Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.
A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Vindrola
O
O
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Lindberg
I
I
D000818:Animals; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects; D015151:Immunoblotting; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000201:enzymology*; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000378:metabolism
D003346:Corticotropin-Releasing Hormone; D008985:Monensin; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1