Srilata Bagchi

The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F

Recently, we identified an inhibitory protein, E2F-I, that blocks the DNA-binding activity of the transcription factor E2F. We also showed that the adenovirus E1A protein reverses this inhibitory activity of E2F-I, thereby restoring the DNA-binding activity of E2F. We have now further purified this inhibitory activity and show that the most purified preparation of E2F-I contains a 105 kd E1A-binding protein. This 105 kd E1A-binding protein cross-reacts with two different antibodies against the retinoblastoma (RB) gene product. Moreover, the RB gene product copurifies with E2F-I activity. Taken together, we conclude that the product of the RB gene is a part of E2F-I and is involved in the regulation of E2F activity.

The Human Papillomavirus E7 Oncoprotein Functionally Interacts with the S4 Subunit of the 26 S Proteasome

Human papillomaviruses (HPV) have been etiologically linked to human cervical cancer. More than 90% of cervical cancer tissues express two HPV-encoded oncoproteins E6 and E7. Both E6 and E7 proteins possess transformation activity. and together they cooperate to transform primary human keratinocytes, fibroblasts. and epithelial cells. The transforming activity of E7 is associated with its ability to bind the retinoblastoma tumor suppressor protein (Rb). However, the carboxyl-terminal mutants of E7 are also defective for transformation, suggesting that other cellular targets for E7 might exist. We screened a human placenta cDNA library by yeast two-hybrid assay using HPV 16 E7 as a bait and identified the subunit 4 (S4) ATPase of the 26 S proteasome as a novel E7-binding protein. E7 binds to S4 through the carboxyl-terminal zinc binding motif, and the binding is independent of E7 sequences involved in binding to Rb. The interaction between S4 and E7 can be easily detected by in vitro protein binding assays. Moreover, we found that E7 increases the ATPase activity of S4. A recent study has shown that, in epithelial cells, E7 degrades Rb through the 26 S proteasome pathway. We hypothesize that E7 might target Rb for degradation by 26 S proteasome through its interaction with the subunit 4 of the proteasome.

The p48 subunit of the damaged-DNA binding protein DDB associates with the CBP/p300 family of histone acetyltransferase

DDB has been implicated in DNA repair as well as transcription. Mutations in DDB have been correlated with the repair-deficiency disease, xeroderma pigmentosum group E (XP-E). The XP-E cells exhibit deficiencies in global genomic repair, suggesting a role for DDB in that process. DDB also possesses a transcription stimulatory activity. We showed that DDB could function as a transcriptional partner of E2F1. But the mechanism by which DDB stimulates E2F-regulated transcription or carry out its DNA repair function is not understood. To investigate the mechanisms, we looked for nuclear proteins that interact with DDB. Here we show that DDB associates with the CBP/p300 family of proteins, in vivo and in vitro. We suggest that DDB participates in global genomic repair by recruiting CBP/p300 to the damaged-chromatin. It is possible that the histone acetyltransferase activities of the CBP/p300 proteins induce chromatin remodeling at the damaged-sites to allow recruitment of the repair complexes. The observation offers insights into both transcription and repair functions of DDB.

Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex.

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.

Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2F- and Sp1-dependent transcription.

The retinoblastoma-related protein p107 has been shown to be a regulator of the transcription factor E2F. p107 associates with E2F via its pocket region and represses E2F-dependent transcription. In this study, we provide evidence for a novel interaction between p107 and the transcription factor Sp1. We show that p107 can be found endogenously associated with Sp1 in the extracts of several different cell lines. Moreover, in transient transfection assays, expression of p107 represses Sp1-dependent transcription. This repression of Sp1-dependent transcription does not require the DNA-binding domain of Sp1. Transcription driven by a chimeric protein containing the Ga14 DNA-binding domain and the Sp1 activation domains is inhibited by p107. Interestingly, unlike the repression of E2F-dependent transcription, the repression of Sp1-dependent transcription does not depend on an intact pocket region. We show that distinct regions of p107 are involved in the control of Sp1 and E2F.

