Negative charge at the casein kinase II phosphorylation site is important for transformation but not for Rb protein binding by the E7 protein of human papillomavirus type 16.

Abstract

The human papillomavirus E7 protein is phosphorylated at the two serines in positions 31/32, which are part of a consensus sequence for casein kinase II (CKII). In this study, we have investigated the effect of CKII phosphorylation site mutations, all of which lead to unphosphorylated E7 proteins. The replacement of the two serines by uncharged alanine residues drastically reduced the ability of E7 to cotransform primary cells with ras, whereas negatively charged aspartic acid at the same positions produced only a slight effect. This difference was not reflected in the plO5Rb binding or the E2 promoter transactivation capability of these two mutants.Mutations that changed the CKII consensus without altering the serine residues also resulted in a loss of phosphorylation and transformation. This indicated that negative charge at positions 31/32 provided either by phosphorylation or by a negatively charged amino acid is necessary for efficient transformation without significantly affecting plO5Rb binding or transactivation.Human papillomavirus type 16 (HPV16) is believed to be involved in the etiology of human cervical cancer (1). In tumors, the viral DNA is frequently integrated (2) and only the early reading frames E6 and E7 are expressed (3). Recent studies have demonstrated that the expression of E6 together with E7 is necessary for the transformation of primary human cells (4-6). In addition, it was shown that the expression of the E7 open reading frame (ORF) in the absence of E6 is capable of transforming rodent fibroblast cell lines (7-10).The E7 protein shares several functional properties with the ElA protein of adenovirus type 5 in that E7 is able to cooperate with an activated ras gene product in the transformation of primary rodent cells (11-13) and it can transactivate the adenovirus E2 promoter (12). In addition, it was recently shown that the E7 protein, like ElA, participates in a complex with the retinoblastoma (Rb) gene product plO5Rb (Rb;.The E7 ORF of HPV16 encodes a nuclear, zinc-binding phosphoprotein with a calculated molecular mass of 11 kDa (17)(18)(19). The E7 protein is phosphorylated in vivo at only one site, 20). In this region, which lies just C terminal to the Rb binding region (residues 17-26), a consensus sequence for phosphorylation by casein kinase II (CKII; Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu; residues 30-37) can be found. In this study, we have explored the effects of sitespecific mutations within the CKII phosphorylation region on cotransformation, transactivation, and in vivo interaction with Rb.

PKN Binds and Phosphorylates Human Papillomavirus E6 Oncoprotein

“…Polyoma middle T phosphorylated on tyrosine residues mediates its association with SHC, phosphatidylinositol 3-kinase, and phospholipase C␥1 (73-78), whereas its serine phosphorylation mediates binding to 14-3-3 protein (50). Notably, HPV E7 phosphorylation by casein kinase II on serines 31 and 32 appeared to be important for its ability to transform primary cells when cointroduced with Ras (53,54). These studies suggest that phosphorylation of E6 may also play a role either in E6-induced immortalization or in the regulation of viral life cycle.…”

“…In this regard, it is noteworthy that the short form of cottontail rabbit papillomavirus E6 and HPV E7 oncoprotein have been shown to be phosphorylated (51)(52)(53)(54)(55). To assess potential phosphorylation of E6, we co-transfected PKN or kinase-defective PKN (K644E) with HPV16 E6 into 293T cells.…”

The binding domain structure of retinoblastoma‐binding proteins

“…DeCaprio et al, 1988Moran, 1988Dyson et al, 1990Dyson et al, 1990Dyson et al, 1990Dyson et al, 1990Dyson et al, 1990Munger et al, 1989Munger et al, 1989Firzlaff et al, 1991Firzlaff et al, 1991Firzlaff et al, 1991Munger et al, 1989Barbosa et al, 1990Barbosa et al, 1990Barbosa et al, 1990 Barbosa et al, 1990Barbosa et al, 1990Munger et al, 1989Moran, 1988Whyte, 1989 structure with a characteristic charged-group profile. First, a Glu or Asp appears one to three residues before the conserved Leu.…”

“…Although c-myc binds Rb, the interaction is weak (Rustgi et al, 1991), and the sequen,e similarity between c-myc and the other Rb-binding proteins is distant . The gene encoding E2F, a cellular transcription factor, has recently been cloned (Helin et al, 1992;Kaelin et al, 1992) (Barbosa et al, 1990;Firzlaff et al, 1991).…”

“…Genetic mapping Whyte et al, 1989;Barbosa et al, 1990;Firzlaff et al, 1991;Heck et al, 1992) and peptide-binding competition experiments (Jones et al, 1990;Kaelin et al, 1990) have demonstrated that this segment encodes a high-affinity Rb-binding domain. Short peptides (9-14 residues) containing the Rb-binding domain from SV40 T or papilloma virus E7 can compete against their fulllength counterparts for specific Rb-binding (Jones et al, 1990;Kaelin et al, 1990), implying that a short segment contains the necessary structural information to govern specific binding.…”