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 calci um-binding proteins of the S100 family, translocate to the membrane du ring human neutrophil activation with stimuli known to require extrace llular calcium for activity. When phorbol 12-myristate 13-acetate (PMA , an extracellular calcium-independent stimulus) is used, no transloca tion is observed. To characterize further the mechanisms involved in t heir translocation, phosphorylation of these proteins was studied. Thr ee isoforms of MRP-14 were markedly phosphorylated in the membrane and in the cytosol upon activation with extracellular calcium-dependent s timuli, such as opsonized zymosan, the calcium ionophore A23187, N-for mylmethionylleucylphenylalanine in the presence of cytochalasin B and arachidonic acid, or upon extracellular calcium-independent stimulatio n (PMA). In no case were p6 and a fourth, more basic isoform of MRP-I4 , phosphorylated. In PMA-activated cells: a phosphorylated acidic isof orm 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 phospho rylation of MRP-8, and decreased cytosolic MRP-14 phosphorylation. To test whether phosphorylated MRP-8 could translocate, A23187, which ind uces translocation of the three S100 proteins, was added after PMA act ivation. This resulted in translocation of 18%+/-5% of phosphorylated MRP-14 and 19%+/-1% of only nonphosphorylated MRP-8. However, upon inh ibition 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 p hosphorylation and/or of PKC in the modulation of MRP-14 and MRP-8 tra nslocation to the membrane.