Proc. Nati. Acad. Sci. USA Vol. 88, pp. 5187-5191, June 1991 Biochemistry

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 (in vitro mutagenesis/ras/protein structure)

JULIANE M. FIRZLAFF*, BERNHARD LUSCHER*, AND ROBERT N. EISENMAN Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98104

Communicated by Edwin G. Krebs, March 25, 1991 (receivedfor review August 24, 1990)

The human papillomavirus E7 protein is ABSTRACT 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; refs. 14-16). The E7 ORF of HPV16 encodes a nuclear, zinc-binding phosphoprotein with a calculated molecular mass of 11 kDa (17-19). The E7 protein is phosphorylated in vivo at only one site, Ser-31/32 (16, 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. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5187

MATERIALS AND METHODS Construction of Mutants. A 4.4-kilobase (kb) HindIII/ EcoRI fragment of plasmid p1059 (nucleotides 79-4468 of HPV16; ref. 12) was cloned into pBS+ (Stratagene). For the site-directed mutagenesis, a kit from Amersham was used according to the manufacturer's recommendations with a minor modification: double-stranded recombinant vector DNA was digested with Nco I (at site 863 in HPV16) and HindIII (at site 932 in pBS+) followed by the isolation and heat denaturation of the large fragment. After the Exo III digestion step, this denatured DNA was added to the mutagenesis reaction mixture to serve as primer for the secondstrand synthesis. The following oligonucleotides were used for the mutagenesis [changes compared to wild type (wt) are underlined]: 5'-ATC CTC CTC CTC TGC AGC GTC ATT TAA TTG CTC AT-3' (alanine), 5'-TC ATC CTC CTC CTC GC GTC GTC ATT TAA TTG CTC AT-3' (aspartic acid), 5'-GC TGG ACC ATC TAT TTG CTG CTG CTQ CTG TGA GCT CTG ATT TAA TTG CTC ATA ACA G-3' (glutamine). A HindIII/Kpn I fragment, encompassing the promoterless E6 and E7 ORFs (nucleotides 79-880 of HPV16) from the wt and mutant clones, was introduced into the mammalian expression vector pEQ176P2. The parent vector pEQ176 (to be described elsewhere; A. Geballe, personal communication) was derived from pON249 (21) and contained the cytomegalovirus immediate early promoter (a 1.1-kb Pst I/Sst I fragment) followed by a polylinker and driving the expression of the bacterial 8-galactosidase gene. In pEQ176P2, the majority of the ,B-galactosidase gene was removed. The HPV16 E6-E7 wt and mutant fragments were inserted into the polylinker of pEQ176P2. Simian virus 40 sequences provided the signal for poly(A) addition and a eukaryotic origin of replication. Double-stranded sequencing of the entire E7 sequence in the recombinant plasmids used for the transfections confirmed the wt and predicted mutant alanine, aspartic acid, and glutamine sequences, respectively. The mutant E7 ORF from plasmid ADLYC (kindly provided by P. Howley, National Cancer Institute), which contains a deletion in the Rb binding site and does not associate with Rb in vitro (15), was excised and cloned into pEQ176P2. Transfections. COS-7 cells were transfected by the method described by Chen and Okayama (22) with 20 ,ug of E7 wt or mutant plasmid DNA per 100-mm plate. After 48 hr, the cells were labeled with [35S]methionine or 32Pi and the radioimAbbreviations: CAT, chloramphenicol acetyltransferase; CKII, casein kinase II; HPV16, human papillomavirus type 16; ORF, open reading frame; Rb, retinoblastoma susceptibility p105 protein; wt, wild type. *Present address: Institute for Molecular Biology, Medizinische Hochschule Hanover, 3000 Hanover 61, Federal Republic of Germany.

