Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants

S M Jong, C S Zong, T Dorai, L H Wang
1992 Journal of Virology  
To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken
more » ... o fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSVand UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68ffws and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain ofsrc or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology. * Corresponding author. t Present address: Department of Pathology, University of Florida, Gainesville, FL 32610. mediating signal transduction at the plasma membrane. Subversion of their normal functions often leads to uncontrolled cell growth and transformation. The oncogene v-ros, which is present in the avian sarcoma virus (ASV) UR2, transforms chicken embryo fibroblasts (CEF) to a rather elongated shape (3). Sequencing and protein analysis have shown that UR2 is unique among ASVs in that its oncogene product, P68'ag-r, is a transmembrane (TM) tyrosine kinase with the gag portion protruding extracellularly (18, 36, 57, 58) . On the other hand, most strains of Rous sarcoma virus (RSV), except the morphf variants (1, 13, 14, 20, 21, 31, 60, 77, 78, 88) , transform CEF to a rounded shape (84). The transforming protein of RSV, p60, is a cytoplasmic protein which is attached to the plasma membrane with the help of its N-terminal myristyl moiety (7, 72, 73) and a presumed membrane-bound receptor (64, 65) . In comparison with the morphf variants of RSV and another ASV, Fujinami sarcoma virus, which also induces a fusiform cell shape (28), UR2 still stands out for its extremely elongated transformation phenotype. This prompted us to investigate the possible underlying genetic differences responsible for the different transformation phenotypes of the two sarcoma viruses RSV and UR2. The differences could be due to the inherent properties of ros and src in their recognition of cellular substrates. Alternatively, the distinct subcellular localiza-4909 on May 9, 2020 by guest
doi:10.1128/jvi.66.8.4909-4918.1992 fatcat:hukxunyc2je57bvkyhrftgdnie