Characterization of phorbol ester-stimulated serine phosphorylation of the human insulin receptor.
Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of the human insulin receptor (IR) was characterized and compared in two cell types of different lineage: normal rat kidney epithelial (NRK) cells and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increased IR beta-subunit phosphorylation to 252 +/- 43 and 25- +/- 47% (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significant differences in the PMA-stimulated phosphorylation of the IR in these two cell types. This phosphorylation of the IR was predominantly located in two tryptic phosphopeptides, and these phosphopeptides were absent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR was immunoprecipitated with an antibody against residues Ser1315 to Lys1329, and this precipitation was blocked with excess unlabelled peptide containing this sequence. Radiosequencing by manual Edman degradation revealed that this tryptic phosphopeptide was phosphorylated at Ser1315. This PMA-stimulated phosphorylation did not inhibit autophosphorylation of the IR in vivo. These results demonstrate that PMA-stimulated phosphorylation of the IR can exhibit significant differences when expressed in different cell types, and that Ser1315 is a major PMA-stimulated phosphorylation site on the human IR.
Feener
E P
EP
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215.
Shiba
T
T
Hu
K Q
KQ
Wilden
P A
PA
White
M F
MF
King
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007668:Kidney; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D014357:Trypsin
D007328:Insulin; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D010768:Phosphoserine; D012694:Serine; D011972:Receptor, Insulin; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate