Retinoblastoma protein binding properties are dependent on 4 cysteine residues in the protein binding pocket.

The retinoblastoma gene product (pRB) participates in regulating mammalian cell replication. The mechanism responsible for pRB's growth regulatory activity is uncertain. However, pRB is known to bind viral transforming proteins including the papilloma virus E7 protein, cellular proteins, and DNA. pRB contains a critical domain termed the "binding pocket" which is required for binding activities. This binding pocket contains 8 cysteine residues. A naturally occurring mutation affecting one of these cysteines is known to eliminate pRB's protein and DNA binding activities. To investigate the cysteine residues in pRB's binding pocket, each residue was mutated to alanine, phenylalanine, or serine. These mutant genes were used to prepare pRBs harboring specific amino acid substitutions. Individual mutations at positions 407, 553, 666, and 706 depressed pRB binding to E7 protein, DNA, and a conformation-specific anti-pRB antibody, XZ133. Combinations of these inhibitory mutations exhibited additive inhibitory effects on pRB's binding properties. Mutations at positions 438, 489, 590, 712, and 853 did not affect pRB binding to E7 protein, DNA, or the XZ133 antibody. Combination of these five neutral mutations yielded a pRB species with full E7 protein, DNA, and XZ133 binding activities. These studies indicate that the cysteine residues at positions 407, 553, 666, and 706 contribute to the E7 protein and DNA binding properties of pRB and appear to do so by maintaining pRB's normal conformation.     

Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7

The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB.     

Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein

Complex formation between the human papilloma virus type-16 E7 protein (HPV-16 E7) and the retinoblastoma suppressor protein (pRB) is believed to be important in the process of cellular transformation that leads to cervical carcinoma. Utilizing an in vitro solution assay as well as a plate binding assay that measures the association between HPV-16 E7 and pRB proteins, we have examined a series of synthetic HPV-16 E7 peptides. HPV-16 E7 peptides which lie between amino acid residues 14 and 32 were found to be potent inhibitors of E7/pRB binding. The minimal peptide structure that possessed full antagonist activity was N-acetyl-E7-(21-29)-peptide amide. This peptide inhibited 100% of E7/pRB binding and exhibited an IC50 of 40 nM in the plate binding assay. A purified beta-galactosidase-E7 fusion protein exhibited an IC50 of 2 nM in the same assay. These results suggest that other regions of the E7 molecule in addition to amino acids 21-29 may contributed to E7/pRB interaction. Analysis of E7-(20-29)-peptides containing single amino acid substitutions suggests that Cys24, Tyr23, Tyr25, Asp21, and Glu26 are important residues for maintaining maximal antagonist activity. This series of peptides should prove useful in analyzing the biological consequences of E7/pRB binding in HPV-infected cells.     

The B-domain lysine patch of pRB is required for binding to large T antigen and release of E2F by phosphorylation

Cell cycle-dependent, site-specific phosphorylation of the retinoblastoma protein, pRB, is mediated by cyclin-dependent kinases (CDKs) and regulates the binding of pRB to many proteins. We previously showed that the interaction of pRB with E2F on DNA was regulated by the accumulation of phosphate groups on pRB. Here we show that positively charged lysine residues in the B domain of pRB are necessary for the release of pRB from E2F on DNA following phosphorylation by cyclin E-cdk2 kinase. These lysine residues are also important in the binding of the simian virus 40 large T antigen (TAg) to pRB, and mutation of these lysines to arginines alters the dependency of the pRB-TAg interaction on phosphorylation of pRB.     

Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity.

Adenovirus E1A transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the cellular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of E1A, which is located near the N terminus. Transformation-defective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton E1A-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by E1A is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton E1A-associated protein plays a major role in E1A-mediated cell growth control mechanisms.     

