Human Papillomavirus Type 16 E7 Oncoprotein Binds and Inactivates Growth-Inhibitory Insulin-Like Growth Factor Binding Protein 3

Abstract

The E7 protein encoded by human papillomavirus type 16 is one of the few viral genes that can immortalize primary human cells and thereby override cellular senescence. While it is generally assumed that this property of E7 depends on its interaction with regulators of the cell cycle, we show here that E7 targets insulin-like growth factor binding protein 3 (IGFBP-3), the product of a p53-inducible gene that is overexpressed in senescent cells. IGFBP-3 can suppress cell proliferation and induce apoptosis; we show here that IGFBP-3-mediated apoptosis is inhibited by E7, which binds to IGFBP-3 and triggers its proteolytic cleavage. Two transformation-deficient mutants of E7 failed to inactivate IGFBP-3, suggesting that inactivation of IGFBP-3 may contribute to cell transformation.Human papillomaviruses (HPVs) of the high-risk group (e.g., HPV-16) cause cancers in humans, while papillomaviruses of the low-risk group (e.g., HPV-11) cause benign epithelial hyperproliferation (90). Cell transformation by highrisk HPVs requires expression of the viral genes E6 and E7 (for a review, see reference 1). Coexpression of HPV-16 E6 and E7 is sufficient to immortalize primary human keratinocytes (35, 57), the natural host cells for papillomavirus infection. The E6 protein of HPV-16 interacts with the p53 tumor suppressor, which leads to recruitment of the ubiquitin ligase E6AP (39), resulting in the ubiquitination and subsequent degradation of p53 (72). Consequently, p53-dependent upregulation of growth-inhibitory genes, such as p21WAF-1 (27), is abrogated. A major target for the E7 oncoprotein of HPV-16 appears to be the p16/Rb pathway, as it is known that E7 binds to all three members of the retinoblastoma protein family and abrogates their growth-suppressive function (for a review, see reference 83); consequently, E7-expressing cells are refractory to growth inhibition by the cyclin-dependent kinase (cdk) inhibitor p16INK4 (49, 50). The identification of specific target proteins for E6 and E7 suggests that both viral oncoproteins target nonidentical regulatory pathways and that immortalization depends on the combined action of both gene products. However, it is known that expression of E7 alone is sufficient to immortalize human cells (85), albeit at reduced efficiency, compared to the simultaneous expression of both E6 and E7 (35). This indicates that E7 may also impinge on growth regulatory pathways that are principal targets for E6. In support of this hypothesis, it was shown that E7 binds and inactivates the cdk inhibitor p21WAF-1 (28, 42), which is encoded by a p53-inducible gene (27). This observation provides an explanation for how p53-mediated growth arrest can be undermined by E7 in the absence of E6, i.e., in cells where p53 is present and functional.Immortalization of mammalian cells is considered the first step in tumorigenesis (88) which abrogates a cellular senescence program that is characterized by irreversible cell cycle exit after extended passaging. There is evidence that mitogenic signal transduction is ...

Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A

“…Two of these mutants, p2Pro in the conserved domain 1 of E7 and p24Gly in the pocket protein-binding domain of conserved domain 2 have already been described (Banks et al, 1990) as has D79-83 in the cysteine loop of E7 . Mutant D79-83 has been demonstrated to be defective for binding to IGFBP-3 (Mannhardt et al, 2000), whereas mutant p24Gly is defective for binding to pRB. Additionally, mutant p24 has previously been suggested to be defective for increasing PKB/Akt phosphorylation HPV-16 E7 induces PKB phosphorylation on both thr308 and ser473 in part in a PI3K-independent manner.…”

“…In addition, when the PI3K/ PKB pathway is activated by insulin-like growth factor-1 (IGF-1), the activation can be inhibited by insulin-like growth factor binding protein 3 (IGFBP-3) (Huynh et al, 2002). Interestingly IGFBP-3 has previously been demonstrated to be a target of the E7 proteins from high-risk mucosal HPVs (Mannhardt et al, 2000). The pathway is also believed to be antagonized by RAF, an upstream component of the mitogen-activated protein kinase (MAPK) pathway (Westbrook et al, 2002), which has been demonstrated to be activated in HPVinfected keratinocytes, in response to expression of the E5 gene (Pim et al, 1992;Gu and Matlashewski, 1995;Crusius et al, 1997;Johnston et al, 1999).…”

Insulin-Like Growth Factor-Binding Protein 3 Expression Increases during Immortalization of Cervical Keratinocytes by Human Papillomavirus Type 16 E6 and E7 Proteins

“…23 This study had two major differences from ours: they used foreskin keratinocytes rather than cervical cells, and they transfected the isolated HPV E7 oncogene rather than the combined E6/E7 oncogenes. These results suggest that either cell type or HPV oncogene expression could account for the observed difference.…”

Solution structure of the partially folded high-risk human papilloma virus 45 oncoprotein E7

“…In studies addressing functional E7 interactions with cellular proteins, for example, IGFBP3, pCAF, HDAC1 and PML4 (Brehm et al, 1999;Mannhardt et al, 2000;Avvakumov et al, 2003;Bischof et al, 2005), several hydrophobic residues of the monomer core or the dimerization interface were deleted (e.g. L87-F91) or exchanged against charged residues, for example, L75, L90 and F91 to Arg.…”