Activation induces dephosphorylation of cofilin and its translocation to plasma membranes in neutrophil-like differentiated HL-60 cells.
We suggested that a cytosolic 21-kDa phosphoprotein played an important role in opsonized zymosan-trigered activation of superoxide-generating enzyme in neutrophil-like HL-60 cells through dephosphorylation (Suzuki, K., Yamaguchi, T., Oshizawa, T., Yamamoto, Y., Nishimaki-Mogami, T., Hayakawa, T., and Takahashi, A (1995) Biochim. Biophys. Acta 1266, 261-267). In the present study, we characterized the phosphoprotein and studied changes in it localization upon activation of phagocytes. The 21-kDa phosphoprotein was rapidly dephosphorylated upon activation not only wit opsonized zymosan but also with formyl-Met-Leu-Phe and arachidonic acid. The peptide fragments derived from the 21-kDa phosphoprotein were found to have the same amino acid sequences as those of cofilin, an actin-binding protein. The phosphoprotein reacted exclusively with anti-cofilin antibody on two dimensional immunoblots. Accordingly, together with its apparent molecular weight, isoelectric point, and detection of phosphoserine as a phosphoamino acid, we concluded that the 21-kDa phosphoprotein was a phosphorylated form of cofilin. The amount of cofilin in membranous fractions was increased upon activation. Furthermore, confocal laser scanning microscopy showed that cofilin existed diffusely in the cytosol and nuclear region of the resting cells, while in the activated cells, it was accumulated at the plasma membrane area, forming ruffles or endocytic vesicles on which O2.- should be produced. These results suggested that in resting cells cofilin exists as a soluble phosphoprotein in the cytosol and nuclei, while upon stimulation a large portion of cofilin is dephosphorylated and translocated to the plasma membrane regions.
Suzuki
K
K
National Institute of Health Sciences, Tokyo, Japan.
Yamaguchi
T
T
Tanaka
T
T
Kawanishi
T
T
Nishimaki-Mogami
T
T
Yamamoto
K
K
Tsuji
T
T
Irimura
T
T
Hayakawa
T
T
Takahashi
A
A
D051339:Actin Depolymerizing Factors; D000595:Amino Acid Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins