Immunological characterization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus

M E Linder, J G Burr
1988 Journal of Virology  
Phosphotyrosine antibodies were used to identify tyrosine-phosphorylated proteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. A large number of tyrosine phosphoproteins were detected. A similar set of proteins was observed in RSV-transformed murine cells. An 85,000-dalton protein, however, was present in transformed avian cells but missing in transformed murine cells. Neither the 85,000-dalton protein nor any of the other tyrosine phosphoproteins appeared to be viral
more » ... ctural proteins. Use of RSV mutants encoding partially deleted src gene products enabled us to identify a 60,000-dalton cellular tyrosine phosphoprotein that comigrated with wild-type pp6O-src. With the exception of calpactin I, the major tyrosine phosphoproteins detected in immunoblots appeared to be different from several previously characterized substrates of pp60-src with similar molecular masses (ezrin, vinculin, and the fibronectin receptor). Oncogenic transformation of cells infected with Rous sarcoma virus (RSV) is initiated and maintained by the src gene product, pp6Ov-src (27) , which is a protein kinase with specificity for tyrosine residues (30). A number of approaches have been used to identify substrates for pp60vsrc (reviewed in reference 37). Proteins that have been identified as presumptive substrates for pp60v-src include the cytosolic protein p50 (4, 25, 31), which may be involved in transport of newly synthesized pp60v-src to its membrane location; three enzymes of the glycolytic pathway: enolase, phosphoglycerate mutase, and lactate dehydrogenase (12); the cytoskeleton-associated proteins vinculin (53), calpactin I (p36) (46), and ezrin (26); and p42 (10), which is also a substrate for several growth factor receptor tyrosine kinases (2, 13, 24). In addition, talin (16, 44), calmodulin (20), and the fibronectin receptor (FNR) (28) are phosphorylated on tyrosine in a transformation-specific manner in cells infected with RSV. So far, there has been no clear demonstration of how phosphorylation on tyrosine within any of these proteins is related to the process of transformation. More recently, antiphosphotyrosine antibodies (Ptyr antibodies) have been used to detect cellular proteins phosphorylated on tyrosine in response to viral transformation or treatment of cells with growth factors (51). These antibodies recognize the tyrosine-phosphorylated forms of the receptors for epidermal growth factor (18), platelet-derived growth factor (18), and insulin (42). The transforming proteins of the src (9, 17, 58), abl (17, 19, 51, 58), fes (17), and fps (17) oncogenes are also detected by Ptyr antibodies. In this study, we used immunoblots with Ptyr antibodies to compare the tyrosine phosphoproteins found in RSVtransformed chicken embryo fibroblasts (RSV-CEF) with those found in RSV-transformed murine cells. Except for an 85,000-dalton protein, the tyrosine phosphoproteins detected in the two types of cells had very similar molecular masses. None of the major proteins identified in Ptyr immunoblots appeared to be viral structural proteins. A previ-* Corresponding author. t Present address:
doi:10.1128/jvi.62.8.2665-2673.1988 fatcat:ag47vtjaazhkrpdjlpo4oh2mgq