Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein.

Hagemeier, Christian; Caswell, Richard; Hayhurst, Graham P.; Sinclair, John; Kouzarides, Tony

doi:10.1002/j.1460-2075.1994.tb06584.x

Cited by 169 publications

(190 citation statements)

References 45 publications

Supporting

Mentioning

184

Contrasting

Unclassified

Order By: Relevance

“…Whereas the IE proteins of several other DNA viruses interact with the pocket proteins, IE72 additionally interacts with the E2Fs (14). Although IE72 cannot interact with pRB (14), it does interact with p107 (35), and IE86 associates with pRB (14).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of a Viral Kinase That Phosphorylates Specific E2Fs and Pocket Proteins

Pajovic

Wong

Black

et al. 1997

Molecular and Cellular Biology

View full text Add to dashboard Cite

The transcription factor E2F and its regulation by pRB and related pocket proteins are central to cell cycle control in higher eukaryotes. Much of our knowledge of this regulation has come from studies using immediate-early proteins of DNA tumor viruses. Previously, we reported that the 72-kDa immediate-early region 1 gene product of the human cytomegalovirus, IE72, transactivates the dihydrofolate reductase promoter through the E2F site and that it physically interacts with E2F1 (M. J. Margolis, S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and J. C. Azizkhan, J. Virol. 69:7759-7767, 1995). In this study, we further characterized the mechanism by which IE72 modulates E2F-dependent transcription. In vitro phosphorylation reactions using gel-purified bacterially expressed proteins revealed that IE72 is a kinase that autophosphorylates and phosphorylates E2F1, -2, and -3 (but not E2F4 or -5) and the RB-related pocket proteins p130 and p107 (but not pRB). The region of IE72 spanning amino acids 173 to 197 shows a high level of homology to the ATP binding sites in over 500 kinases. The kinase-negative protein IE72⌬ATP, from which this region has been deleted, cannot activate E2F-dependent transcription. The kinase activity of IE72 is also required for its ability to reduce the association of E2F4 with p107 and p130. Taken together, these data suggest that the kinase activity of IE72 is required for E2F-dependent transcriptional activation and that this is likely to result from phosphorylation of specific members of the E2F and pocket protein families by IE72.E2F was originally identified as a transcription factor required by E1A for activation of the adenovirus E2 promoter. The gene encoding dihydrofolate reductase (DHFR) was the first cellular gene shown to contain a binding site for E2F (6), and transactivation of its promoter by adenovirus E1A was found to be E2F dependent (18). Subsequently, E2F was shown to be involved in the cell cycle regulation of several genes important in cellular growth control (for reviews, see references 23, 24, and 38). E2F activity is regulated by its interaction with the product of the tumor suppressor retinoblastoma susceptibility gene, pRB, and the related pocket proteins, p107 and p130, which are themselves tightly regulated during differentiation (36) and the cell cycle (for a review, see reference 32). With the cloning of E2F1, followed by the cloning of other, related proteins, E2F was shown to consist of a family with five members that heterodimerize with DP1 or DP2, which are members of a related protein family (for a review, see reference 23). The different E2F family members show specificity in their interactions with the pocket proteins; whereas E2F4 is able to interact with all of the pocket proteins, E2F1, -2, and -3 appear to interact only with pRB and E2F5 apparently interacts only with p130 (4,13,16,19,21,25,29). Modulation of transcription from promoters containing E2F sites can be achieved through increased E2F transactivation activity by release of free E...

show abstract

Section: Discussionmentioning

confidence: 99%

“…Although IE72 cannot interact with pRB (14), it does interact with p107 (35), and IE86 associates with pRB (14). As such, HCMV major IE proteins together may fulfill a role similar to that of the IE proteins of other DNA viruses, and the kinase activity of IE72 appears to be involved.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Viral Kinase That Phosphorylates Specific E2Fs and Pocket Proteins

Pajovic

Wong

Black

et al. 1997

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…Recombinant proteins were expressed in, and puri®ed from, E. coli as reported previously (Bannister et al, 1991). Pull-down assays were performed as described previously (Hagemeier et al, 1994).…”

Section: Gst Fusion Proteins and Pull-down Assaymentioning

confidence: 99%

“…Thirty-six hours after transfection cells were harvested by scraping into PBS, pelleted then lysed by rotation in EBC bu er containing 200 mM NaCl as described (Hagemeier et al, 1994). Immunoprecipitations were carried out using an anti RB monoclonal antibody (G3-245, Pharmingen) or anticyclin B2 antibody (gift from J Pines) in the presence of protein G-Sepharose and protein A-agarose (Sigma).…”

Section: Coimmunoprecipitation Of Rb and Hbp-1 Proteinsmentioning

confidence: 99%

“…Immunoprecipitations were carried out using an anti RB monoclonal antibody (G3-245, Pharmingen) or anticyclin B2 antibody (gift from J Pines) in the presence of protein G-Sepharose and protein A-agarose (Sigma). Precipitates were washed ®ve times in NETN bu er as described (Hagemeier et al, 1994), separated by SDS ± PAGE and blotted onto nitrocellulose. Western blotting was performed with anti-HA monoclonal antibody (12CA5, Boehringer).…”

Section: Coimmunoprecipitation Of Rb and Hbp-1 Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A

et al. 1997

View full text Add to dashboard Cite

A yeast two-hybrid screen has identi®ed HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1 ± a high a nity binding site, mediated by an LXCXE motif and a separate low a nity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N-and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP coactivator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture.

show abstract

The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts

et al. 2000

View full text Add to dashboard Cite

Human cytomegalovirus (HCMV) infection is associated with excessive proinflammatory immune responses such as cytokine/chemokine production or upregulation of adhesion molecules on the host cells. It is assumed that these features of HCMV-related immunopathology can not be treated effectively with currently available anti HCMV drugs. In the present study the efficacy of ganciclovir (GCV), foscarnet (PFA), cidofovir (HPMPC), and ISIS 2922, an antisense oligonucleotide complementary to HCMV immediate-early (IE) mRNA, was investigated on HCMV-induced secretion and functional activity of the C-X-C chemokine IL-8 and the expression of the intercellular adhesion molecule-1 (ICAM-1). As compared with mock-infected cells IL-8 production was increased up to 9-fold and ICAM-1 expression up to 4-fold in HCMV-infected fibroblasts. Treatment of infected cells with GCV (40 microM), PFA (200 microM) or HPMPC (2 microM) suppressed completely virus replication as demonstrated by quantification of late (L) antigen expression and infectious virus production. These drugs, however, failed to inhibit IE antigen expression and did not prevent HCMV-induced upregulation of IL-8 and ICAM-1. In contrast, ISIS 2922 (1 microM) suppressed both IE and L antigen expression by 99% and inhibited infectious virus production by 10(4)-fold. Moreover, ISIS 2922 significantly suppressed HCMV-induced upregulation of both IL-8 and ICAM-1 expression on the transcriptional and on the protein level. Our results indicate that ISIS 2922 but not inhibitors of HCMV DNA prevents HCMV-induced upregulation of IL-8 and ICAM-1, both hallmarks of inflammatory processes. Thus, inhibition of HCMV IE expression with ISIS 2922 may be an important strategy for the treatment of HCMV-related immunopathogenesis.

show abstract

Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein.

Cited by 169 publications

References 45 publications

Identification of a Viral Kinase That Phosphorylates Specific E2Fs and Pocket Proteins

Identification of a Viral Kinase That Phosphorylates Specific E2Fs and Pocket Proteins

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A

The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts

Contact Info

Product

Resources

About