Prohormone processing and secretory vesicle formation in vitro: Roles for calcium and phosphotyrosine metabolism

Abstract

To determine the role of calcium in prosomatostatin (proSR1F) processing and vesicle formation from the trans-Golgi network (TGN), we treated permeabilized rat anterior pituitary GH{dollar}\sb3{dollar} cells with the calcium ionophore A23187 (Austin and Shields, 1996). In the presence of the calcium chelator EGTA, proSR1F cleavage was markedly inhibited. Submillimolar free calcium ({dollar}\sim{dollar}15 {dollar}\mu{dollar}M) was sufficient to maintain efficient proSR1F cleavage, a value far lower than that estimated for total calcium in the TGN or secretory granules. Experiments using both A23187 and the protonophore CCCP revealed that free calcium is absolutely required for efficient proSR1F cleavage, even at the optimal pH of 6.1. Secretory vesicle formation by contrast was not inhibited by calcium chelation but rather, by millimolar extralumenal calcium. These observations demonstrate that proSR1F processing and budding from the TGN can be uncoupled in vitro and have distinct biochemical requirements. Interestingly, basal secretion of proSR1F from intact cells was largely calcium-dependent, which is a characteristic of the regulated secretory pathway.;Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. To determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized GH, cells with inhibitors of either tyrosine phosphatases or tyrosine kinases (Austin and Shields, in press). The tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrosine kinase inhibitors such as tyrphostin A25 also prevented secretory vesicle budding, suggesting that vesicle formation requires both phosphatase and kinase activities. Anti-phosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors in a manner that correlated with inhibition of vesicle budding. Subcellular fractionation identified phosphoproteins of {dollar}\sim{dollar}175, {dollar}\sim{dollar}130, and 90-110 kD, which are tightly membrane-associated, are enriched in TGN-containing Golgi fractions, and whose tyrosine phosphorylation correlated with inhibition of vesicle budding. These polypeptides serve as candidates for regulators of secretory vesicle budding. Our results suggest that in endocrine cells protein tyrosine phosphorylation and dephosphorylation are required for secretory vesicle release from the TGN.