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Cell Growth & Differentiation Vol. 11, 425-435, August 2000
© 2000 American Association for Cancer Research

Casein Kinase II Phosphorylation of the Human Papillomavirus-18 E7 Protein Is Critical for Promoting S-Phase Entry1

Wei-Ming Chien, Jacqueline N. Parker2, Delf-Christian Schmidt-Grimminger, Thomas R. Broker and Louise T. Chow3

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase {alpha} or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The productive phase of HPV4 infections takes place only in keratinocytes that are undergoing terminal squamous differentiation in hyperproliferative benign lesions of cutaneous or mucosal epithelia, variably called warts, papillomas, or condylomata. Viral DNA and RNA are very low in abundance in basal and parabasal cells that maintain the ability to proliferate but increase dramatically in a fraction of differentiated spinous cells. Progeny virus is then produced in some of the superficial cells prior to programmed cell death (reviewed in Ref. 1 ). A small fraction of infections by certain HPVs in the anogenital epithelia can lead to dysplasias and carcinomas that no longer support viral reproduction. Thus, the mucosotropic HPVs are grouped into the non-oncogenic types, typically HPV-6 and HPV-11, and the oncogenic genotypes, including HPV-16 and HPV-18. Viral E7 and E6 genes are invariably up-regulated in cycling cells in high-grade dysplasias and cancers, relative to productively infected cells, implicating them in oncogenesis (reviewed in Ref. 1 ).

The purpose of the HPV E7 protein is to promote S-phase reentry in postmitotic, differentiated keratinocytes (2) , so that the cellular DNA replication machinery becomes available to support virus DNA replication (Ref. 3 ; reviewed in Ref. 4 ). The underphosphorylated pRb associates with and represses the family of E2F/DP transcription factors that control the expression of many genes essential for S-phase entry and progression. In normal cycling cells, inactivation of pRb and derepression of E2F is accomplished primarily by G1-phase D-type cyclins in complex with cdk4 and cdk6 (reviewed in Ref. 5 ). The E7 protein binds to and inactivates the underphosphorylated pRb and promotes its degradation by the proteasome pathway, bypassing the requirement for cyclin D/cdk4 or cdk6 for S-phase entry (reviewed in Ref. 6 ; see also Refs. 7-10 ). The oncogenic HPV E6 protein and a cellular protein E6AP function together as an ubiquitin ligase for the tumor suppressor protein p53 and promotes its degradation (6) . Thus, overexpression of the oncogenic HPV E6 and E7 genes can efficiently immortalize human keratinocytes and transform certain rodent cell lines and initiate oncogenesis in transgenic mice (reviewed in Ref. 11 ).

The E7 protein shares structural and functional homologies with other small DNA virus proteins such as adenovirus E1A and SV40 T-antigen. A CR2 shared among these oncoproteins includes the RB interaction motif LxCxE (512) . The ability of E7 to bind pRb is critical to its immortalization function, and the relative affinities of the E7 proteins from different HPV types for pRb correlate with the efficiency of their immortalization or transformation functions (13-15) . However, some reports suggest that E7 binding to pRb is not necessary for the immortalization function (16) or for stimulating proliferation (17) , whereas another showed that HPV-1 E7 has no transformation activity despite a high affinity for pRb (18) . E7 also binds to pRb-related protein p107 and can transactivate genes (1920) . E7 binding to p130 is very weak and has rarely been reported (21) . In addition, E7 interacts with other cellular proteins, including cdk inhibitors p21cip1 and p27kip1 (22-24) , the AP-1 proteins (2526) , cyclin A (27) , hTid-1, a human homologue of the Drosophila tumor suppressor protein Tid56 (28) , the TATA-box binding protein (29-31) , a component of NURD histone deacetylase complex Mi2ß (3233) , and p300 (34) . The roles of these additional interactions in the viral life cycle are not understood but may represent aspects of checks and balances in the regulatory network that potentiates viral reproduction. Adjacent to the pRb binding motif in CR2 is a CKII recognition site consisting of consecutive acidic residues and one or more serine residues as substrates (Fig. 1ACitation ). Mutations in the CKII recognition or phosphorylation sites do not affect pRb binding in vitro, but again, consequences of these mutations have not been consistent (1335-37) . The discrepancies among these investigations may have arisen because different mutations and assays were used.



