NEUTROPHILS STIMULATED WITH A VARIETY OF CHEMOATTRACTANTS EXHIBIT RAPID ACTIVATION OF P21-ACTIVATED KINASES (PAKS) - SEPARATE SIGNALS ARE REQUIRED FOR ACTIVATION AND INACTIVATION OF PAKS

Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to r eceptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattr actant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B- 4, interleukin-8, or the chemokine RANTES exhibited a rapid and transi ent activation of the 63- and 69-kDa Paks. These kinases exhibited max imal activation,vith each of these agonists within 15 s followed by si gnificant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activ ation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor, Addition of fMLP to neutrophils already stimulated wit h C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agoni sts that elicited only shape changes and chemotaxis in neutrophils. Wh ile all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the ot her stimuli in triggering activation of the c-Jun N-terminal kinase (J NK) and the p38 mitogen-activated protein kinase (MAPK). These data in dicate that separate signals are required for activation and inactivat ion of Paks and that, in contrast to other cell types, activated Pak d oes not trigger activation of JNK or p38-MAPK in neutrophils. These re sults are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by th e alpha and beta gamma subunits of complex G proteins.