The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex

NIH3T3 cells were transfected with 9/~g of MLV constructs plus 1 #g pZAP, and supernatants collected and used for infection of fresh NIH3T3 cells. 2 d after infection, the 60 mm plates of ceils were serum starved in DME for 24 h, then treated for 5 rain with 100 ng/ml PDGF-BB (Amgan, Thousand Oaks, CA) or left untreated. Cells were lysed in NP-40 lysis buffer (20 mM Tris, pH 7.5, 137 mM NaCI, 1% NP-40, 1 mM sodium orthovanadate, 5 mM EDTA, 10 t~g/ml Aprotinin, and 10% glycerol) and scraped from plates with rubber policemen. The lysates were clarified by centrifugation, then incubated for 2 h with a rabbit antiserum specific for the mouse PDGF-B receptor (Upstate Biotechnology Inc., Lake Placid, NY). Immune complexes were collected with protein A-Sepharose beads, spun through a 10% sucrose in NP-40 lysis buffer solution, washed twice with NP-40 lysis buffer, and once with 20 mM Tris, pH 7.5. 40 t~l of kinase buffer (20 mM Tris, pH 7.5, 10 mM MnCI2, 10 mM MgCI2) containing 5 #Ci ['y-32P]ATP was then added to the beads, and reactions were incubated for 10 min at 37°C. Reaction products were separated by SDS-PAGE (7.5 %) and visualized by autoradiography. Figure 1 . Structure of Golgilocalized v-sis derivatives. All constructs used the first 239 amino acids of the v-sis protein, which includes: a signal sequence; a pmpeptide with N-linked oligosaccharide addition site and dibasic proteolytic processing site; and the 82-amino acid MTR. The sis-G-ER + and sis-G-ERconstructs were produced by fusing a known ER retention signal (DEKKMP) or a scrambled signal (DEMPKK) from an adenoviral protein, E3/19K, onto the cytoplasmic end of sis-G fusions (lee and Donoghue, 1992). The next six constructs contain residues 21-45 of El, a glycoprotein from an avian coronavirus. These residues encode the first transmembrahe domain of El, which has been shown to confer cis-Golgi localization. The last three constructs in the E1 set have a 39-amino acid section of the glycoprotein G from the vesicular stomatitis virus (VSV-G) fused to the cytoplasmic tail. The mutant forms of these constructs,