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Biochemistry: Firz1aff et al.

munoprecipitations followed by SDS/15% PAGE were performed as described (20). The anti-HPV16E7 antibody was raised against a bacterial trpE-E7 fusion protein (20). The preparation of primary baby rat kidney (BRK) cells and the transfections were carried out as described (23) using 1 ug of E7 (mutant) plasmid DNA, 1 Lg -of pT24 (24) expressing an activated ras gene, and 8 ,ug of salmon sperm DNA per 60-mm plate. After 3-4 weeks, the plates were scored for morphologic transformants. For the chloramphenicol acetyltransferase (CAT) assays, NIH 3T3 cells were transfected in duplicate as described above with 15 Ag of E7 (or E7 mutant) plasmid DNA, 3 ;Lg of pEC plasmid (25) containing the E2 promoter of adenovirus driving the CAT gene, and 2 ixg of pEQ176 plasmid per 100-mm plate. Forty-eight hours after transfection, the CAT assays were performed essentially as described by Gorman et al. (26). f-Galactosidase expression was measured in a colorimetric assay using 4-methylumbelliferyl j-D-galactosidase (27), and the amount of input cell lysate was adjusted for transfection efficiency using the P3-galactosidase expression levels for normalization. The extent of acetylation was assessed by densitometry using the bioimage system (BioImage, Ann Arbor, MI). All cells were maintained in Dulbecco's modified Eagle's medium with 10o fetal bovine serum. In Vivo Binding of E7 to Rb. Two days after transfection, the COS-7 cells were labeled for 1 hr with 0.5 mCi of [35S]methionine (1 Ci = 37 GBq) as described above. The cells were then Iysed in L buffer (250 mM NaCl/50 mM Hepes, pH '7.5/5 mM EDTA/0.5 mM dithiothreitol/0.1% Nonidet P-40/0.2 mM phenylmethylsulfonyl fluoride/0.5% aprotinin/5 mM NaF/10 mM /3-glycerophosphate) and centrifuged, and the supernatant was incubated with C36 anti-Rb monoclonal antibody (a gift from P. Whyte; see ref. 28) and 2 ul of rabbit anti-mouse IgG antiserum. Alternatively, a polyclonal rabbit antiserum raised against a synthetic peptide encompassing amino acids 896-913 of Rb (from P. Whyte) was used. The incubation was carried out in the presence of protein A-Sepharose CL-4B beads (Sigma) for 1 hr at 4°C. The beads were washed three times with L buffer and the proteins were separated by SDS/15% PAGE. An aliquot of each cell lysate was incubated with 10 ,ul of rabbit polyclonal anti-HPV16E7 antiserum and protein A-Sepharose CL-4B beads in the presence of 0.5% each of Nonidet P-40, deoxycholate, and SDS. After incubation for 1 hr at 4°C, the- beads were washed three times in RIPA buffer (20) and analyzed as described above.

Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 1. Structure of the wt and mutated E7 ORF of HPV16. (Upper) Diagram represents the E7 ORF. The first methionine, the position ofthe sequences necessary for Rb binding (15), and the CKII site (20) are indicated. (Lower) The wt and mutant DNA sequences at the CKII site are 'shown, with altered nucleotides in italics. The corresponding amino acid sequence is depicted underneath with the substituted amino acids highlighted.

could be detected in the cells transfected with the vector pEQ176P2 (lane 1). The E7 protein does not migrate according to its calculated molecular mass of 11 kDa in SDS/PAGE and the 19-kDa protein is of the size expected from previous studies (3, 20). The glutamine mutant protein had a slightly A

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RESULTS Generation and Expression of Mutant E7 Proteins. The E7 protein of HPV16 is phosphorylated in vivo at Ser-31/32 within the CKII consensus sequence (16, 20). To study the effect ofalterations in the CKII site on the transformation and transactivation functions of E7, three mutations (Fig. 1) were generated by site-directed mutagenesis. The two serines in the CKII site were replaced by two nonpolar (alanine) and two acidic (aspartic acid) amino acids. For the third mutation (glutamine), the six acidic residues of the CKII site were substituted by glutamines. The wt and mutant E7 sequences together with E6 were cloned into the mammalian expression vector pEQ176P2 under the control of a cytomegalovirus immediate early promoter. To monitor the expression of the E7 proteins, COS-7 cells were transiently transfected with the E7-expressing vectors, labeled with V35S]methionine, and immunoprecipitated with a previously described antiHPV16E7 antibody (20). As shown in Fig. 2A, the immune serum precipitated a protein of =19 kDa (lanes 2-4) in the cells transfected with the E7 wt, alanine, and aspartic acid expression plasmids, respectively, whereas no such protein

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FIG. 2. Expression of E7 wt and mutant proteins in COS-7 cells. COS-7 cells were transfected with the vector alone; plasmids expressing the wt E7 protein; or the alanine, aspartic acid, and glutamine mutant proteins as indicated. The cells were labeled with [35S]methionine (A) or [32P]phosphate (B) and immunoprecipitated with anti-HPV16E7 antiserum. The positions of molecular mass markers are indicated (in kDa) on the right.