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Fig. 1. CR2 of Ad E1A, SV40 T antigen, and E7 of HPV-11, HPV-16, and HPV-18, and the binding of wild-type or mutated HPV-18 E7 proteins to pRb and p107. A, amino acid sequence alignments and conserved residues (in boxes). Amino acid positions are given for orientation. Residues in the panel of HPV-18 E7 mutations described in the text are marked with *, including DLLC deletion and substitutions of single (C27, S32, S24), double (S32, S34), or triple (E35, 36, 37) residues. The replacement amino acids at each of these positions are described in the text and figures. B, detection of wild-type HPV-18 E7 and mutations by immunoblots and pull-down assays with GST-pRb pocket protein. Upper panel, an immunoblot to reveal wild-type and mutated HPV-18 E7 proteins in the same amount of total cell extracts from COS cells transfected with expression vectors. Extracts were prepared from cells transfected with pMTX vector (Lane 1), wild-type HPV-18 E7 (Lane 2), mutations dDLLC (Lane 3), C27S (Lane 4), or E35,36,37Q (Lane 5). Lower panel, an immunoblot to reveal E7 proteins recovered from the same amount of transfected COS cell extracts in pull-down assays with glutathione-Sepharose beads saturated with GST-pRb pocket domain fusion protein expressed in E. coli. These experiments were performed multiple times, and no significant and consistent differences were observed. C, pRb and p107 pull-down assays with bacterially expressed GST fusion with wild-type or mutated HPV-18 E7 proteins. Increasing amounts of glutathione-Sepharose beads (2, 6, and 10 µl) saturated with GST-E7 proteins were used to bind pRb or p107 protein from K562 and C33A cell extracts, respectively. The recovered proteins were then revealed by immunoblots.

 
All of the above functional assays of HPV E7 were conducted in proliferating human or rodent cells in culture, whereas the ability of the mutations to reactivate various cellular genes leading to S-phase reentry by differentiated keratinocytes has not been investigated. The HPV enhancer and promoter located in the URR control the expression the viral oncogenes. The transcriptional activity of URR is differentiation dependent in organotypic cultures of PHKs from neonatal foreskin (abbreviated as raft cultures; Ref. 38 ), simulating the pattern of viral gene expression in productively infected benign lesions. When driven by HPV-18 URR, the HPV-18 E7 gene induces unscheduled cellular DNA synthesis in postmitotic, differentiated keratinocytes in raft cultures, recapitulating observations made in patient papillomas caused by the non-oncogenic HPV-11 (2) . Thus, the raft culture provides a unique opportunity for dissecting the E7 functional domains and also for probing the mechanisms by which S-phase entry is controlled.

In this study, we prepared and characterized E7 mutations and showed that E7 binding to pRb and derepressing the E2F pathway is necessary but not sufficient to reactivate unscheduled DNA replication in differentiated PHKs. Specifically, a number of mutations blocked in CKII phosphorylation were capable of binding to pRb in vitro and transactivating the Mr 180,000 subunit of DNA polymerase {alpha} (pol {alpha} p180) or cyclin E in vivo, genes controlled by the E2F pathway, some as efficiently as the wild-type E7. However, these mutations failed to promote unscheduled DNA synthesis, in part because of a failure to induce the PCNA, the processivity factor of DNA polymerase {delta} essential for DNA replication. Other mutations induced both PCNA and cyclin E effectively but promoted S-phase reentry very inefficiently or not at all. These observations imply that transcription derepression of the E2F pathway is necessary but not sufficient to induce S-phase entry and that E7 phosphorylation by CKII is critical to this function.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Binding to pRb and p107 Proteins in Vitro by E7 Mutated in the pRb Binding Motif or CKII Recognition Site
The sequences spanning CR2 from the adenovirus E1A protein, the SV40 T antigen, and HPV E7 proteins, as well as residues altered in the panel of HPV-18 E7 mutations characterized in this study are depicted in Fig. 1ACitation . We first tested whether the E7 proteins mutated in pRb binding motif, the DLLC deletion mutation (dDLLC) and substitution mutation (C27S), or in the CKII recognition site (E35,36,37Q) were present in cells in similar steady-state levels as the wild-type protein. Expression vectors of wild-type and mutated E7 genes were each transfected into COS cells. There was no significant difference in the abundance of these E7 proteins in immunoblots (Fig. 1BCitation ).

To examine the ability of the wild-type and mutated E7 proteins to associate with pRb in vitro, a bacterially expressed fusion protein of glutathione S-transferase and the pRb-pocket domain (GST-pRb) was used to pull-down E7 proteins expressed in COS cells, as revealed by immunoblots. The wild-type E7 and the E35,36,37Q mutation were able to bind the pRb pocket domain, whereas the dDLLC and C27S mutations did not (Fig. 1BCitation ). The same results were obtained using the reciprocal GST-E7 wild-type or GST-E7 mutations to pull-down pRb from K562 cell extracts (Fig. 1CCitation ). To test for the E7 interaction with p107, we prepared extracts of C33A cells. These cells contain a mutated pRb gene (39) and are thus a good and clean source for p107. In a pull-down assay, the interactions between GST-E7, GST-E7E35,36,37Q, and GST-E7dDLLC with p107 paralleled those with pRb (Fig. 1CCitation ). Interestingly, C27S was able to bind p107, although it did not bind pRb (Fig. 1CCitation ).

Induction of Unscheduled Cellular DNA Synthesis by E7 Mutated in pRb Binding or CKII Recognition
We then prepared a vector-only retrovirus and recombinant retroviruses that contain wild-type E7 or E7 mutations, each under the control of the native differentiation-dependent HPV-18 URR. Bulk uninfected and infected PHKs were developed into epithelial raft cultures and were labeled for 12 h with BrdUrd immediately prior to harvest. All raft cultures exhibited proper morphological and molecular differentiation in the suprabasal cells as described previously, with four distinct strata: the proliferating basal/parabasal strata, the differentiated spinous and granular strata, and multiple layers of superficial cornified envelope (2) . In situ hybridization showed similar amounts of HPV E7 transcripts in all of the E7-containing raft cultures but not in the control cultures (data not shown).

Thin sections of raft cultures were then probed with antibody to BrdUrd to reveal DNA synthesis. In the uninfected PHKs or PHKs infected with vector-only virus, signals were confined to nuclei of proliferating basal and occasionally parabasal cells. Positive basal cells were also observed in all raft cultures transduced with wild-type or mutated E7 viruses. The wild-type E7 protein additionally reactivated unscheduled host DNA replication in the differentiated keratinocytes (Fig. 2ACitation ), as described previously (2) . The dDLLC mutation failed to bind pRb, or p107 (Fig. 1B and C)Citation did not induce unscheduled DNA replication. Neither did the C27S mutation, which bound to p107 but not pRb in vitro (Fig. 1BCitation and C, and Fig. 2ACitation ). Unexpectedly, the E35,36,37Q mutation failed to promote S-phase reentry (Fig. 2ACitation ), despite its ability to bind both pRb and p107 (Fig. 1B and C)Citation . These results demonstrate that the ability of E7 protein binding to pRb in vitro is necessary but not sufficient to promote S-phase entry in vivo and that binding of E7 to p107, but not pRb, had no effect in this in vivo assay. To investigate the molecular basis for the inability of the E7 mutations to induce unscheduled cellular DNA synthesis in differentiated keratinocytes, we performed in situ hybridization to examine the activation of representative genes necessary for DNA synthesis.



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Fig. 2. Induction of cellular replication genes and unscheduled cellular DNA synthesis in postmitotic, differentiated keratinocytes by wild-type HPV-18 E7 and mutations in pRb binding and in CKII recognition. Raft cultures were derived from uninfected PHKs or PHKs infected with vector-only virus, virus containing HPV-18 URR-E7, or URR-E7 mutations as indicated in each panel. The cultures were exposed to BrdUrd for 12 h immediately before harvest on day 9. A, induction of cellular DNA synthesis as revealed by immunohistochemical staining with antibody against BrdUrd. B, transcription of the pol {alpha} p180 gene in raft cultures (top and middle rows) and in a benign laryngeal papilloma caused by HPV-11 (bottom) for confirmation in vivo. In situ hybridization was conducted with 35S-labeled antisense-strand riboprobes for the pol {alpha} p180 mRNA except for two negative controls in which sense-strand riboprobes were used, as indicated. All photomicrographs were taken with dark-field illumination except for the left panel in the bottom row, which was a bright-field view of the laryngeal papilloma of the right panel to reveal histology. The pol {alpha} p180 transcription was highly induced only in differentiated keratinocytes expressing the wild-type E7 and the E35,36,37Q mutation. C, induction of PCNA in raft cultures. Immunohistochemistry was conducted to detect PCNA. Only the wild-type E7 induced PCNA in the differentiated keratinocytes.

