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. 1993 Dec 15;296(Pt 3):675–683. doi: 10.1042/bj2960675

Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: possible role of protein kinase M.

Z Al 1, C M Cohen 1
PMCID: PMC1137750  PMID: 8280066

Abstract

Human erythrocytes contain cytosolic protein kinase C (PKC) which, when activated by phorbol 12-myristate 13-acetate (PMA), induces the phosphorylation of the membrane skeletal proteins band 4.1, band 4.9 and adducin. We found that brief treatments of erythrocytes with PMA resulted in a decrease in cytosolic PKC content and in the transient appearance in the cytosol of a Ca(2+)- and phospholipid-independent 55 kDa fragment of PKC, called PKM. Prolonged treatment with PMA resulted in the complete and irreversible loss of erythrocyte PKC. To investigate the possible role of calpain in this process, the calpain inhibitors leupeptin and E-64 were sealed inside erythrocytes by reversible haemolysis. Both inhibitors prolonged the lifetime of PKC in PMA-treated cells, and leupeptin was shown to block the PMA-stimulated appearance of PKM in the cytosol. Significantly, leupeptin also completely blocked PMA-stimulated phosphorylation of membrane and cytosolic substrates. This effect was mimicked by other calpain inhibitors (MDL-28170 and calpain inhibitor I), but did not occur when other protease inhibitors such as phenylmethanesulphonyl fluoride, pepstatin A or chymostatin were used. In addition, the phosphorylation of exogenous histone sealed inside erythrocytes was also blocked by leupeptin. Immunoblotting showed that leupeptin did not prevent the PMA-induced translocation of PKC to the erythrocyte membrane. Thus inhibition of PKC phosphorylation of membrane skeletal proteins by calpain inhibitors was not due to inhibition of PKC translocation to the membrane. Our results suggest that PMA treatment of erythrocytes results in the translocation of PKC to the plasma membrane, followed by calpain-mediated cleavage of PKC to PKM. This cleavage, or some other leupeptin-inhibitable process, is a necessary step for the phosphorylation of membrane skeletal substrates, suggesting that the short-lived PKM may be responsible for membrane skeletal phosphorylation. Our results suggest a potential mechanism whereby erythrocyte PKC may be subject to continual down-regulation during the lifespan of the erythrocyte due to repeated activation events, possibly related to transient Ca2+ influx. Such down-regulation may play an important role in erythrocyte survival or pathophysiology.

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