Proc. Natl. Acad. Sci. USA 88 (1991)

Biochemistry: Firz1aff et al. slower electrophoretic mobility compared to wt. In cells expressing the E7 aspartic acid mutant, an additional band of smaller molecular mass was detected. In pulse-chase experiments, no precursor product relationship between these two forms was observed. The half-lives of all the E7 wt and mutant proteins were the same (data not shown). To assess whether the mutations had the predicted effect on the ability of the proteins to be phosphorylated in vivo, a second set of COS-7 cells transfected in parallel with the different E7 constructs was labeled with [32P]phosphate and immunoprecipitated with anti-E7 antiserum. As shown in Fig. 2B, phosphate could only be incorporated into the wt E7 protein, whereas no phosphorylation was detected in the proteins in which the acceptor serines were mutated to alanine or aspartic acid or in which the consensus sequence around the acceptor serines was altered (glutamine mutant). Cotransformation of BRK Cells. The E7 protein of HPV16 is capable of transforming primary BRK cells in cooperation with an activated ras oncogene product (12, 13). We therefore examined the transforming potential of the mutant E7 proteins in this assay. Primary cultures of kidney cells from 6-day-old Fischer rats were cotransfected with pT24, expressing the activated T24 Ha-rasl gene, and the different E7-encoding plasmids. After 3-4 weeks, the cells were stained and foci of transformed cells were counted (Fig. 3). The transformation of the BRK cells was not greatly affected by the aspartic acid mutation relative to wt, whereas the alanine and glutamine mutations severely impaired the cooperation of the mutant E7 protein with the ras oncoprotein. Two types of transformed BRK cells transfected with the wt and aspartic acid mutant constructs were observed, one of fibroblastoid and the other of epithelioid morphology (data not shown). From both cell types, immortalized cell lines could be established and expression of either wt or aspartic acid mutant E7 protein was detected (data not shown). In contrast, none of the alanine, glutamine, or vector transfected cells gave rise to immortalized cell lines. In Vivo Binding of wt and Mutant E7 to Rb. We next assessed whether the mutations at the CKII consensus site affected the ability of the different E7 proteins to bind to Rb. Since Rb is constitutively expressed in COS-7 cells (data not shown; ref. 29), we introduced wt and mutant E7 proteins into these cells and determined whether these proteins formed a complex with Rb. Two days after transfecting the different constructs into COS-7 cells, the cells were lysed under mild conditions to preserve potential protein-protein interactions. The E7 wt (lane 2) as well as the three mutant E7 proteins (lanes 3-5) were found to be coprecipitated with (O

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an anti-Rb monoclonal antibody C36 (Fig. 4A) consistent with previous work, demonstrating association of Rb with wt E7 (14-16). The total amount of E7 proteins present in the lysate was determined by precipitation with an anti-E7 antiserum (lanes 7-10). The amount of E7 proteins coprecipitated with Rb in three independent experiments was compared to the total amount of E7 protein by densitometric scanning. Between 1% and 10% of the wt, alanine, and aspartic acid mutant proteins were bound but no consistent, statistically significant difference in the binding between these three proteins was found. However, we cannot rule out the possibility of minor differences in the binding affinities. In contrast, we consistently detected only between 0.3% and 0.5% of the glutamine mutant protein present in the Rb complex, indicating that the rather extensive mutations in the glutamine protein had a more general effect on the structure of the protein and prevented efficient binding. To demonstrate the specificity of E7 binding to Rb in our assay, we also transiently expressed in COS-7 cells a mutant E7 protein that has been previously shown to be unable to bind plO5Rb in vitro (15). The construct was derived from plasmid ADLYC, containing a deletion of four amino acids in the Rb binding domain of E7 (15). After immunoprecipitation under mild conditions, using a rabbit polyclonal antiserum against an Rb peptide (Fig. 4B) or the monoclonal antibody C36 (data not shown), only the wt E7 protein was coprecipitated (lane 3), whereas ADLYC mutant E7 protein was not (lane 2), even though approximately equal amounts of the two proteins were present in the transfected cells (lanes 4 and 5, respectively). In addition, E7 was not immunoprecipitated under mild conditions with a monoclonal anti-myc antibody or with a polyclonal rabbit anti-mouse IgG antiserum (data not shown). Furthermore, no coprecipitation of other transiently expressed proteins such as c-myc and erbA, which are not known to associate with Rb, was observed with the C36 anti-Rb antibody (data not shown). Taken together, we conclude that phosphorylation of E7 is not required for Rb