 
Transactivation of the Mr 180,000 Subunit of DNA Polymerase {alpha}.
DNA pol {alpha} is essential for initiation of DNA replication. The expression of the gene for the p180 catalytic subunit is known to be cell cycle regulated (4041) and activated by E2F (42) . Thus, it serves as a good marker for derepression of the E2F pathway. The results of in situ hybridization are shown in Fig. 2BCitation (upper and middle rows). Background level signals were found in the raft cultures infected with vector-only virus. The wild-type HPV-18 E7 protein induced highly abundant pol {alpha} p180 transcripts throughout the differentiated strata but not in the basal cells, consistent with the URR differentiation-dependent activation. As anticipated, the dDLLC mutation, which no longer bound pRb or p107, did not induce p180 transcription, neither did C27S, which bound to p107 but not pRb. In contrast, the E35,36,37Q mutation, which did bind to pRb and p107, induced p180 transcription in differentiated strata as well as did the wild-type E7. The sense strand probe produced no signal. An identical pattern of p180 transcription induction was observed in an HPV-11 infected laryngeal papilloma (Fig. 2Citation B, bottom row). Thus, the pol {alpha} p180 mRNA is normally present at a very low level in cycling basal/parabasal cells and is strongly activated by E7 in postmitotic, differentiated keratinocytes. Furthermore, the ability of E7 to bind to pRb in vitro is necessary for this transcriptional transactivation, but binding to p107 is insufficient.

Induction of PCNA.
Because E7 binding to pRb is not sufficient to reactivate host DNA replication in differentiated cells despite the activation of the E2F pathway, we surmised that other cellular DNA replication genes were not activated by these E7 mutations. To test this hypothesis, we examined the induction of PCNA by immunohistochemical staining. We demonstrated previously that PCNA is strongly induced in differentiated keratinocytes in benign patient lesions regardless of infecting HPV genotype, in raft cultures of explanted HPV-11 infected foreskin xenografts recovered from nude mice, and in raft cultures of PHKs transduced with HPV-18 URR-E7 (243-46) . However, the human PCNA gene does not contain a known E2F binding site in the promoter region. Thus, the activation of this gene may reveal additional requirements.

The results of immunohistochemical staining for PCNA are shown in Fig. 2CCitation . In the control cultures, PCNA was detected only in the basal and infrequently in parabasal cells, whereas the wild-type E7 induced PCNA in a subset of differentiated cells. None of the mutated E7 proteins did. Therefore, binding of E7 to pRb is necessary but not sufficient for PCNA induction. Because binding of E7 to p107 but not pRb was ineffective in all three assays described above, this protein-protein interaction was not further considered in additional E7 mutations described below.

Phosphorylation and Steady State of E7 Mutations in CKII Phosphorylation Sites
Because the CKII recognition site mutation E35,36,37Q did not activate PCNA (Fig. 2CCitation ), it is possible that E7 phosphorylation is critical for PCNA induction. We then prepared single and double E7 mutations at the adjacent CKII substrates, S32 and S34. In the single mutations, one of the serine residues was replaced with a negatively charged (D or E), a neutral (Q, N, or P), or a positively charged (K or R) residue. In the double mutations, both residues were replaced with either neutral (S32,34Q) or acidic (S32,34D) amino acids. These mutations should provide insight as to whether the negative charge or the stereochemistry of phosphoserine is important for E7 functions.

Immunoprecipitation of the total proteins from transfected COS cells followed by immunoblot showed that all mutations had a similar steady-state concentration as the wild-type E7 protein (Fig. 3ACitation ). We then examined E7 phosphorylation by metabolic labeling with ortho-32P-phosphate in transfected COS cells. All single mutations were phosphorylated (Fig. 3ACitation ), whereas mutations at the CKII recognition site (E35,36,37Q) or at both substrates (S32,34Q, S32,34D) were not (Fig. 3ACitation ). These results show that S32 and S34 constitute the primary if not the only phosphorylation sites, that CKII is the primary kinase that phosphorylates E7, and that the two serine residues are independent substrates. Similarly, the HPV-16 E7 mutation in the CKII recognition site was no longer phosphorylated in COS cells (37) . Moreover, bacterially expressed wild-type E7 is phosphorylated by CKII in vitro, but substitutions of both serine substrates are not (13) . To test whether phosphorylation site mutations maintain the ability to bind pRb, pull-down assays with GST-pRb pocket fusion protein expressed in Escherichia coli were performed. All phosphorylation site mutations expressed in COS cells bound to pRb pocket domains in a manner similar to the wild-type E7 protein (Fig. 3BCitation ).



