Phosphorylation of myeloid-related proteins MRP-14 and MRP-8 during human neutrophil activation.
The myeloid-related proteins MRP-14 and MRP-8 and also p6, three calcium-binding proteins of the S100 family, translocate to the membrane during human neutrophil activation with stimuli known to require extracellular calcium for activity. When phorbol 12-myristate 13-acetate (PMA, an extracellular calcium-independent stimulus) is used, no translocation is observed. To characterize further the mechanisms involved in their translocation, phosphorylation of these proteins was studied. Three isoforms of MRP-14 were markedly phosphorylated in the membrane and in the cytosol upon activation with extracellular calcium-dependent stimuli, such as opsonized zymosan, the calcium ionophore A23187, N-formylmethionylleucylphenylalanine in the presence of cytochalasin B and arachidonic acid, or upon extracellular calcium-independent stimulation (PMA). In no case were p6 and a fourth, more basic isoform of MRP-14, phosphorylated. In PMA-activated cells, a phosphorylated acidic isoform of MRP-8 was detected in the cytosol only. However, phosphorylated MRP-8 represented only a small fraction of total MRP-8. Cgp 41251, an inhibitor of protein kinase C (PKC), completely inhibited the phosphorylation of MRP-8, and decreased cytosolic MRP-14 phosphorylation. To test whether phosphorylated MRP-8 could translocate, A23187, which induces translocation of the three S100 proteins, was added after PMA activation. This resulted in translocation of 18% +/- 5% of phosphorylated MRP-14 and 19% +/- 1% of only nonphosphorylated MRP-8. However, upon inhibition of PKC, translocation of MRP-14 and MRP-8 was increased up to 38% +/- 7% and 34% +/- 3% respectively. This suggests a putative role of phosphorylation and/or of PKC in the modulation of MRP-14 and MRP-8 translocation to the membrane.
Central Laboratory of Clinical Chemistry, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
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Guignard
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Mauel
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Markert
D000943:Antigens, Differentiation / Q000378:metabolism*; D016718:Arachidonic Acid / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D040501:Calgranulin A; D040502:Calgranulin B; D002462:Cell Membrane / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015151:Immunoblotting; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000378:metabolism; D010766:Phosphorylation; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D040502:Calgranulin B; D016718:Arachidonic Acid; D000001:Calcimycin; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019311:Staurosporine; C059539:midostaurin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium