Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain.
The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.
Gallis
B
B
Lewis
A
A
Wignall
J
J
Alpert
A
A
Mochizuki
D Y
DY
Cosman
D
D
Hopp
T
T
Urdal
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004848:Epithelium; D006801:Humans; D007163:Immunosorbent Techniques; D008321:Mammary Glands, Animal; D051379:Mice; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D015375:Receptors, Interleukin-2; D012694:Serine / Q000378:metabolism; D014162:Transfection
D004274:DNA, Recombinant; D010446:Peptide Fragments; D010710:Phosphates; D011971:Receptors, Immunologic; D015375:Receptors, Interleukin-2; D012694:Serine; D004247:DNA; D011493:Protein Kinase C