A rabbit polyclonal antibody raised against myeloid-related protein 8 (MRP-8), a protein of the S100 family, recognized another S100 protein (MRP-14) as well as a protein of 6.5 kDa (p6) in the cytosol of resting neutrophils. p6 was found to be a novel member of the S100 family. It consisted of two isoforms with pI values of 6.2 (the minor form, p6a) and 6.3 (the major form, p6b) and constituted 5% of the total cytosolic proteins. Both isoforms were also demonstrated in the cytosol of monocytes, but not in lymphocytes, as previously shown for MRP-8 and MRP-14. Only the major isoform bound radioactive Ca2+, as also observed for MRP-8, whereas the different variants of MRP-14 were all labelled. On neutrophil activation with opsonized zymosan, a stimulant known to require extracellular Ca2+, 58% of p6a and 42% of p6b was translocated to the membrane. With phorbol 12-myristate 13-acetate, a Ca(2+)-independent stimulant, no translocation was detected. This translocation pattern was similar to that observed with MRP-8 and MRP-14. In addition, p6, MRP-8 and MRP-14 were specifically associated with the cytoskeletal fraction of the membrane. The Ca(2+)-dependent translocation of the novel S100 protein in parallel with MRP-8 and MRP-14 suggests a role for these proteins in regulating the Ca2+ signal to the membrane cytoskeleton and thus in regulating neutrophil activation.
The myeloid-related proteins MRP-14 and MRP-8 and also p6, three calcium-binding proteins of the SlOO 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, A231 87, which induces translocation of the three SlOO proteins, was added after PMA activation. This resulted in translocation of 18 % 2 5 % of phosphorylated MRP-14 and 19 % 2 1 % of only nonphosphorylated MRP-8. However, upon inhibition of PKC, translocation of MRP-14 and MRP-8 was increased up to 38% 1-7% and 34% 2 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.
Nucleoside diphosphate kinase (NDP kinase) catalyses the phosphate transfer between nucleoside triphosphates and nucleoside diphosphates. As formation of guanosine triphosphate could be dependent on ATP in neutrophils, the presence of NDP kinase was tested in these phagocytic cells. Both membrane and cytosolic fractions of human neutrophils were found to contain NDP kinase activity. The specific activity measured in the cytosol appeared 10-fold higher than in the membrane and was not modified when the cells were activated with phorbol 12-myristate 13-acetate. Interestingly, stimulation with N-formylmethionyl leucylphenylalanine in the presence of cytochalasin B showed an increase in membrane NDP kinase activity together with the translocation of the enzyme from the cytosol to the membrane, suggesting a possible role of NDP kinase in regulating G-proteins as previously reported. In addition, activation with opsonized zymosan induced an increase in cytosolic activity, suggesting different regulation depending on the signal transduction pathway. The neutrophil enzyme consisted of two subunits of 21 kDa (NDPKA) and 18 kDa (NDPKB) again essentially present in the cytosol of the cell. Separation of proteins by two-dimensional PAGE demonstrated that each subunit consisted of at least four isoforms, indicating post translational modifications. A characteristic of this family of enzymes is the stability of the phosphorylated intermediate. In neutrophils, only one acidic isoform of each NDPKA and NDPKB was labelled in the presence of EDTA. In addition, non-denatured complexes were apparent between 91 and 130 kDa, suggesting a hexameric structure as was also proposed for NDP kinases from other eukaryotic cells. These complexes were found to differ in their isoelectric points, indicating the existence of various isoenzymes probably resulting from combination between several isoforms of each subunit.
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