binding. Transactivation of the Adenovirus E2 Promoter by E7 Mutant Proteins. The E7 protein is also functionally similar to the ElA protein of adenovirus in its ability to transactivate the E2 promoter of adenovirus (12). To test the mutant E7 proteins for this function, we used plasmid pEC (25), which expresses the CAT gene under the control of the adenovirus E2 promoter. As an internal control for variations in trans-

fection efficiency, the plasmid pEQ176, which encodes j3-galactosidase, was used. These plasmids were cotransfected in duplicate into NIH 3T3 cells with the different E7-expressing plasmids or the vector pEQ176P2. A CAT assay was performed from the cell lysates (Fig. 5A) and the percentage of acetylation was measured by densitometry. Fig. SB represents the combined results of four independent experiments done in duplicate. The transactivation potential of the E7 protein did not appear to be grossly affected by the alanine (lanes 3 and 4) or aspartic acid (lanes 5 and 6) mutation in the CKII site with 44.5% and 35.6% acetylation, respectively, compared to 38.5% for the wt protein (lanes 1 and 2). The transactivation of the glutamine mutant (lanes 9 and 10) was reduced to 14.5% acetylation, which was still well above the value for the vector (1.2%), which represents the basal level of the E2 promoter (lanes 7, 8, 11, and 12).

DISCUSSION In this study, we have determined that the phosphorylation of the E7 protein of HPV16 at Ser-31/32 is important for its transformation function. The presence of negatively charged amino acids such as aspartic acid can substitute for the two serines at this position, whereas uncharged alanine residues are unable to do so. In addition, altering the consensus

Biochemistry: Firz1aff et al.

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sequence for CKII phosphorylation around Ser-31/32 prevented phosphorylation and severely impaired transformation by E7. The binding of wt and the mutant E7 proteins to Rb was retained but a reduction in the binding affinity of the E7 glutamine mutant protein was observed. The transacti-

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different E7 proteins. NIH 3T3 cells were transfected in duplicate with the vector; E7 wt; and mutant alanine, aspartic acid, and

glutamine protein-expressing plasmids as indicated, together with plasmids pEC and pEQ176. (A) Cell extracts were normalized for their ,3-galactosidase expression and assayed for CAT activity. (B) The average percentage acetylation from four independent experiments done in duplicate is shown and the standard deviation is indicated.

COS-7 cells were transfected with the different E7-expressing constructs as indicated (see also Fig. 2), labeled with [35S]methionine, and immunoprecipitated with C36 anti-Rb antibody (lanes 1-5) or with anti-E7 antiserum (lanes 6-10). (B) COS-7 cells were transfected with constructs expressing the wt E7 or the ADLYC E7 mutant protein as indicated, labeled with [35S]methionine, and immunoprecipitated with a rabbit anti-Rb

