Products of phosphoinositide specific phospholipase C can trigger dephosphorylation of cofilin in chemoattractant stimulated neutrophils

The signal transduction pathways that trigger dephosphorylation of cofilin in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) were investigated with a phospho-specific antibody that recognized cofilin only when this protein was phosphorylated on ser-3. Unlike earlier studies that monitored changes in P-32-labeled cofilin, this Ab allowed us to monitor changes in the total mass of phosphorylated cofilin during neutrophil stimulation. Neutrophils stimulated with fMLP (1.0 muM) for 1.0 min exhibited a massive loss (> 85%) of phosphate from cofilin, which was blocked by an antagonist of phosphoinositide-specific phospholipase C (PI-PLC) (1.0 muM U73122). Products of PI-PLC, sn-1,2-diglyceride and inositol. (1,4,5)-trisphosphate, are known to activate protein kinase C (PKC) and increase intracellular Ce2+, respectively. Treatment of neutrophils with agents that selectively activate PKC [4beta-phorbol 12-myristate 13-acetate (PMA)] or cellular Ca2+ (ionophore A23187) also triggered dephosphorylation of cofilin. Both a nonspecific (100 nM staurosporine) and a highly selective antagonist of PKC (200 nM bisindolytmaleimide I) blocked dephosphorylation of cofilin in neutrophils stimulated with PMA but not with fMLP or ionophore A23187. The calmodulin (CaM) antagonists trifluoperazine (15 muM) and W-7 (50 muM) blocked dephosphorylation of cofilin in stimulated neutrophils whereas inactive/less-active analogs of these inhibitors (15 muM promethazine, 50 muM W-5) were substantially less effective. Calyculin A (40 nM), an antagonist of type 1 and 2A protein phosphatases, also triggered a massive dephosphorylation of cofilin in unstimulated neutrophils through a pathway that was insensitive to inhibitors of type 2B phosphatases. These data suggest that both PKC-dependent and independent pathways can trigger dephosphorylation of cofilin in neutrophils with the latter pathway predominating in fMLP-stimulated cells. These pathways may also contain CaM and a type 2C and/or novel phosphatase (e.g., slingshot).