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Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A.

ABSTRACT: The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.

SUBMITTER: Varro A

PROVIDER: S-EPMC1134634 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Project description:We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.

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Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A.

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