Activation of polymorphonuclear leukocytes (PMN)by chemotactic peptides initiates a series of functional responses that serve to eliminate pathogens. The intermediate steps that link engagement of the chemoattractant receptor to the microbicidal responses involve protein kinases that have yet to be identified. In this study we detected in human PMN the presence of p38 mitogenactivated protein kinase (MAPK), which became rapidly tyrosine phosphorylated and activated in response to the chemotactic peptide N-formyl-methionyl-leucylphenylalanine (fMLP). Pretreatment of PMN with wortmannin, a phosphatidylinositol 3-kinase inhibitor, or bis-indolylmaleimide, a protein kinase C antagonist, resulted in partial inhibition of p38 phosphorylation upon fMLP stimulation. Similarly, phosphorylation of p38 was only partially inhibited when the fMLP-induced cytosolic calcium transient was prevented. Stimulation of PMN by the chemoattractant also resulted in the rapid phosphorylation and activation of MAPK-activated protein kinase-2 (MAPKAPK-2), which was completely inhibited by the specific p38 inhibitor, SB203580. The physical interaction of p38 with MAPKAPK-2 was studied by coimmunoprecipitation. These two kinases were found to be associated in unstimulated PMN but dissociated upon activation of the cells by fMLP. Together these findings demonstrate the activation of p38 by chemotactic peptides in human PMN by a process involving phosphatidylinositol 3-kinase, protein kinase C, and calcium. p38, in turn, is an upstream activator of MAPKAPK-2. Polymorphonuclear leukocytes (PMN)1 respond rapidly to invading microorganisms or tissue injury by activation of numerous effectors, including the generation of superoxide anions, secretion of lytic enzymes, and phagocytosis of particles. These responses serve to neutralize and destroy the invading pathogens (1). The recruitment of PMN to sites of bacterial infection and their subsequent activation are initiated by binding of chemoattractants to specific cell surface receptors, which are coupled to heterotrimeric G proteins (2). Receptor engagement triggers a complex cascade of biochemical events which culminates in the activation of the microbicidal responses. Many of these intervening steps have yet to be defined. Increased phosphorylation of several proteins has been found to correlate with the stimulation of PMN effectors, thereby suggesting a causal role in the activation process (3-6). Additional support for a central role of phosphorylation was provided by the finding that pharmacological agents that interfere with protein kinases and phosphatases are also potent modulators of PMN responsiveness (7-10). Indeed, there is abundant evidence that protein kinase C isoforms are essential to the microbicidal response (11,12), and activation of tyrosine phosphorylation seems to be equally important (7, 9). Studies in PMN stimulated with the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) led to the identification of Erk-1 and Erk-2 as major targets of tyrosine phosphoryl...
The ubiquitous isoform of the Na ؉ /H ؉ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59 fgr and p56/59 hck were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.
The distribution of cytosolic phospholipase A, (cPLA,), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA, and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A231 87, both proteins accumulated primarily in nucleicontaining fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5-lipoxygenase and cPLA, were localized in a fraction that contained nuclear membranes. In comparison, 5-LAP was localized to the nuclearmembrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffinity labeling. In agreement with the immunoblot data, A231 87 stimulation markedly enhanced 5-lipoxygenase enzymatic activity in the nuclear-membrane fraction, which was accompanied by decreased cytosolic 5-lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA,, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state.
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