolated from a Flat Revertant of ras ‐Transformed Cells
Author(s) -
Fujita Hisakazu,
Laham Lorraine E.,
Janmey Paul A.,
Kwiatkowski David J.,
Stossel Thomas P.,
Banno Yoshiko,
Nozawa Yoshinori,
Müllauer Leonhard,
Ishizaki Akira,
Kuzumaki Noboru
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.0615j.x
Subject(s) - gelsolin , phosphatidylinositol , phosphatidylinositol 4,5 bisphosphate , biology , wild type , actin , mutant , biochemistry , phospholipase c , intracellular , microbiology and biotechnology , kinase , enzyme , gene
A mutant gelsolin, [His321]gelsolin, was isolated from R1, a flat revertant of human activated c‐Ha‐ ras oncogene‐transformed NIH/3T3 cells (EJ‐NIH/3T3) produced by ethylmethanesulfonate treatment. [His321]Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ‐NIH/3T3 cells when it is constitutively expressed [Müllauer, L., Fujita, H., Ishizaki, A. & Kuzumaki, N. (1993) Oncogene 8 , 2531–2536]. To investigate the biochemical consequences of the amino acid substitution of His321, we expressed the [His321]gelsolin and wild‐type gelsolin in Escherichia coli , purified them, and analyzed their effects on actin, polyphosphoinositol lipids and phospholipase C. [His321]Gelsolin has decreased actin‐filament‐severing activity and increased nucleating activity compared with wild‐type gelsolin in vitro. Furthermore, compared to wild‐type gelsolin both nucleation and severing by [His321]gelsolin are inhibited more strongly by the phosphoinositol lipids phosphatidylinositol 4‐phosphate (PtdIns P ) and phosphatidylinositol 4,5‐bisphosphate (PtdIns P 2 ). In addition, [His321]gelsolin inhibits PtdIns P 2 hydrolysis by phospholipase Cγ1 more strongly than wild‐type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Gelsolin has six homologous amino acid repeats called S1–S6. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin‐binding domains are in segments 1, 2, and 4–6, and that the region around the residue 321 may contain a phosphoinositol‐lipid‐binding site. Altered functions of [His321]gelsolin might be important for the loss of tumorigenicity of the ras ‐transformed cells.