THE V-SIS ONCOPROTEIN LOSES TRANSFORMING ACTIVITY WHEN TARGETED TO THE EARLY GOLGI-COMPLEX

Citation
Kc. Hart et al., THE V-SIS ONCOPROTEIN LOSES TRANSFORMING ACTIVITY WHEN TARGETED TO THE EARLY GOLGI-COMPLEX, The Journal of cell biology, 127(6), 1994, pp. 1843-1857
Citations number
63
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
0021-9525
Volume
127
Issue
6
Year of publication
1994
Part
2
Pages
1843 - 1857
Database
ISI
SICI code
0021-9525(1994)127:6<1843:TVOLTA>2.0.ZU;2-X
Abstract
The location of autocrine interactions between the v-sis protein and P DGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constr ucted fusion proteins that anchor v-sis to specific intracellular memb ranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 gl ycoprotein to v-sis protein completely abolished its transforming abil ity when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimeriz e properly. Derivatives of some of these constructs were also construc ted bearing the cytoplasmic tail from the glycoprotein of vesicular st omatitis virus (VSV-G). These constructs allowed examination of subcel lular localization by double-label immunofluorescence, using antibodie s that distinguish between the extracellular PDGF-related domain and t he VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi marke rs confirmed its targeting to the early Golgi complex. The sis-E1 cons tructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteol ytic processing. Treatment with suramin, a polyanionic compound that d isrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 construc ts described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Gol gi complex do not result in functional signal transduction. Another v- sis fusion protein was constructed by attaching the transmembrane doma in and COOH-terminus of TGN38, a protein that localizes to the trans-G olgi network (TGN). This construct was primarily retained intracellula rly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated t he TGN-localization, as evidenced by very prominent cell surface local ization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the p roperties of TGN38 itself, which is known to recycle from the cell sur face to the TGN.