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Fig. 3. Protein stability, in vivo phosphorylation, and in vitro pRb binding activity of wild-type and E7 mutations in CKII phosphorylation. A, immunoprecipitation of E7 protein with (top) or without (bottom) metabolic labeling with 32P-Pi in COS cells. B, pull-down of E7 protein from COS cell extracts with a GST-Rb pocket domain fusion protein, as described in Fig. 1Citation B.

 
Induction of Cyclin E by the E7 Mutations in CKII Phosphorylation
Each of these mutations was then transduced into PHKs via a recombinant retrovirus and its properties examined in epithelial raft cultures. We first sought to corroborate in vitro pRb binding data by investigating the expression of a second pRb/E2F-regulated cellular gene, cyclin E (4748) , which is normally synthesized in late G1 and early S-phase in cycling cells. The cyclin E/cdk2 complex is essential for S-phase entry. It also phosphorylates pRb and is suspected to function additionally in the initiation of DNA replication (5) . We have shown recently that HPV-18 E7 concordantly induces cyclin E and p21cip1, an inhibitor of cyclin E/cdk2 and cyclin A/cdk2, in a subset of postmitotic, differentiated keratinocytes in which S-phase reentry is not observed (46) . Thus, cyclin E serves as an excellent reporter for derepression of the E2F pathway.

The results of immunohistochemical staining for cyclin E in raft cultures are presented in Fig. 4ACitation . Untransduced cultures (PHKs) or cultures transduced with the empty vector had no detectable cyclin E in any of the cells. Neither dDLLC nor C27S induced cyclin E (Fig. 4ACitation ). In contrast, the wild-type E7 induced cyclin E in some of the upper spinous and granular cells, as did some of the mutations, including those containing acidic (S32D, S34D, and S32,34D) or neutral residues (S32Q, S32N, S34Q, S34N, and S34P). When a positively charged residue was introduced into one of the phosphorylation substrate (S32K, S34R), cyclin E signal was much reduced. The CKII recognition mutation (E35,36,37Q), which no longer was phosphorylated (Fig. 3)Citation , and the mutation in which both serine substrates were mutated to neutral residues (S32,34Q) had signals that were barely above background. However, by indirect immunofluorescence, cyclin E was detected in the upper spinous cells (data not shown). Thus, there is a qualitative correlation between the ability of E7 mutations to bind pRb in vitro and to induce cyclin E in differentiated cells, but the efficiency of induction appears to depend on negative charges at the CKII phosphorylation sites.



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Fig. 4. Induction of cyclin E and p21cip1 in postmitotic, differentiated keratinocytes by E7 mutations in pRb binding or in CKII phosphorylation. Immunohistochemical staining for cyclin E (A) and p21cip1 (B) was performed in raft cultures expressing wild-type or E7 mutations as indicated. All cultures were positive in some of the differentiated cells except for the dDLLC and C27S mutations and the vector-only control. Cyclin E signals in cultures transduced with S32,34Q and E35,36,37Q mutations were very weak but were confirmed by immunofluorescence (data not shown). A number of raft culture had some nonspecific trapping in the superficial squames.

 
Induction of PCNA by the E7 Mutations in CKII Phosphorylation Sites
We next examined the ability of these E7 CKII phosphorylation site mutations to induce PCNA in raft cultures. Mutations in which one or both serine residues were substituted with an acidic residue (S32D, S34D, and S32,34D) and single substitution mutations with a neutral residue (S32Q, S34Q, S32N, and S34N) maintained the ability to induce PCNA in differentiated keratinocytes with an efficiency comparable with or somewhat lower than that of the wild-type E7 (compare Figs. 5ACitation and 2C). However, when both phosphorylation sites were replaced with neutral residues (S32,34Q) or when one of the serine residues was replaced with a positively charged residue or with the proline helix breaker (S32K, S34R, S34P), PCNA was below the sensitivity of detection in the differentiated strata (Fig. 5ACitation and data not shown). In contrast, PCNA-positive cells were observed in the basal proliferating cells in all of the raft cultures. Collectively, these results suggest that, in addition to the ability to bind pRb, negative charges provided either by phosphorylation or by one or more acidic residues in the CKII phosphorylation sites are critical for PCNA transactivation, and that substitution of either of the two serine substrates with a positively charged amino acid incapacitated E7 in PCNA induction.



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Fig. 5. Induction of PCNA and unscheduled cellular DNA synthesis in postmitotic, differentiated keratinocytes by E7 mutations in one or both serine substrates of CKII. The cultures were exposed to BrdUrd for 12 h immediately before harvest on day 9. Immunohistochemical staining for PCNA (A) or BrdUrd (B) was performed in raft cultures expressing E7 mutations as indicated. Having negative charges at one or both CKII sites was sufficient for PCNA activation, whereas a positive substitution incapacitated it. However, none was able to induce efficient unscheduled cellular DNA synthesis in differentiated keratinocytes. See wild-type E7 in Fig. 2Citation , A and C.

 
Promotion of S-Phase Reentry by E7 Mutations in CKII Phosphorylation Sites
Because E7 mutations with negative charges at CKII phosphorylation sites activated the PCNA gene efficiently, and many of them, such as S32Q, S34Q, and S34D, also appeared to retain the ability to activate the pRb/E2F pathway, as demonstrated by cyclin E induction (Figs. 4ACitation and 5A), we were curious whether these and other phosphorylation site mutations were capable of inducing unscheduled DNA synthesis in the differentiated strata. As before, the raft cultures were exposed to BrdUrd for 12 h prior to harvest to label cells that re-entered the S phase.

The results after immunohistochemical staining the tissue sections with an antibody to BrdUrd are shown in Fig. 5BCitation . As expected, substitutions (S32K, S34R, S32,34Q, and S34P) that were unable to induce PCNA did not promote S-phase reentry. The S34N mutation, which had a significantly reduced ability to induce PCNA, activated DNA synthesis very poorly. Interestingly, single or double substitutions by acidic residues (S32D, S34D, and S32,S34D) or single substitutions with a neutral residue (S32Q, S32N, and S34Q) all had dramatically reduced ability to induce unscheduled DNA synthesis, including mutations that activated both cyclin E and PCNA effectively (S32Q, S34Q, and S34D; compare Fig. 5A and BCitation ). These data strongly suggest that phosphoserines influence E7 interaction with cellular proteins that are critical to efficient induction of S-phase reentry in postmitotic differentiated keratinocytes.

Induction of the p21cip1 Protein by E7 Mutations in pRb Binding or in CKII Phosphorylation
HPV-18 E7 induces the accumulation of p21cip1 in an inactive complex containing cyclin E/cdk2 (46) .5 To examine the possibility that p21cip1 protein was more efficiently induced by E7 phosphorylation mutations than by the wild-type E7 via some unforeseen mechanisms, we examined p21cip1 protein accumulation. The mutations (S32D, S32Q, S34D, and S34Q) induced p21cip1 to levels comparable with or lower than that achieved by the wild-type E7 in parallel with the induction of cyclin E. For the dDLLC, C27S, E35,36,37Q, and S32,34Q mutations, which did not induce cyclin E or did so extremely poorly, p21cip1 was below the sensitivity of detection by immunohistochemistry (Fig. 4Citation , compare A and B, and data not shown). These data suggest that the E7 phosphorylation site mutations are deficient in inducing some critical proteins necessary for S-phase entry.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The importance of pRb in controlling S-phase entry underscores the remarkable convergent evolution among the small DNA tumor viruses, such that HPV E7, the adenovirus E1A proteins, and the SV40 and polyomavirus T antigens all target this cellular protein to activate genes required to support DNA synthesis (49) . In this study, we dissected the E7 functional domains and used the E7 mutations as a tool to probe the mechanisms involved in controlling genes required for S-phase entry. To complement biochemical analyses, we examined the replication gene induction and S-phase reentry in postmitotic, differentiated cells in PHK raft cultures. Although more labor intensive than analyses in cycling cells, this raft culture system has certain advantages. It is the natural function of E7. The proliferating cells in the basal strata provide internal controls. Our investigation has revealed two major classes of mutations that are incapable of stimulating S-phase reentry or do so very inefficiently and show that derepression of the pRb/E2F pathway is necessary but not sufficient to promote S-phase entry.

