Phorbol ester- and Ca2+-dependent phosphorylation of human red cell membrane skeletal proteins.
The addition of the tumor promoting phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to intact human red blood cells activates protein kinase C and stimulates the phosphorylation of the membrane skeletal proteins band 4.1 and band 4.9 as well as two proteins of molecular mass 115 and 110 kDa. We show that 12-O-tetradecanoyl phorbol 13-acetate promotes the association of cytosolic protein kinase C with the red cell membrane and that the enzyme is present on ghost membranes but is largely absent from inside-out vesicles. We show that micromolar Ca2+ added to ghosts also promotes the phosphorylation of band 4.1 and the approximately 100-kDa proteins, a reaction which has not been described previously. Digestion and extraction studies show that the 100-kDa proteins are unrelated to band 3 since they are absent from NaOH stripped membranes, but are found in Triton-prepared cytoskeletons. Digestion of intact red cells with chymotrypsin or neuraminidase, which attack principally band 3 and glycophorin, respectively, markedly inhibits protein kinase C phosphorylation of band 4.1 in red cells and ghosts and of the 100-kDa proteins in ghosts. These enzymes have no effect upon the activity of the Ca2+-activated phosphorylation reaction, suggesting that it does not involve protein kinase C. These results shed light on two phosphorylation reactions which act exclusively on red cell membrane skeletal proteins. Our findings suggest that digestion of the integral membrane proteins band 3 and glycophorin, the principal targets of external protease digestion, affects the activity or specificity of protein kinase C. Finally we have described two apparently novel approximately 100-kDa phosphorylated proteins which are components of Triton-prepared red cell membrane skeletons.
C M
CM
Cohen
S F
SF
Foley
D002118:Calcium / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D008970:Molecular Weight; D010704:Phorbols / Q000494:pharmacology*; D010710:Phosphates / Q000097:blood; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D008565:Membrane Proteins; D010704:Phorbols; D010710:Phosphates; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium