Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product.

The SV40 T antigen (T)/adenovirus E1A-binding domain of the retinoblastoma gene product (pRB) has been fused to S. japonicum glutathione S-transferase, and the chimera, bound to insoluble glutathione, was used to search for cellular proteins that can interact specifically with pRB. At least seven such proteins were detected in extracts of multiple human tumor cell lines. These proteins failed to bind to a family of pRB fusion proteins that harbor inactivating mutations in the T/E1A-binding domain and to the wild-type fusion protein in the presence of a peptide replica of the pRB-binding domain of T. Therefore, the binding of one or more of these proteins may contribute to the growth-suppressing function of pRB.     

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.     

The role of the J domain of SV40 large T in cellular transformation.

SV40 large T antigen (TAg)-mediated transformation is dependent on binding to p53 and the retinoblastoma tumor suppressor protein (pRB) and inactivating their growth suppressive functions. Transformation minimally requires three regions of TAg: a C-terminal domain that mediates binding to p53; the LXCXE motif (residues 103-107), necessary for binding to pRB and the related proteins p107 and p130; and an N-terminal domain (residues 1-82) that contains homology to the J domain found in cellular DnaJ/Hsp40 molecular chaperone proteins. We have found that the N-terminal J domain of T Ag cooperates with the LXCXE motif to inactivate the growth suppressive functions of the pRB-related proteins.     

Involvement of pRB family in TGF beta-dependent epithelial cell hypertrophy

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK- 52E cells (a renal epithelial cell line), transforming growth factor beta 1 (TGF beta) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGF beta did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF beta blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGF beta-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF beta-induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF beta-induced hypertrophy was inhibited in E7- and E6E7-expressing, but not E6- expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF beta-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF beta-induced blockade at G1/S, the latter due to maintained activity of pRB or a related protein.