Posttranslational processing of proenkephalin in SK-N-MC cells: evidence for phosphorylation.
SK-N-MC cells have recently been shown to be a rich source of proenkephalin and/or the proenkephalin-derived peptide, peptide B. We have investigated the synthesis and the posttranslational processing of proenkephalin in these cells. SK-N-MC cells retain very little of the proenkephalin synthesized; greater than 99% of the immunoreactive enkephalin synthesized within a 48-h period is secreted into the medium rather than contained intracellularly. When medium samples were subjected to gel filtration and assayed for the various enkephalins present within proenkephalin, only two major molecular-weight classes of peptides, with molecular weights and immunoreactive profiles consistent with those of proenkephalin and the 3.6-kDa carboxyl-terminal fragment peptide B, were observed. The proenkephalin-like peptide present in medium samples was shown by western blot procedures to consist of a 32-kDa protein with a slight amount of a higher-molecular-weight immunoreactive component above it. Only proenkephalin-sized peptides were present within cell extracts. Radiolabeled proenkephalin added to cell cultures was also cleaved to products similarly sized to those found in medium extracts; radiolabeled proenkephalin incubated in the absence of cells was not cleaved. Cleavage of exogenous proenkephalin thus probably at least partially occurs following secretion. Cell radiolabeling experiments with [32P]orthophosphate demonstrated that SK-N-MC proenkephalin is phosphorylated. Microheterogeneity of proenkephalin was also observed using isoelectric focusing coupled with western blotting. Our results suggest that the SK-N-MC cell line represents a useful model to study the earliest steps of the posttranslational processing of human proenkephalin in a neuronal cell type.
Lindberg
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I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical School, New Orleans 70112.
Shaw
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E
D002460:Cell Line; D003470:Culture Media; D004744:Enkephalin, Methionine / Q000378:metabolism; D004745:Enkephalins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D014415:Tunicamycin / Q000494:pharmacology
D003470:Culture Media; D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D014415:Tunicamycin; D004744:Enkephalin, Methionine