Leah A. Lipsich

Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation

We examine the interaction between polyoma-virus-encoded middle tumor antigen and the cellular src gene product, pp60c-src, using a series of monoclonal antibodies that recognize mammalian pp60c-src. Our results show that infection of mouse cells with transformation-competent strains of polyoma virus results in the stimulation of pp60c-src kinase activity severalfold over that observed in uninfected mouse cells and mouse cells infected with transformation-deficient polyoma virus. A similar degree of enhancement of pp60c-src kinase activity was found in polyoma-virus-transformed rodent cells. No differences were detected in the level of pp60c-src synthesis in polyoma-virus-infected and uninfected mouse cells or polyoma-virus-transformed and normal rodent cells. These studies demonstrate that polyoma-virus-encoded middle tumor antigen is associated with pp60c-src in lysates of polyoma-virus-infected and polyoma-virus-transformed cells and suggest a novel mechanism for the functional activation of a cellular proto-oncogene product, namely, that the interaction between middle tumor antigen and pp60c-src leads to a stimulation of pp60c-src tyrosyl kinase activity.

Association of the transforming proteins of Rous, Fujinami, and Y73 avian sarcoma viruses with the same two cellular proteins.

Two forms of the transforming proteins of Fujinami (pp140fps) and Yamaguchi 73 (pp94yes) sarcoma viruses were detected in lysates of chicken cells transformed by these viruses; the majority of pp140fps and pp94yes molecules were present as monomers; however, a small percentage of these proteins was associated in a complex with two cellular proteins of Mr 90,000 and 50,000. These cellular proteins were shown to be identical to those previously found to be complexed with the transforming protein of Rous sarcoma virus, pp60src. These results suggest a common role for the interaction of pp90 and pp50 with viral transforming proteins encoding tyrosyl-protein kinases.

Differentiation and transformation of neural plate cells

The developmental potential of presumptive neural plate cells of prestreak chick embryos (stage 1) and neural plate cells from definitive streak chick embryos (stage 4) has been examined in cell culture using specific markers that identify the major cell types in the vertebrate central nervous system. The prestreak presumptive neural plate (PSPNP) cells, stage 1, assume an epithelial appearance in vitro and synthesize cellular fibronectin, but do not express markers for the neuronal, astrocytic, melanocytic, or oligodendrocytic lineages. Conversely, definitive-streak neural plate (DSNP) cells contain precursors which express cell-type-specific markers for terminally differentiated neurons, astrocytes, and melanocytes, and synthesize an extracellular matrix of cellular fibronectin. Differentiation of DSNP cells in vitro can be prevented by infection with a temperature-sensitive mutant of Rous sarcoma virus (RSV), tsNY68. Differentiation of DSNP cell transformants can be resumed by a temperature shift to the nonpermissive temperature. The morphological and biochemical changes associated with tsNY68 transformation are accompanied by alterations in pp60src kinase activity in the transformed cells.

Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.