FIMBRIN LOCALIZED TO AN INSOLUBLE CYTOSKELETAL FRACTION IS CONSTITUTIVELY PHOSPHORYLATED ON ITS HEADPIECE DOMAIN IN ADHERENT MACROPHAGES

被引:58
作者
MESSIER, JM
SHAW, LM
CHAFEL, M
MATSUDAIRA, P
MERCURIO, AM
机构
[1] HARVARD UNIV,DEACONESS HOSP,SCH MED,CANC BIOL LAB,50 BINNEY ST,BOSTON,MA 02115
[2] HARVARD UNIV,SCH MED,COMMITTEE CELL & DEV BIOL,BOSTON,MA 02115
[3] MIT,WHITEHEAD INST BIOMED RES,CAMBRIDGE,MA 02139
[4] MIT,DEPT BIOL,CAMBRIDGE,MA 02139
来源
CELL MOTILITY AND THE CYTOSKELETON | 1993年 / 25卷 / 03期
关键词
ACTIN-BUNDLING PROTEIN; PHOSPHORYLATION; MACROPHAGE FRACTIONS;
D O I
10.1002/cm.970250303
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
The actin-bundling protein fimbrin is homologous to l-plastin, a 65 kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069-1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton-X-100 (soluble fraction), Tween 40-deoxycholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone-extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC-extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. MetaboliC (PO4)-P-32 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10 kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins.
引用
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页码:223 / 233
页数:11
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[41]   IMMUNOCYTOCHEMICAL DISTRIBUTION OF EXTRACELLULAR-MATRIX RECEPTORS IN HUMAN OSTEOCLASTS - A BETA-3 INTEGRIN IS COLOCALIZED WITH VINCULIN AND TALIN IN THE PODOSOMES OF OSTEOCLASTOMA GIANT-CELLS [J].
ZAMBONINZALLONE, A ;
TETI, A ;
GRANO, M ;
RUBINACCI, A ;
ABBADINI, M ;
GABOLI, M ;
MARCHISIO, PC .
EXPERIMENTAL CELL RESEARCH, 1989, 182 (02) :645-652