Functions of [His321]gelsolin isolated from a flat revertant of ras-transformed cells.
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 (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). In addition, [His321]gelsolin inhibits PtdInsP2 hydrolysis by phospholipase C gamma 1 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.
Fujita
H
H
Laboratory of Molecular Genetics, Hokkaido University School of Medicine, Sapporo, Japan.
Laham
L E
LE
Janmey
P A
PA
Kwiatkowski
D J
DJ
Stossel
T P
TP
Banno
Y
Y
Nozawa
Y
Y
Müllauer
L
L
Ishizaki
A
A
Kuzumaki
N
N
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D018260:Gelsolin / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D015870:Gene Expression; D006639:Histidine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015689:Oncogene Protein p21(ras) / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism
D000199:Actins; D017931:DNA Primers; D018260:Gelsolin; D010716:Phosphatidylinositols; D006639:Histidine; D010738:Type C Phospholipases; D015689:Oncogene Protein p21(ras)