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

A normal response in cells that are chronically exposed to PDGF is the downregulation of PDGF receptors (Garrett et al., 1984) . This downregulation occurs via endocytosis of Arrows indicate regions of each cell that stain positively for both the sis-E1-G fusion protein and the Golgi complex. To analyze if such a process occurred in cells expressing any of the v-sis fusion proteins described in this paper, PDGF-/3 receptors were immunoprecipitated from serum-starved cells expressing the fusion proteins either before or after stimulation with PDGF-BB. These receptors were then subjected to an in vitro kinase assay, and incorporation of labeled phosphate from [3,-32P]ATP into the receptor protein was visualized by SDS-PAGE. As seen in Fig. 8 , in the absence of stimulation with PDGF, little or no activatable receptors were detected (B, lanes 1-9). After stimulation with PDGF-BB, however, NIH3T3 cells, mock-transfected cells, and cells expressing sis-E1 all exhibited a significant level of cell surface activatable receptors (A, 1-3), as demonstrated by the phosphorylation of PDGF-# receptors of ~180 kD. Expression of the mutant sis-El(Ql) and sis-El(ins) constructs, which reach the cell surface, led to downregulation of cell surface activatable receptors, and thus there was little detectable kinase activity in these samples (Fig. 8A, lanes 4 and 5) . Similar results were obtained with sis-TGN38, sis-TGN38A and v-sis, which also reach the cell surface ( Fig. 8/1, lanes 6, 7, and 8) . These results demonstrate that when v-sis is forced to remain in an intracellular compartment, such as the early Golgi, it is unable to downregulate cell surface PDGF receptors. (B, D, F, H, J, and L) . A and B, C and D, E and F, G and H, 1 and J, K and L,