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vation potential of the E7 protein was not the determining factor for the transformation ability. It is not known at this point whether both Ser-31/32 are phosphorylated simultaneously or only one at a time with a possible preference of one over the other. After mutagenesis of either serine in trpE-E7 fusion proteins, the remaining one can still be phosphorylated by CKII in vitro (16), suggesting that both serines can be CKII targets. Similar conclusions have been drawn for the Myc oncoprotein, which can be phosphorylated by CKII at multiple adjacent sites (30). In E7, upon substitution of the two serines at positions 31/32 by two alanines, this region is only slightly altered compared to having unphosphorylated serine at this position (data not shown) when analyzed for protein conformation and hydropathy (31, 32). Therefore, the alanine mutant can be considered as a constitutively unphosphorylated E7 protein. To mimic a fully phosphorylated state, two aspartic acids were introduced in place of the two serines. This raises the negative charge of this region by 2 but not as much as phosphorylation of the two serines, which would add the equivalent of 3 negative charges. In our tests of the above-mentioned mutants for cotransformation of BRK cells in conjunction with an activated ras gene product, it was evident that the aspartic acid mutant transformed almost as efficiently as the wt E7 protein, whereas the alanine mutant transformed 10 times less well (Fig. 3). Therefore, it can be concluded that the introduction of negative charge at positions 31/32 is necessary for efficient transformation by E7 and it seems likely that CKII phosphorylation may act to positively regulate this function of the E7 protein. Evidence is accumulating that a phosphorylated serine or threonine sometimes can be functionally replaced by a negatively charged amino acid (33-35). In the glutamine mutant protein, the acceptor serines were in place but the 6 acidic amino acids around them that constitute the CKII consensus site were changed into glu-

Biochemistry: Firz1aff et al. tamines. As predicted from studies with synthetic peptides (36, 37), no in vivo phosphorylation was detectable at the site with the altered consensus sequence. This result strengthens the argument that such sites are targets for CKII in vivo. While these studies were under way, it was reported (38, 39) that CKII site mutations have only a limited effect on transformation. Since these were single amino acid changes that are likely to interfere only partially with phosphorylation, the small effect on transformation is not surprising. In contrast, in two other reports mutations of the two serines at

positions 31/32 to Arg/Pro (16) or only arginine (40) reduce the ability of the E7 protein to induce transformation but these changes introduce amino acids that are structurally very different from serine. Nonetheless, taken together with our data they provide strong support for the importance of phosphorylation at this site. Although CKII phosphorylation can alter the transformation potential of E7, it is clear from recent studies on both E7 (16, 38) and ElA (41) that the binding of Rb is another factor possibly involved in the transforming potential of these two proteins. Since the CKII site is adjacent to the Rb binding site (see Fig. 1), it was suggested that CKII might regulate association with Rb (20). However, in this study no consistent difference between wt and alanine and aspartic acid mutant E7 proteins in their in vivo binding to Rb was found. Only the rather extensive glutamine mutant protein showed

decreased binding efficiency. This result is in agreement with reports on other E7 mutants showing that mutations in and around the CKII site do not abolish in vitro Rb binding (16). The possibility that binding of Rb protein is not sufficient for transformation is also supported by the fact that mutations in the N- and C-terminal regions of the E7 protein, outside of the Rb binding site and CKII recognition sequence, can affect the transformation capacity of E7 (38). Comparison of the wt with the alanine and aspartic acid mutants indicates that no gross alteration in the transactivation was detected, indicating that phosphorylation may not be important for the regulation of this activity. As the alanine mutant as well as a mutant described by Edmonds and Vousden (p35/36 Asp/His; ref. 38), whose transactivation potential was highly impaired, are both able to transform, it can be concluded that transformation is not mediated through transactivation by the E7 protein. Up to now, no cellular targets for transactivation by E7 have been reported. Our data show that there is an additional function of the E7 protein separate from binding to Rb but critical for transformation, which is mediated by a negative charge at the CKII site. Therefore, it would be of interest to know what the levels of CKII expression are in cells that are targets for HPV transformation and to what extent environmental signals might influence both CKII activity and E7 function. We are grateful to Drs. A. Geballe, P. Howley, and J. Nevins for plasmids and P. Whyte for anti-Rb antibodies. We thank J. Cooper, P. Kaur, and P. Whyte for critically reading the manuscript; E. Tolentino for oligonucleotide synthesis; and P. Goodwin and S. Spyropoulos of the Image Analysis Laboratory for densitometry.

This work was supported by Grant PO1CA28151 to R.N.E.

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