Stimulus-induced selective association of actin-associated proteins (α-actinin) and protein kinase C isoforms with the cytoskeleton of human neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein a-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal alpha-actinin and actin. Increased association of PKC beta I and beta II with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, alpha-actinin, and PKC beta II induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal alpha-actinin and PKC beta II showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 mu M) completely blocked PMA-induced increases in cytoskeletal alpha-actinin but reduced cytoskeletal recruitment of PHC beta II only by 16%. Higher concentrations of latrunculin A (4 mu M), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKC beta II by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli. (C) 1999 Academic Press.