The first class is the pRb binding-negative mutations, such as dDLLC and C27S (Fig. 1)Citation . Neither promoted S-phase reentry in postmitotic cells. Neither was able to transactivate pol {alpha} p180 nor cyclin E (Figs. 2BCitation and 4A), in agreement with the previous reports that the E2F site in their respective promoter confers the cell cycle-dependent regulation (4748) . Interestingly, PCNA was not induced in the differentiated keratinocytes either (Fig. 2)Citation , suggesting that the pRb/E2F pathway is also involved in its regulation, despite the absence of a known E2F site in the promoter region immediately upstream of the coding region (50) . Collectively, our data demonstrate that E7 binding to pRb is critical to activate S-phase entry. The C27S mutation binds p107 but not pRb in vitro (Fig. 1)Citation , indicating that binding to p107 is not sufficient, but high background in immunoblots of raft culture extracts with p107 antibody precluded a direct confirmation. These results agree with comparable HPV-16 E7dDLLC and C24G mutations (Fig. 1)Citation , except that the C24G mutation does not bind either pRb or p107 (375152) .

The second class of mutations is altered in CKII recognition sequence or in one or both CKII serine substrates. Each of these mutations is deficient in promoting S-phase reentry, some not at all (Fig. 5)Citation , but each binds to pRb in vitro, apparently as well as the wild-type E7 under our assay conditions (Fig. 3)Citation . They reactivated pol {alpha} or cyclin E expression in differentiated strata to levels comparable with or lower than that achieved by the wild-type E7 (Figs. 2 and 4)Citation Citation . To a certain extent, the induction of cyclin E and, in particular, PCNA is dependent on negative charges at the residues 32 and S34. These mutations can be further divided into subgroups on the basis of their ability to activate the PCNA gene. The first subgroup did not induce PCNA and hence was DNA synthesis negative (Figs. 4 and 5Citation Citation , and data not shown). Mutations in this subgroup are no longer phosphorylated in COS cells (E35,36,37Q, S32,34Q) or contain a positively charged residue (S32K, S34R) or a helix breaker (S34P) at one of the CKII phosphorylation sites. These mutations induced cyclin E, but the signals were much weaker or fewer cells were positive, relative to the wild-type E7. Because cyclin E accumulation is attributable to protein costabilization with the endogenous p21cip1 protein, the reduced induction then reflects a less than complete activation of cyclin E transcription. This defect may have contributed to the failure or deficiency in S-phase reentry. The second subgroup includes mutations that are negatively charged at one or both CKII substrates because of acidic or neutral group substitution or phosphorylation (S32D, S34D, S32,34D, S32N, S32Q, and S34Q). They induced PCNA to a similar extent as the wild-type E7 (Fig. 5ACitation ), and some also activated cyclin E effectively, such as S32Q, S34Q, and S32D (Fig. 4ACitation ). But all promoted S-phase reentry very inefficiently or not at all. S34N has a reduced ability to induce PCNA and cyclin E and a correspondingly poor ability to activate unscheduled cellular DNA synthesis (Figs. 4 and 5)Citation Citation . Two additional mutations, S34A and S34G, also were even less effective than S34N on inducing S-phase reentry (data not shown), supporting the interpretation that this residue is particularly critical for E7 activity. But we cannot rule out the possibility that the titer of these viruses or the stability of the E7 protein was reduced. We excluded the possibility that the mutations induced the cdk2 inhibitor p21cip1 protein more efficiently than the wild-type E7 (Fig. 4BCitation ). Thus, it seems probable that additional S-phase gene or genes must not be adequately activated (compare Figs. 24and 5Citation Citation Citation B). In this regard, the observation that the extent of p21cip1 induction correlated with that of cyclin E (Fig. 4)Citation supports our interpretation that their accumulation is attributable to protein costabilization into replication-incompetent complex with cdk2 (46) .5

We, as well as others (this study; Refs. 36 and 37 ), performed E7 kinase assays in COS cells, thus, one might question whether S32 or S34 is phosphorylated in raft cultures. The fact that basic or neutral amino acid substitutions are more defective than acidic residue substitutions argues strongly that one or probably both serine residues are indeed phosphorylated in raft cultures. Regardless of whether E7 is additionally phosphorylated by other kinases in differentiated keratinocytes, our results indicate that S32 and S34 that are known substrates of CKII in vitro and in COS cells are critical to its function. Similarly, HPV-16 E7 mutations in CKII phosphorylation also have reduced activities in transactivating the E2F-regulated Ad E2 promoter and in transforming rodent cell lines (1335-3753) . Our results in organotypic cultures can begin to provide a molecular basis for these phenotypes.

