Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros

Abstract

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was deleted; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity. The proto-oncogene c-ros is the cellular counterpart of the oncogene v-ros of avian sarcoma virus (ASV) UR2 (30, 31). Sequence analysis of the chicken and human genomic DNA clones representing the 3' portion of c-ros revealed that it is a receptor-like protein tyrosine kinase (PTK) (28, 30). Recently, the c-ros cDNAs from rat (29), human (3), and chicken (5) have been isolated and sequenced. Those results confirm the initial suggestion that c-ros codes for a receptor-like PTK. However, it differs from other well-characterized receptor PTKs (RPTKs) such as the insulin receptor (IR) and insulinlike growth factor I receptor (IGFR) (8, 40, 42), epidermal growth factor receptor (41), platelet-derived growth factor receptor (47), and colony-stimulating factor receptor (6, 1...

Oncogenic Kinase Signaling in Human Neoplasms

“…Such mutations were found on c‐erbB‐2/neu 9,10. Mutation of the transmembrane domain was also found in other viral oncogenes such as v‐ROS, which obtains a broad substrate specificity 11…”

“…9,10 Mutation of the transmembrane domain was also found in other viral oncogenes such as v-ROS, which obtains a broad substrate specificity. 11 Alternatively, tyrosine kinases can become oncogenic by mutations that make them active and independent of ligand binding or dimerization. Nonreceptor tyrosine kinases are also activated by mutations that affect their negative regulation, such as the mutation on tyrosine 527 of Src that leads to deregulation of its activation.…”

The ortholog of the human proto‐oncogene ROS1 is required for epithelial development in C. elegans

“…Expression of ROL‐3 in epidermal tissues is consistent with the murine model, demonstrating the potential for a conserved function between these proteins. It has been demonstrated that ROS1 directly binds SH2 domain containing tyrosine phosphatase SHP‐1 (Keilhack et al, ), and has been shown to activate both SHP‐1 and SHP‐2, the mitogen‐activated protein kinase ERK1/2, insulin receptor substrate 1 (IRS‐1), phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) and the STAT3 and VAV3 signaling pathways (Zong et al, ; Xiong et al, ; Zeng et al, ; Charest et al, ). Orthologs of several of these proteins are present in the C. elegans genome, leading to potential future studies to test the conserved nature of these interactions.…”

Effect of Dimerization on Signal Transduction and Biological Function of Oncogenic Ros, Insulin, and Insulin-like Growth Factor I Receptors

“…DNA Transfections-Transfections were done using the calcium phosphate co-precipitation method (31). Primary CEFs were plated at a density of 1 ϫ 10 6 cells/6-cm dish and incubated for 18 -20 h at 37°C.…”

“…The medium was then replaced with fresh medium containing 10% calf serum. 50 l of 2.5 M CaCl 2 was added to 450 l of sterile H 2 O containing 10 g each of transforming viral plasmid DNA and SacI-digested UR2AV helper viral DNA (31). This solution was slowly added to 500 l of 2 ϫ Hepes-buffered saline (50 mM Hepes, pH 7.2, 280 mM NaCl, 10 mM KCl, 1.5 mM Na 2 HPO 4 , and 12 mM dextrose) that was being aerated.…”