Posttranslational processing of proenkephalin in AtT-20 cells: evidence for cleavage at a Lys-Lys site.
Proteolytic processing of proenkephalin was examined in several subclones of AtT-20 cells stably transfected with rat proenkephalin cDNA (AT/PE cells). Proenkephalin is synthesized in both N-glycosylated and unglycosylated forms, as demonstrated by treatment with tunicamycin. RIAs and Western blot studies showed that AT/PE clones process proenkephalin at some, but not all, Lys-Arg sequences in a limited processing profile reminiscent of bovine adrenal chromaffin cells. Pulse-chase studies using Met5-enkephalin-Arg-Gly-Leu antiserum demonstrated that 50% of the precursor is processed within 1 h, and processing is complete after 2.5 h with the production of the 5.3-kilodalton (kDa) peptide. Further cleavage to the octapeptide Met5-enkephalin-Arg-Gly-Leu is minimal. Radiosequencing results verified the efficient cleavage of a Lys-Lys site within proenkephalin that resulted in the production of the 5.3-kDa peptide. Proenkephalin cleavage products stored within cells, which included the 5.3-kDa peptide, could be released upon stimulation of cells with BaCl2 (2-fold above basal levels), 8-bromo-cAMP or CRF (7- and 8-fold above basal levels, respectively), and a mixture of BaCl2 and 8-bromo-cAMP (20-fold above basal levels). An important difference between the processing of proenkephalin and the ACTH/endorphin precursor (POMC) in AtT-20 cells is efficient cleavage of a Lys-Lys site in proenkephalin and not in POMC. The ability of AT/PE to process proenkephalin in a natural manner makes it a suitable model system to investigate elements involved in the processing of proenkephalin at Lys-Lys sites.
Mathis
J P
JP
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Lindberg
I
I
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D004745:Enkephalins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000032:analysis*; D051379:Mice; D010911:Pituitary Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D011233:Precipitin Tests; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011863:Radioimmunoassay; D014162:Transfection; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D008239:Lysine