Abstract
The prohormone convertases play important roles in the maturation of neuropeptides and peptide hormone precursors. Prohormone convertase-2 (PC2) is the only convertase that requires the expression of another neuroendocrine protein, 7B2, for expression of enzyme activity. In this study, we determined that 7B2 can be phosphorylated in Rin cells (a rat insulinoma cell line) and cultured chromaffin cells, but not in AtT-20 cells (derived from mouse anterior pituitary). Phosphoamino acid analysis of Rin cell 7B2 indicated the presence of phosphorylated serine and threonine. Phosphorylation of Ser115 (located within the minimally active 36-residue peptide) was confirmed by mutagenesis, although Ser115 did not represent the sole residue phosphorylated. Two independent assays were used to investigate the effect of phosphorylated 7B2 on PC2 activation: the ability of 7B2 to bind to pro-PC2 was assessed by co-immunoprecipitation, and activation of pro-PC2 was assessed in a cell-free assay. Phosphorylated 7B2 was unable to bind pro-PC2, and the phosphorylated 7B2 peptide (residues 86-121, known to be the minimally active peptide for pro-PC2 activation) was impaired in its ability to facilitate the generation of PC2 activity in membrane fractions containing pro-PC2. In vitro phosphorylation experiments using Golgi membrane fractions showed that 7B2 could be phosphorylated by endogenous Golgi kinases. Golgi kinase activity was strongly inhibited by the broad-range kinase inhibitor staurosporine and partially inhibited by the protein kinase C inhibitor bisindolylmaleimide I, but not by the other protein kinase A, Ca2+/calmodulin-dependent kinase II, myosin light chain kinase, and protein kinase G inhibitors tested. We conclude that phosphorylation of 7B2 functionally inactivates this protein and suggest that this may be analogous to the phosphorylating inactivation of BiP, which impairs its ability to bind substrate.
Highlights
Chinese hamster ovary (CHO) cells) may still undergo propeptide cleavage, but in this case, propeptide removal does not result in the production of enzymatically active species [4]
Phosphorylation of secretory proteins occurs at serine or threonine residues within the consensus sequence of a Golgi apparatus casein kinase-like enzyme, (Ser/Thr)-Xaa-(acidic residue/Ser(P)) [19]
7B2 Is Phosphorylated in Rin Cells and Primary Bovine Adrenal Chromaffin Cells, but Not in AtT-20 Cells—To determine whether 7B2 can be phosphorylated in endocrine cells, we labeled RinPE/7B2, AtT-20/ Prohormone convertase-2 (PC2)/7B2, and primary cultures of bovine adrenal chromaffin cells with [32P]orthophosphate and immunoprecipitated labeled cell extracts using antiserum against 7B2
Summary
Chinese hamster ovary (CHO) cells) may still undergo propeptide cleavage, but in this case, propeptide removal does not result in the production of enzymatically active species [4]. Structure-function studies have revealed that the minimal portion of 7B2 required to effect capacitation of pro-PC2 consists of a 36-residue disulfide-bonded peptide located within the 21-kDa domain (residues 1–151), which contains a putative polyproline helix and an ␣-helix (4 – 6). Secretory proteins undergo phosphorylation within the lumen of the Golgi apparatus; these include casein [11], vitellogenin [12, 13], osteopontin [14], proline-rich protein [15], and chromogranins [16]. Phosphorylation of secretory proteins occurs at serine or threonine residues within the consensus sequence of a Golgi apparatus casein kinase-like enzyme, (Ser/Thr)-Xaa-(acidic residue/Ser(P)) [19]. Reversible phosphorylation within the secretory pathway has far been described only for chaperone proteins such as BiP and GRP94 (20 –23), in which phosphorylation is thought to regulate chaperone-substrate interaction in a negative manner. We demonstrate that phosphorylation of 7B2 occurs in neuroendocrine cells at multiple sites, that it can be carried out by endogenous Golgi kinases, and that this modification results in its functional inactivation
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