FORMATION OF NASCENT SECRETORY VESICLES FROM THE TRANS-GOLGI NETWORK OF ENDOCRINE-CELLS IS INHIBITED BY TYROSINE KINASE AND PHOSPHATASE INHIBITORS

Citation
Cd. Austin et D. Shields, FORMATION OF NASCENT SECRETORY VESICLES FROM THE TRANS-GOLGI NETWORK OF ENDOCRINE-CELLS IS INHIBITED BY TYROSINE KINASE AND PHOSPHATASE INHIBITORS, The Journal of cell biology, 135(6), 1996, pp. 1471-1483
Citations number
63
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
0021-9525
Volume
135
Issue
6
Year of publication
1996
Part
1
Pages
1471 - 1483
Database
ISI
SICI code
0021-9525(1996)135:6<1471:FONSVF>2.0.ZU;2-R
Abstract
Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-bind ing proteins, heterotrimeric G proteins, inositol phospholipid metabol ism, and protein serine/threonine phosphorylation. At the cell surface , protein phosphorylation and dephosphorylation on tyrosine residues c an rapidly modulate cytosolic signaling pathways in response to extrac ellular stimuli and have been implicated in the internalization and so rting of signaling receptors, To determine if phosphotyrosine metaboli sm might also regulate secretory vesicle budding from the TGN, we trea ted permeabilized rat pituitary GH(3) cells with inhibitors of either tyrosine phosphatases or tyrosine kinases, We demonstrate that the tyr osine phosphatase inhibitors pervanadate and zinc potently inhibited b udding of nascent secretory vesicles, Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, s uggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonize d the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphot yrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhib itors. Subcellular fractionation identified several tyrosine phosphory lated polypeptides of similar to 175, similar to 130, and 90-110 kD th at were enriched in TGN-containing Golgi fractions and tightly membran e associated. The phosphorylation of these polypeptides correlated wit h inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphorylation and dephosphorylation are req uired for secretory vesicle release from the TGN.