Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

Abstract

Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of ∼63 and 69 kDa (γ- and α-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17β-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)-5-chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC50 values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca2+/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.

UVA Induces Ser381 Phosphorylation of p90RSK/MAPKAP-K1 via ERK and JNK Pathways

“…We provide evidence that a selective p38 kinase inhibitor, SB202190, and DNM-p38 had no effect on p90 RSK Ser 381 phosphorylation and its kinase activity induced by UVA. Although the studies of Lian et al (46) and Horstmann et al (67) suggested that the p38 kinase inhibitor-sensitive pathway was involved in activation of S6 kinases, our data suggest that UVA-induced Ser 381 phosphorylation may not be dependent on p38 kinase. The discrepancy may be related to cell type and the kind of stimuli.…”

Ligation of Cell Surface-Associated Glucose-Regulated Protein 78 by Receptor-Recognized Forms of α2-Macroglobulin: Activation of p21-Activated Protein Kinase-2-Dependent Signaling in Murine Peritoneal Macrophages

“…Increased intracellular calcium is a ubiquitous second messenger of downstream signaling. Heterotrimeric G proteins can activate Ras via phosphatidylinositol-specific phospholipase C and Ca 2ϩ /calmodulin complex (61)(62)(63). Inhibition of intracellular calcium channels, Ca 2ϩ /calmodulin complex, and Ca 2ϩ /calmodulin kinase blocked PAK activation in fMLP-stimulated neutrophils (62).…”

p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases

“…31). Preparation of phosphorylated GST-Pak 3 and in vitro dephosphorylation of this protein catalyzed by PP 1, PP 2A, PP 2B, and PP 2C.…”