Neuroendocrine Protein 7B2 Can Be Inactivated by Phosphorylation within the Secretory Pathway

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 Ser 115 (located within the minimally active 36-residue peptide) was confirmed by mutagenesis, although Ser 115 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, Ca 2؉ /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.Prohormone convertase-2 (PC2), 2 a member of the family of eukaryotic subtilisins, exhibits an interesting but atypical cell biology compared with other convertases. The PC2 zymogen spends an unusually long time in the endoplasmic reticulum (ER) compared with other convertases and then binds to a partner protein, 7B2, which escorts it to the Golgi (1, 2). In the Golgi, a 7B2-mediated "capacitation" process renders pro-PC2 capable of autoactivation to an active enzyme molecule (reviewed in Ref. 3). Pro-PC2 expressed in cells lacking 7B2 (such as 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). The biochemical mechanism underlying the 7B2 capacitation effect is still enigmatic: however, it can be reproduced in a cell-free environment using Golgi membranes (5). Our current hypothesis is that 7B2 prevents pro-PC2 aggregation and/or facilitates its binding to membranes during intracellular transit. Structure-function studies have revealed that the minimal portion of 7B2 required to e...

Radial nerve cord protein phosphorylation dynamics during starfish arm tip wound healing events

“…This was achieved using a 2D differential phosphoproteomics approach to analyze the RNC‐soluble proteins enriched fraction. Among the proteins found to be modulated by phosphorylation in this study, some are particularly relevant, namely: (i) calpain, a serine protease that appears to be phosphorylated exclusively in starfish injured RNC, indicating that this PTM might be responsible for the modulation of its proteolytic activity during starfish nerve cord regeneration events, similar to what was previously described in other organisms ; (ii) spectrin, one of calpain's preferred substrate, which was found to be dephosphorylated in starfish injured nerves (48 h and 13 days PAA), thus increasing its vulnerability toward calpain‐mediated degradation , a regenerative event that triggers cell membrane rearrangements toward the formation of the growth cone; (iii) Notch homologue, which was dephosphorylated in injured RNC, thus acting as a possible inductor of cell differentiation during RNC regeneration ; (iv) phosphorylation‐mediated re‐organization of the cytoskeleton and regulation of filaments and microtubules dynamics; (v) phosphorylation of a START domain‐containing protein, known to be involved in important lipid‐signaling events during neuronal regeneration, and finally, (vi) dephosphorylation‐induced activation of a neuroendocrine convertase that might be involved in the maturation of signaling neuropeptides during RNC regeneration events.…”

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins

“…Thus, 7B2 appears to have novel anti-aggregation domains. The role of the known post-translational modifications of this protein, sulfation (49) and phosphorylation (50, 51) (see Fig. 4 A ), in anti-aggregation remains to be established.…”

Characterization of an Endoplasmic Reticulum-associated Silaffin Kinase from the Diatom Thalassiosira pseudonana

“…In membrane fractions from mammalian cells, ER-and Golgi apparatus-associated casein kinase-like activities have been identified that are believed to be involved in the biogenesis of secretory phosphoproteins including proteins involved in biomineralization of bone and teeth (e.g. osteopontin, bone sialoprotein, and dentin sialophosphoprotein) (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). However, to date the primary structures of these kinases have remained elusive.…”