ACTIVATION INDUCES DEPHOSPHORYLATION OF COFILIN AND ITS TRANSLOCATIONTO PLASMA-MEMBRANES IN NEUTROPHIL-LIKE DIFFERENTIATED HL-60 CELLS

We suggested that a cytosolic 21-kDa phosphoprotein played an importan t role in opsonized zymosan-triggered activation of superoxide-generat ing enzyme in neutrophil-like HL-60 cells through dephosphorylation (S uzubi, K., Yamaguchi, T., Oshizawa, T., Yamamoto, Y., Nishimaki-Mogami , T., Hayakawa, T., and Takahashi, A (1995) Biochim, Biophys, Acta 126 6, 261-267). In the present study, we characterized the phosphoprotein and studied changes in its localization upon activation of phagocytes . The 21-kDa phosphoprotein was rapidly dephosphorylated upon activati on not only with opsonized zymosan but also with formyl-Met-Leu-Phe an d arachidonic acid. The peptide fragments derived from the 21-kDa phos phoprotein were found to have the same amino acid sequences as those o f cofilin, an actin-binding protein. The phosphoprotein reacted exclus ively with anti cofilin antibody on two dimensional immunoblots. Accor dingly, 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. T he amount of cofilin in membranous fractions was increased upon activa tion. Furthermore, confocal laser scanning microscopy showed that cofi lin 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 O-2(rad ical) (anion) should be produced. These results suggested that in rest ing cells cofilin exists as a soluble phosphoprotein in the cytosol an d nuclei, while upon stimulation a large portion of cofilin is dephosp horylated and translocated to the plasma membrane regions.