Antagonists of calcium fluxes and calmodulin block activation of the p21-activated protein kinases in neutrophils

Neutrophils stimulated with fMLP or a variety of other chemoattractants tha t bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecula r masses of similar to 63 and 69 kDa (gamma- and alpha -Pak). Previous stud ies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks, We now report that a varie ty of structurally distinct compounds which interrupt different stages in c alcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Pa ks in fMLP-stimulated neutrophils. These antagonists included selective inh ibitors of phospholipase C (1-[6-((17 beta -3-methoxyestra-1,3,5(10)-trien- 17-yl)amino)hexyl]-1H-pyrrole- 2,5-dione), the intracellular Ca2+ channel ( 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl) -5chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenes ulfonamide: trifluoperazine), and CaM-activated protein kinases (N- [2-(N-( chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxyben zenesulfonamide). This inhibition was dose-dependent with IC50 values very similar to those that interrupt CaM-dependent reactions in vitro. In contra st, less active analogues of these compounds (1-[6-((17 beta -3-methoxyestr a-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrro- lidinedione; N-(6-aminohexy l)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; pro methazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N- (4-chlorocinnamyl)-N-me thylbenzyl-amine]) did not affect activation of Paks in these cells. CaM an tagonists (N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide: trifluoperaz ine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesu lfonamide; promethazine), were also found to block activation of the small GTPases Pas and Pac in stimulated neutrophils along with the extracellular signal-regulated kinases, These data strongly suggest that the Ca2+/CaM com plex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.