Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C.
Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain.
D000595:Amino Acid Sequence; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011990:Receptors, Transferrin; D012694:Serine; D013379:Substrate Specificity; D014168:Transferrin / Q000378:metabolism*
D010446:Peptide Fragments; D010455:Peptides; D010748:Phosphopeptides; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; D012694:Serine; D011493:Protein Kinase C