COFILIN UNDERGOES RAPID DEPHOSPHORYLATION IN STIMULATED NEUTROPHILS AND TRANSLOCATES TO RUFFLED MEMBRANES ENRICHED IN PRODUCTS OF THE NADPHOXIDASE COMPLEX - EVIDENCE FOR A NOVEL CYCLE OF PHOSPHORYLATION AND DEPHOSPHORYLATION

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid depho sphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-ph orbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin b y peptide sequencing and immunoblotting with specific antibodies. Evid ence is presented that in neutrophils cofilin is regulated by a contin ual cycle of phosphorylation and dephosphorylation, and that the phosp hatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinas e that participates in these reactions may be a novel enzyme. The kine tics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O-2(-)) rel ease. Immunofluorescent studies revealed that cofilin was present thou roughout the cytosol of resting neutrophils and underwent rapid transl ocation to the F-actin-rich, ruffled membranes of stimulated cells. Cy tochemical analysis further revealed that the ruffled membranes also c ontained large amounts of hydrogen peroxide (H2O2), a product of the O -2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase) . Cofilin is therefore well placed to participate in the continual pol ymerization and depolymerization of F-actin that is thought to give ri se to the oscillatory pattern of H2O2 production observed under certai n conditions.