Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function

Abstract

Activation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca 2؉ /calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34 ؉ -derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca 2؉ [Ca 2؉ ] i . To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK 297-321 ) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK 297-321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK 297-321 in a dosedependent manner. Furthermore, fMLPinduced neutrophil migration on albumin-coated surfaces was perturbed, as well as ␤2-integrin-mediated adhesion. IntroductionHuman granulocytes, which include neutrophils and eosinophils, play an important role in host defense against invading pathogens. 1 During induction of inflammatory reactions, granulocytes in the peripheral blood become primed or preactivated, leave the bloodstream by diapedesis through the endothelium, and migrate toward the site of inflammation. 2 At the inflammatory site, granulocytes become highly activated, secrete proteolytic enzymes by degranulation, and form cytotoxic reactive oxygen species (ROS) to eliminate invading microorganisms. 3 These granulocyte effector functions are initiated by inflammatory mediators, such as chemoattractants, which activate the G-protein-coupled receptors (GPCRs). One of the signaling events that occurs after ligand binding to GPCRs, is the elevation of intracellular Ca 2ϩ ([Ca 2ϩ ] i ). Ligand binding to GPCRs initially activates trimeric G-proteins and subsequent dissociation of the G␣ and G␤␥ subunits. The dissociation of the G␤␥ subunit results in activation of phosphatidylinosytol-3-OH kinase (PI3K) and phospholipase C␤ (PLC␤). PLC␤ activation in turn leads to the hydrolysis of phosphatidylinositol4,5phosphate (PIP 2 ), producing diacylglycerol (DAG) and inositol-3 phosphate (IP 3 ). 4 Subsequently, IP 3 binds to its receptor on the Ca 2ϩ stores, resulting in Ca 2ϩ release into the cytoplasm. Elevation of [Ca 2ϩ ] i in granulocytes has been observed during activation of several granulocyte effector functions, for example in N-formyl-methionylleucyl-phenylalanine (fMLP)-induced respira...

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…These results contrast with those initially reported by Verploegen et al, where continuous CKLiK expression was identified in G-CSF-induced umbilical cord blood-derived CD34 + cells [13]. However, later studies identified low CKLiK protein expression in human peripheral blood despite high level CKLiK activity in Ca 2+ /calmodulin-stimulated cells [14]. Furthermore, both CKLiK and CaMKKα gene expression levels initially increase but then diminish during ATRA-induced differentiation of human promyelocytic NB4 cells (see Fig.…”

“…The proposed antagonistic role of CKLiK during neutrophil differentiation seems to conflict with recently identified positive roles for CKLiK in neutrophil functions, including chemotaxis and the respiratory burst [14]. Furthermore, the calmodulin inhibitor W7 disrupts these same neutrophil functions, and our data demonstrate that the respiratory burst is inhibited by KN-93 ( Fig.…”

“…Studies by our and Dr. Paul Coffer's laboratories have shown that i) both CaMKKα and the CaMKI homologue, CaM kinase I-like Kinase (CKLiK), are upregulated during neutrophil development, ii) CKLiK activity is enhanced by CaMKKα in vitro, and iii) CKLiK activity is required for certain neutrophil functional responses [12][13][14]. However, other studies have demonstrated that CaMK inhibitors KN-93 and KN-62 can induce the differentiation of multiple leukemic myeloid cells (e.g.…”

Inhibitory effect of plant phenolics on fMLP-induced intracellular calcium rise and chemiluminescence of human polymorphonuclear leukocytes and their chemotactic activityin vitro

“…This inhibitory effect involved among other things, a diminished tyrosine phosphorylation of phospholipase CG2 (Wright et al, 2010). Quercetin can also bind to calmodulin (Nishino et al, 1984), a multifunctional calcium-dependent signal transducer that in human granulocytes activates various kinases and is responsible for the modulation of their functions (Verploegen et al, 2005). Moreover, quercetin, in the same manner as wortmannin (a specific inhibitor of phosphoinositide-3-kinase), modulated the function of human basophils (Chirumbolo et al, 2010).…”

Pharmacogenetics for T2DM and Anti-Diabetic Drugs

“…CDC123 gene encodes a protein involved in cell cycle regulation and nutritional control of gene transcription [23,49]. CAMK1D regulates granulocyte function [50], it is also possible that a causative variant in this region is related to CAMK1D and affects pancreatic -cell function through increased apoptosis. SNP rs12779790 is found associated with T2DM in several studies.…”