This proposal intends to examine the mechanism by which cell-transforming proteins come to reside in the nucleus. The adenovirus E1A gene product is required for the transcription of other adenovirus genes and is involved in cell transformation and immortalization. This protein acts at the level of transcription and has been shown to be concentrated in the nucleus of cells. We have used the microinjected Xenopus laevis oocyte to examine transcriptional activity and nuclear targeting of this protein. When overproduced in bacteria to yield a bona fide (nonfusion) protein, E1A stimulates the transcription of an adenovirus gene in injected oocytes. When examined for its ability to enter oocyte nuclei following cytoplasmic injection, however, we show that the protein undergoes a modification such that its apparent molecular weight increases by about 2,000 daltons. Only this modified form of the protein is found in nuclei. A second transforming nuclear protein, that encoded by the human c-myc gene, also undergoes a similar modification in the cytoplasm before it can enter the nucleus.
The specific aims of this proposal are to determine the nature of the modification, the amino acid residue(s) it modifies, and its relationship to nuclear targeting and possible transcriptional regulation. The modification will be determined by oocyte microinjection of small E1A peptides, followed by HPLC and amino acid sequencing. Further studies include the construction of fusion proteins between E1A and myc and other nonnuclear proteins via recombinant DNA technology. The ability of the fusion proteins to be modified and enter nuclei will be assessed. The long-term goals of this project are to determine the signal on proteins which target them to the nucleus and to investigate determinants on proteins which might be responsible for transcriptional regulation and transformation. Thus, these studies are of basic importance in cellular and molecular as well as developmental biology. The gene products which are under investigation have been implicated in the two-step model for cancer. Thus, this proposed project has a direct bearing on our ultimate understanding of cancer. (K)
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