Regulation of p21rac activation in human neutrophils

The small guanosine triphosphate (GTPase) p21rac is highly expressed in hum an neutrophils where it is thought to play a role in cytoskeletal reorganiz ation and superoxide production. Using the p21rac binding domain of PAK (PA K-RBD) as an activation-specific probe, we have investigated agonist-stimul ated activation of p21rac. Stimulation of neutrophils with the chemoattract ants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an ext remely rapid and transient p21rac activation, being optimal within 5 second s. This activation correlates with the rapid changes of intracellular free Ca2+ ([Ca2+](i)) stimulated by fMLP; however, changes in [Ca2+](i) were nei ther sufficient nor required for p21rac activation. Furthermore, fMLP-induc ed p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, o r phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-m acrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alph a did not cause p21rac activation or modulate fMLP-induced p21rac activatio n. AlF-, a potent activator of heterotrimeric G-protein cu-subunits, howeve r, was found to activate p21rac. Stimulation of neutrophils with phorbol my ristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, act ivate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca2+](i), tyrosine phosphorylation, or PI3K. (C) 1999 by The American Society of Hematology.