Rb-independent induction of apoptosis by bovine papillomavirus type 1 E7 in response to tumor necrosis factor alpha

Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that causes fibropapillomas of the host. BPV-1 has served as the prototype for studies of the molecular biology of the papillomaviruses. BPV-1 efficiently induces anchorage-independent growth and focus formation in murine C127 cells. The transforming properties of BPV-1 primarily reside in two genes, E5 and E6. Each of these genes is sufficient to transform cells. Although no independent transformation activity has been detected for E7, it was shown to be required for full transformation of C127 by BPV-1. We investigated the biological activities of BPV-1 E7 in several assays. Our results indicate that expression of BPV-1 E7 sensitizes cells to tumor necrosis factor alpha (TNF)-induced apoptosis. The TNF-induced apoptosis in E7-expressing cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A(2) was activated. Unlike the E7 proteins from the cancer-related human papillomaviruses, the BPV-1 E7 protein does not associate efficiently with the retinoblastoma protein (pRB) in vitro, nor does it significantly affect the pRB levels in cultured cells. Furthermore, BPV-1 E7 sensitizes Rb-null cells to TNF-induced apoptosis. These studies indicate that BPV-1 E7 can sensitize cells to apoptosis through mechanisms that are independent of pRB.     

Simian virus 40 large T antigen's association with the CUL7 SCF complex contributes to cellular transformation

Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that delta69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not delta69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and delta69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.     

Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins.

Studies of adenovirus E1A oncoprotein mutants suggest that the association of E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated transformation. Mutational analysis of E1A indicates that two regions of pRB are required for E1A to form stable complexes with the retinoblastoma protein. In addition to pRB binding, these regions are necessary for E1A association with several other cellular proteins, including p130, p107, cyclin A, and p33cdk2. Here we show that short synthetic peptides containing the pRB-binding sequences of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7 protein of human papillomavirus type 16 contains an element that binds to pRB and appears to be functionally homologous to the E1A sequences. Peptides containing this region of the E7 protein were able to interact with p107, cyclin A, and p130 in addition to pRB. These findings suggest that the common mechanism of transformation used by these viral oncogenes involves their association with a set of polypeptides.     

Protein kinase C inhibits binding of diacylglycerol kinase-zeta to the retinoblastoma protein

We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-zeta (DGKzeta) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKzeta that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKzeta binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKzeta-MARCKS phosphorylation-site domain also prevented DGKzeta binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKzeta and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCalpha isoform inhibits DGKzeta binding to pRB, whereas dominant-negative PKCalpha neutralized this inhibition. PKCalpha activation thus prevents DGKzeta regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.     

DNA-binding properties of the E1A-associated 300-kilodalton protein.

One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.     

Structure-function analysis of the THAP zinc finger of THAP1, a large C2CH DNA-binding module linked to Rb/E2F pathways

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of approximately 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel beta-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.     

Exploring the structural and functional effect of pRB by significant nsSNP in the coding region of RB1 gene causing retinoblastoma

In this study,we identified the most deleterious nsSNP in RB1 gene through structural and functional properties of its protein (pRB) and investigated its binding affinity with E2F-2.Out of 956 SNPs,we investigated 12 nsSNPs in coding region in which three of them (SNPids rs3092895,rs3092903 and rs3092905) are commonly found to be damaged by I-Mutant 2.0,SIFT and PolyPhen programs.With this effort,we modeled the mutant pRB proteins based on these deleterious nsSNPs.From a comparison of total energy,stabilizing residues and RMSD of these three mutant proteins with native pRB protein,we identified that the major mutation is from Glutamic acid to Glycine at the residue position of 746 of pRB.Further,we compared the binding efficiency of both native and mutant pRB (E746G) with E2F-2.We found that mutant pRB has less binding affinity with E2F-2 as compared to native type.This is due to sixteen hydrogen bonding and two salt bridges that exist between native type and E2F-2,whereas mutant type makes only thirteen hydrogen bonds and one salt bridge with E2F-2.Based on our investigation,we propose that the SNP with an id rs3092905 could be the most deleterious nsSNP in RB1 gene causing retinoblastoma.