Bartholomew M. Sefton - US grants

The biochemical basis of cellular transformation by the avian sarcoma viruses Rous sarcoma virus, Y73 virus and PRCII virus will be studied. Particular attention will be paid to the role of the phosphorylation of cellular proteins on tyrosine. The role of 1) the phosphorylation of the cytoskeletal protein vinculin and 2) the abundance of fibronectin in the determination of the morphology of transformed cells will be evaluated using viruses which induce atypical morphological transformation. The metabolism and the state of phosphorylation of the two cellular proteins, 80K and 50K, which are found bound to the transforming protein of Rous sarcoma virus will be studied in uninfected cells, in cells transformed by Rous sarcoma virus, and in cells transformed by other agents. Antisera specific to proteins which are substrates of tyrosine protein kinases will be prepared and used to identify cellular proteins which are regulated by the phosphorylation of tyrosine. finally, the state of phosphorylation and the activity of cellular tyrosine protein kinases in uninfected cells and in a variety of transformed cells will be determined. Immediate attention will be paid to the cellular homologue of the transforming protein of Rous sarcoma virus. Antisera useful for the study of the cellular homologues of the transforming proteins of Y73 virus and of PRCII virus will be developed. The structure and the mode of replication of the avian coronavirus infections bronchitis virus will be studied. The polypeptides present in the virion will be enumerated, compared in primary structure, and localized in the virion. The mRNA which is translated to yield each viral structural protein will be determined by in vitro translation of fractionated viral mRNAs. The post-translational processing of each protein will be studied by immunoprecipitation of the viral proteins from infected cells. Finally, the transcriptional mechanism which generates the family of subgenomic mRNAs will be characterized by UV target size analysis.

Tyrosine protein kinases play a central role in the regulation of cell multiplication. This project is directed at understanding the function of the tyrosine protein kinase encoded by the lck proto-oncogene, p56lck. This regulatory molecule is of interest because it is expressed exclusively in lymphoid cells and because it is implicated in lymphomagenesis. The work has three parts. One is designed to define precisely the transforming potential of wild-type lck and of genetically-activated lck. This will be done by the infection of neo-natal mice and cultured lymphoid cells with retroviruses expressing p56lck. The identity of transformed cells will be determined by the analysis of surface markers. This should reveal the types of cells that this protein can transform. Additionally, the potential role of p56lck in human leukemia will be examined by characterization of the protein in leukemic cell lines. Second, the role of p56lck in normal lymphoid function will be examined. Agents that induce the dephosphorylation and activation of p56lck will be sought and their mode of action determined. Additionally, the effect of ectopic or excessive expression of p56lck on the function of hematopoietic cell lines will be examined by infection with retroviruses encoding p56lck and determination of the functional properties of the infected cells. From perturbations of function, it is hoped that the normal function of the protein can be inferred. Finally, because lck is expressed only in lymphoid cells, the structure and properties of the promoters and enhancers driving the expression of the gene are of considerable interest. These will be isolated by molecular cloning and the regions conferring specificity will be identified through the use of luciferase as a reporter gene.

These studies are designed in part to identify and characterize the crucial polypeptide substrates of viral transforming proteins which possess tyrosine protein kinase activity. Novel substrates will be sought by "Western Blotting" using anti-phosphotyrosine antibodies. Since there are indications that the phosphorylation of many of the substrates of the viral protein kinases does not contribute materially to cellular transformation, advantage will be taken of mutants of Rous sarcoma virus which encode a non- fatty acylated transforming protein. These mutant proteins posses undimished protein kinase activity but cannot transform cells. The small subset of the substrates of the wild-type protein which do not undergo phosphorylation in cells infected with these mutants will be identified and studied. In addition, a variety of immune reagents--tumor sera and sera produced by immunization with proteins expressed in bacteria-- will be prepared and used to proto-oncogenes. The properties and structure of these proteins will be determined and the cells in which they are expressed will be identified. The effect of growth factors on the protein kinase activity and the phosphorylation of these cellular proteins will be examined.

This project is designed to understand the role of tyrosine protein phosphorylation in the activation of B lymphocytes by antigen. The binding of antigen or anti-immunoglobulin antibody to immunoglobulin M or D on the surface of mature B cells induces the rapid phosphorylation of approximately 10 proteins on tyrosine. Surface immunoglobulin therefore must be coupled in some manner to one or more tyrosine protein kinases or phosphatases. Because an inhibitor of tyrosine phosphorylation renders B cells unresponsive to stimulation through the antigen receptor, tyrosine phosphorylation is apparently important in B cell activation. B cells that do not express the tyrosine protein phosphatase CD45 are unresponsive to the cross-linking of surface immunoglobulin. This insensitivity could be due to increased inhibitory phosphorylation of a tyrosine protein kinase. We will therefore study the phosphorylation of the several src-family tyrosine protein kinases found associated with the antigen receptor complex. Additionally, we will ask whether the insensitivity of CD45-negative B cells is reversed by introduction into them of a constitutively-activated version of a src-family kinase. We will also study the interaction of the src-family kinases with the antigen receptor complex. We will identify the domain of the src kinases responsible for their interaction by introduction into B cells of mutated src kinases. Additionally, we will ask whether the cytoplasmic domains of either or both of the immunoglobulin-associated proteins, Ig-alpha and Ig-beta, can interact with tyrosine kinases directly. If so, we will ask whether these domains can participate in signal transduction. Finally, we will exploit newly-isolated monoclonal antibodies to two novel tyrosine phosphorylated proteins in activated B cells. The 95K and 140K proteins that these antibodies recognize will be characterized and their genes cloned. These studies have the potential to reveal the identities of tyrosine protein kinases and substrates that are important in the regulation of the proliferation of normal B cells and may play a role in the unregulated growth of malignant B cells such as those transformed by Abelson murine leukemia virus.