Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils

Abstract

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca²⁺ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105 kDa protein (Rel-1) requires Ca²⁺-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10⁻⁹ to 10⁻⁶ M), becoming maximal after 30 s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca²⁺ but chelation negated the effects, including the cytosolic Ca²⁺ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca²⁺ decreased the fMLP-mediated Ca²⁺ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca²⁺. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca²⁺-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.

Introduction

Neutrophils (PMNs) are an essential part of the host defense against pathogenic organisms. PMNs accomplish microbicidal functions through adhesion to and diapedesis through the vascular endothelium, chemotaxis to the site of infection, phagocytosis of invading organisms into a phagolysosome, and production and release of both superoxide anion and toxic, granular proteins into the phagolysosome [1], [2], [3]. Chemoattractants, especially formyl-Met-Leu-Phe (fMLP), a component of bacterial cell walls, functions to both attract PMNs to the site of infection and, at higher concentrations, activate the microbicidal arsenal to eradicate pathogens [4], [5], [6].

In addition to the rapid activation of tyrosine kinases during transmembrane signaling, most physiologic chemoattractants, including fMLP, elicit activation of protein kinases and cause rapid increases in cytosolic Ca²⁺ concentration through the release of Ca²⁺ from intracellular stores and the influx of Ca²⁺ through receptor-linked channels [7], [8], [9], [10], [11]. Increases in cytosolic Ca²⁺ are vital for normal PMN functions, including oxidase assembly, chemotaxis, and degranulation [12]; furthermore, chelation of cytosolic Ca²⁺ results in the inhibition of these PMN functions [7], [8], [9], [11], [12].

NFκB is a heterodimeric transcription regulatory factor that is important in immune and inflammatory responses of many different cell types, including neutrophils [13], [14], [15], [16]. The inactive form of NFκB is found mainly in the cytoplasm as either its precursor, a 105 kDa protein (Rel-1), or as part of the IκBα complex, composed of the subunits p50 and p65 [13], [14], [17], [18], [19]. When cells are stimulated with LPS, TNF-α, fMLP, or other inflammatory mediators, the inactive forms of NFκB go through proteolytic degradation and ubquitination. The C-terminal region of the p105 precursor is phosphorylated on its serine residues by IKKs, which leads to ubquitination and degradation [17], [18], [19], [20], [21], [22]. The degradation of the p105 releases the p50 subunit, where it then complexes with a p65 subunit, forming NFκB1, and is translocated to the nucleus. The second process that occurs is the degradation of the IκB complex which holds the NFκB1 heterodimer inactive in the cytoplasm. The IκB complex is also phosphorylated on its serine residues, polyubquinated, and degraded to release the active NFκB heterodimer, which is then translocated to the nucleus [17], [18], [19], [20], [21], [22]. Serine phosphorylation of both p105 and IκB are necessary events in the activation of NFκB/p50, but so is the tyrosine phosphorylation process [23]. The p105 protein has been shown to be a tyrosine kinase substrate: when tyrosine phosphorylation is inhibited by genistein or other protein tyrosine kinase inhibitors, the activation of NFκB is inhibited [23], [24]. Moreover, when PMNs were stimulated with ionomycin there was an increase in p38 MAPK phosphorylation and a phosphorylation of a 105 kDa protein that was inhibited by SB203580, a p38 MAPK inhibitor [25].

The following study describes fMLP-mediated tyrosine phosphorylation of a 105 kDa protein identified by immunoprecipitation as Rel-1 in human PMNs. This phosphorylation was rapid, concentration- and Ca²⁺-dependent, and pertussis toxin (Ptx) sensitive. Also, the phosphorylation of the 105 kDa/Rel-1 protein was inhibited by pretreatment of PMNs with an inhibitor of p38 MAP kinase (MAPK) activity; therefore, Rel-1 is activated through the MAPK signaling pathway.