Cul4A and DDB1 Associate with Skp2 To Target p27Kip1 for Proteolysis Involving the COP9 Signalosome

ABSTRACT DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.

Both Rb and E7 are regulated by the ubiquitin proteasome pathway in HPV-containing cervical tumor cells

High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. The E6 and E7 oncoproteins encoded by HPV target host cell tumor suppressor proteins. E6 induces proteolysis of p53 through the ubiquitin-proteasome pathway. Recent studies showed that overexpression of E7 caused proteolytic degradation of the tumor suppressor Rb. However, unlike p53, Rb is not regulated by proteolysis in normal cells. In addition, it was unclear whether in its natural context E7 regulates Rb through the ubiquitin-proteasome pathway. Therefore, we sought to determine whether Rb is regulated by the ubiquitin-proteasome pathway in HPV-containing tumor cells. We carried out a detailed analysis in Caski cells, that are derived from HPV-containing cervical cancer tissues. Studies with various protease inhibitors revealed that Rb is regulated specifically by the ubiquitin-proteasome pathway in HPV-containing cervical tumor cells. Several inhibitors of the 26S proteasome significantly increased the level of Rb in the Caski cells. Rb controls cell growth by forming complexes with the E2F-family transcription factors. Surprisingly, in spite of a significant accumulation of the hypophosphorylated form of Rb, no Rb/E2F complex was detectable in the proteasome inhibitor treated cells. Further analysis revealed that there was an increased accumulation of the E7 oncoprotein. We showed that the proteasome inhibitors simultaneously blocked the proteolysis of E7 and Rb, suggesting that E7 is also regulated by the ubiquitin-dependent proteolysis in cervical cancer cells. Taken together, this study suggests that targeted inhibition of Rb proteolysis will be required for restoring Rb function in HPV-containing cervical cancer cells.

p21 Disrupts the interaction between cdk2 and the E2F-p130 complex.

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculated that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.

Cul4A Physically Associates with MDM2 and Participates in the Proteolysis of p53

The cullin 4A (Cul4A) gene is amplified and overexpressed in breast and hepatocellular carcinomas. Cul4A functions as an E3 ligase and participates in the proteolysis of several regulatory proteins through the ubiquitin-proteasome pathway. Here, we show that Cul4A associates with MDM2 and p53. Depletion of Cul4A leads to an accumulation of p53. Moreover, expression of Cul4A increases the decay-rate of p53 and delays the accumulation of p53 in response to DNA damage. Cul4A fails to increase the decay of p53 in mouse embryonic fibroblasts lacking MDM2. In addition, the Cul4A-mediated rapid decay of p53 is blocked by p19ARF. The results provide evidence for a role of Cul4A in the MDM2-mediated proteolysis of p53.

DDB2 decides cell fate following DNA damage

The xeroderma pigmentosum complementation group E (XP-E) gene product damaged-DNA binding protein 2 (DDB2) plays important roles in nucleotide excision repair (NER). Previously, we showed that DDB2 participates in NER by regulating the level of p21Waf1/Cip1. Here we show that the p21Waf1/Cip1 -regulatory function of DDB2 plays a central role in defining the response (apoptosis or arrest) to DNA damage. The DDB2-deficient cells are resistant to apoptosis in response to a variety of DNA-damaging agents, despite activation of p53 and the pro-apoptotic genes. Instead, these cells undergo cell cycle arrest. Also, the DDB2-deficient cells are resistant to E2F1-induced apoptosis. The resistance to apoptosis of the DDB2-deficient cells is caused by an increased accumulation of p21Waf1/Cip1 after DNA damage. We provide evidence that DDB2 targets p21Waf1/Cip1 for proteolysis. The resistance to apoptosis in DDB2-deficient cells also involves Mdm2 in a manner that is distinct from the p53-regulatory activity of Mdm2. Our results provide evidence for a new regulatory loop involving the NER protein DDB2, Mdm2, and p21Waf1/Cip1 that is critical in deciding cell fate (apoptosis or arrest) upon DNA damage.