The c‐fyn proto‐oncogene is a member of a family of closely related genes of which c‐src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c‐fyn gene product, the c‐fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c‐src and pp62c‐yes, pp59c‐fyn is able to form a stable complex with middle‐T antigen, the transforming protein of polyomavirus. The transformation‐defective middle‐T mutant NG59, which is unable to associate stably with pp60c‐src does not associate with pp59c‐fyn. In contrast to pp60c‐src, complex formation with middle‐T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c‐fyn. These findings lead us to suggest that middle‐T mediated transformation may be a consequence of the deregulation of several members of the src‐family of protein tyrosine kinases.

A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.

Our results indicate that only one type of tyrosine kinase is present within each middle-T antigen-tyrosine kinase complex, suggesting that middle-T antigen forms separate complexes with different tyrosine kinases. Furthermore, we determined that there is only one molecule of middle-T antigen within any one of these complexes. We interpret this to mean that in any given cell, polyomavirus transformation involves, at least in part, the simultaneous deregulation of a number of separate pathways controlling cellular proliferation.Finally, we also demonstrate that the separate middle-T:pp6Oc(r' and middle-T:pp59c-fy complexes are each able to interact with the same cellular p8l/85-kDa phosphoprotein, a possible component of the phosphatidylinositol kinase.Cellular transformation can result from malfunction of a number of different genes or gene products which are otherwise involved in the normal control of cellular proliferation. Proto-oncogenes, for example, can be diverted from their normal role by gene rearrangement, DNA amplification, overexpression, or mutation of their protein products. In these cases, the presence of excess amounts of the wild-type molecule or of a mutant version of the gene product leads to inappropriate activity and a consequent deleterious effect. By contrast, the absence of tumor suppressor genes and/or their gene products are associated with other instances of deregulated cellular proliferation. Although the first examples of lesions resulting in protooncogene activation and in tumor suppressor gene inactivation were recognized at the DNA level, more recent examples of these phenomena have been shown to result from protein-protein interactions. Thus, middle-T antigen, the transforming protein of polyomavirus, interacts with and activates the cellular proto-oncogene pp60-src (8), and simian virus 40 (SV40) large-T antigen interacts with p53 (25), a protein recently recognized as having tumor-suppressor activity (11). Although the existence of such complexes suggested simple models for cellular transformation by the papovaviruses, it is now clear that the viral transforming proteins interact not with one but with many cellular regulatory proteins. Middle-T antigen interacts with several members of the tyrosine kinase class (3,8,17,22,23); with p81/85 (35), a possible component of the phosphatidylinositol kinase pathway; and with protein phosphatases (28, 34). Simian virus 40 large-T antigen also interacts with p53, with the retinoblastoma gene product (9), and with other as yet unidentified cellular proteins. Here, we attempt to characterize the individual middle-T antigen-containing complexes. Our results suggest that middle-T antigen forms separate complexes with different tyrosine kinases and that within any one complex, there is only one molecule of middle-T antigen. Presumably, this means that in any given cell, polyomavirus transformation involves, at least in part, the simultaneous deregulation of a number of separate pathways controlling cellular proliferation.Stoichiometry of tyro...

Except for its unique amino-terminal region (residues 1 through 83), which possibly dictates substrate recognition, pp59c-fYbears a high degree of homology with other members of the src family of tyrosine kinases. Here we show that the carboxy terminus of pp59c-fyn is necessary for stable middle-T-antigen association, that pp59C-fyfis normally phosphorylated on both serine and tyrosine residues, and that Tyr-531 and Tyr-420 are phosphorylation sites in vivo and in vitro, respectively. Analysis of a spontaneously generated mutant encoding a truncated form of pp59c-fyn and of variants specifically mutated at the Tyr-531 and Tyr-420 phosphorylation sites indicates that pp59c-fyn has regulatory elements analogous to those that have already been identified for other src-like tyrosine kinases. However, further examination of the pp59clYfy variants suggests the likelihood of additional means by which its activities might be regulated. Although alteration of Tyr-531 to phenylalanine (531F) in pp59'-fy' results in a protein which is more active enzymatically that the wild type, the enhancement is much less than that for the analogous variant of pp6o-src. Furthermore, contrary to results of similar experiments on other src-like proto-oncogene products, 531F did not induce transformation of NIH 3T3 cells. Studies involving pp59c`fy-pp60csrc chimeras in which the unique amino-terminal sequences (residues 1 through 83) of the two kinases were precisely interchanged implied that the inability of 531F to induce transformation is probably not caused by the absence of substrates for pp59c fYnin NIH 3T3 cells but rather by the insufficient enhancement of pp59c-fyn kinase activity. It is therefore probable that the kinase and transforming activities of pp59C-fyf are repressed by additional regulatory elements possibly located in the amino-terminal half of the molecule.

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