Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9 and other membrane proteins.

Protein kinase C (PK-C) was demonstrated in human erythrocytes using the exogenous substrate histone H1. The enzyme was dependent on the simultaneous presence of micromolar Ca2+ and phosphatidylserine. The Ca2+ requirement was reduced in the presence of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Activity was normally recovered in cytoplasmic extracts, but treatment of intact cells with TPA (EC50 = 40 nM) prior to lysis caused a rapid translocation of activity from the cytoplasm to the membrane. Following translocation, PK-C activity in the isolated membrane was independent of Ca2+ and phosphatidylserine and could not be removed by manipulating ionic strength. Ghosts from TPA-treated cells showed a marked increase in the phosphorylation of five proteins (termed PK-C-1-5) of Mr 120,000, 110,000, 80,000, 78,000, and 49,000. Addition of purified bovine brain PK-C to ghosts from untreated cells resulted in the phosphorylation of the same five proteins. PK-C-3/4 corresponded to Band 4.1 and PK-C-5 to Band 4.9. Both proteins were isolated from ghosts and shown to be substrates for PK-C in vitro. PK-C-1 and -2 appear to be minor peripheral membrane proteins as both were released from the membrane by alkaline solutions. Incubation of 32P-prelabeled erythrocytes with TPA (EC50 = 40 nM) also resulted in a dose-dependent phosphorylation of PK-C-1-5. These results suggest that PK-C may play an important role in erythrocyte membrane function.