Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product

The E7 transforming protein of human papilloma virus-16 binds to the retinoblastoma gene product (pRb) through a nine-amino-acid segment of E7 (21-29). This segment of E7 is homologous to the pRb-binding domains of the simian virus 40 large T and adenovirus E1A transforming proteins. Each of these viral transforming proteins bind to the same region of pRb. To isolate cellular proteins that interact with this viral protein-binding domain on pRb, we used recombinant pRb to screen a human complementary DNA expression library. Two cDNAs were isolated that encode retinoblastoma binding proteins (RBP-1 and RBP-2). We report here that these RBP genes exist in separate loci and produce discrete messenger RNAs. The predicted amino-acid sequence of these genes showed no homology to known proteins, but both RBPs contain the pRb binding motif conserved between E7, large T and E1A14. In vitro expression of the RBP cDNAs yielded proteins that specifically bound to pRb. Recombinant E7 protein, the E7 21-29 peptide and the homologous RBP-1 peptide inhibited RBP-pRb binding. Mutations introduced into the putative pRb-binding segment in RBP-1 impaired its binding activity. These studies indicate that the cellular RBP-1, RBP-2 and viral E7 proteins interact with pRb through similar domains.     

A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product

Tumour-suppressor genes, such as the human retinoblastoma susceptibility gene (Rb), are widely recognized as being vital in the control of cell growth and tumour formation. This role is indicated, in part, by the suppression of tumorigenicity of human tumour cells after retrovirus-mediated Rb replacement. How Rb acts to bring about this suppression is not clear but one clue is that the Rb protein forms complexes with the transforming oncoproteins of several DNA tumour viruses, and that two regions of Rb essential for such binding frequently contain mutations in tumour cells. These observations suggest that endogenous cellular proteins might exist that bind to the same regions of Rb and thereby mediate its function. We report here the identification of one such human cellular Rb-associated protein of relative molecular mass 46,000 (46K) (RbAP46). Two lines of evidence support the notion that RbAP46 and simian virus 40 T antigen have homologous Rb-binding properties: first, several mutated Rb proteins that failed to bind to T also did not associate with RbAP46; and second, both T antigen and T peptide (amino acids 101-118) were able to compete with RbAP46 for binding to Rb. The apparent targeting of the RbAP46-Rb interaction by oncoproteins of DNA tumour viruses strongly suggests that formation of this complex is functionally important.     

Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product

One of the cellular targets implicated in the process of transformation by the adenovirus E1A proteins is a 105K cellular protein. Previously, this protein had been shown to form stable protein/protein complexes with the E1A polypeptides but its identity was unknown. Here, we demonstrate that it is the product of the retinoblastoma gene. The interaction between E1A and the retinoblastoma gene product is the first demonstration of a physical link between an oncogene and an anti-oncogene.     

Polyoma virus transforming protein associates with the product of the c-src cellular gene

Polyoma virus can transform the growth properties of rodent cells grown in culture and form tumours in susceptible animals, an activity largely due to one of the virus-encoded proteins, called middle T. Middle T has an associated tyrosine-specific protein kinase activity in vitro and interacts with cellular membranes, but the biochemical basis of its ability to transform remains unclear. Although there is some correlation between the transforming activity of different polyoma virus mutants and their ability to accept phosphate on tyrosine in middle T in the in vitro kinase reaction, the abundance of phosphotyrosine in protein is not elevated in polyoma virus-transformed cells and no cellular substrates for the putative kinase have been identified. It is also not yet known whether the tyrosine kinase of middle T is an intrinsic activity of the protein itself or the property of an associated enzyme. The experiments described here indicate that a fraction of middle T forms a stable complex with pp60c-src, the product of a cellular oncogene, and lead us to propose that the middle T associated kinase at least in part is a property of pp60c-src rather than middle T itself.     

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

The proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.     

Homology of a candidate spermatogenic gene from the mouse Y chromosome to the ubiquitin-activating enzyme E1.

The Sxr (sex-reversed) region, a fragment of the Y chromosome short arm, can cause chromosomally female XXSxr or XSxrO mice to develop as sterile males. The original Sxr region, termed Sxra, encodes: Tdy, the primary sex-determining gene; Hya, the controlling or structural locus for the minor transplantation antigen H-Y; gene(s) controlling the expression of the serologically detected male antigen (SDMA); Spy, a gene(s) required for the survival and proliferation of A spermatogonia during spermatogenesis; Zfy-1/Zfy-2, zinc-finger-containing genes of unknown function; and Sry, which is probably identical to Tdy. A deletion variant of Sxra, termed Sxrb, which lacks Hya, SDMA expression, Spy and some Zfy-2 sequences, makes positional cloning of these genes possible. We report here the isolation of a new testis-specific gene, Sby, mapping to the DNA deleted from the Sxrb region (the delta Sxrb interval). Sby has extensive homology to the X-linked human ubiquitin-activating enzyme E1. The critical role of this enzyme in nuclear DNA replication together with the testis-specific expression of Sby suggests Sby as a candidate for the spermatogenic gene Spy.