What might be the potential mechanisms affected by E7 mutations blocked in CKII phosphorylation? The affinity of E7 protein for the TATA-box binding protein is enhanced by CKII phosphorylation (3054) . However, we consider the possibility involving TBP unlikely because our mutations clearly distinguish the promoters of cyclin E, pol {alpha} p180, and PCNA. Alternatively, the functions of NH2-terminal CR1 or COOH-terminal CR3 may be affected by the charges at the CKII phosphorylation sites in CR2. In particular, HPV-E7 CR1 is important in PCNA induction and in immortalization of baby rat kidney cells (1655) , whereas CR3 has been implicated in the up-regulation of ATF (56) . In this regard, the Ad E1A 243R regulates the human PCNA promoter through ATF, p107, and RFX-1 in HeLa cells (57-60) . Because HeLa cells are phenotypically negative for p53 and pRb family of proteins because they constitutively express HPV-18 E6 and E7 proteins from integrated viral DNA, the induction of the PCNA promoter by E1A would seem not to be identical to the HPV-18 E7-induced transactivation examined in our study. In addition, for cell cycle-regulated transcription, additional regions of this gene are critical. Two E2F sites in the first intron and additional 3' region of the human PCNA gene are implicated in cell cycle-regulated expression (6162) .

Regardless of whether CKII phosphorylation of S32 and S34 impacts the E7 CR1 and CR3 functions, we believe that it may also significantly influence the E7 interactions with pRb and associated proteins, but those effects are not revealed by our binding assays in vitro. This is because the nature, number, and concentration of interacting proteins as well as the state of protein phosphorylation are all different in the in vitro assays from those occurring in vivo. For instance, all three members of the pocket proteins can recruit histone deacetylases in an E2F-dependent manner (63-66) . Furthermore, a recent study showed that pocket proteins negatively regulate DNA replication by interacting with MCM7, which is a component of the initiation helicase complex at the replication origin (67) . We suggest that, in vivo, even a subtle difference in the affinity of E7 for pRb conferred by the CKII phosphorylation may affect its ability to compete with histone deacetylase for binding to pRb protein or to cause an efficient release from E2F or other host proteins. Thus, the mutated E7 proteins can be efficient in inducing some cellular S-phase genes while deficient in reactivating others, depending on the sequence context of the targeted promoters. In this scenario, the E7 mutations examined in our study could have affected the extent to which the pRb/E2F pathway was derepressed, rather than revealing additional independent pathways.

In summary, we have taken advantage of the ability of E7 to induce S-phase reentry in postmitotic, differentiated cells to dissect the E7 functional domains and to probe the mechanisms by which cellular genes essential for DNA replication are activated. Our results demonstrate that negative charges at one or both of serine CKII phosphorylation sites in the E7 CR2 are additionally required for efficient activation of certain S-phase genes. However, only wild-type E7 phosphorylated by CKII at both serine residues in CR2 promotes efficient S-phase reentry in postmitotic cells.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Plasmids.
To mutate the HPV-18 E7 gene, the HindIII restriction fragment spanning HPV-18 E7 and the downstream SV40 early promoter (E7-SV40P) was excised from the recombinant DNA pLJd-18URRE7 (2) and cloned into pBluescript SK+ (Stratagene) site to generate pBS-E7-SV40P. Mutations were generated by PCR amplification with a combination of T7 promoter primer, an M13 reverse primer, an mutagenic forward (f) or reverse (r) oligonucleotides as follows: for dDLLC, f 5'-644aatgaaattccggtt^cacgagcaattaagc-3' (in which ^ signifies deletion); for C27S, f 5'-665ctatgtcaCcagcaattaagc-3'; r 5'-685gcttaattgctGgtgaca-3'; for E35,36,37Q, f 5'-683agcgactcaCagCaaCaaaacgatgaa-3'; r 5'-709ttcatcgttttGttGctGtgagtcgct-3'; for S32, r 5'- 698cttcctctgagtcStNtaattgctcgtga-3'; for S34: r 5'-704cgtttcttcctcStNgtcgcttaattgc-3' or f 5'-676gcaattaagcgacSSagaggaagttt-3'; r 5'-702ttttcttcctctSSgtcgcttaatttg-3'; for S32,34Q, r 5'-700>ttcttcctcCTGgtcCTGtaattgctcg-3'; and for S32,34D, r 5'-697ttcctcGTCgtcGTCtaattgctcg-3'. Mutated nucleotides are in capital letters with S = G + C; n = A, C, G, or T. The mutated E7 genes with the adjoining SV40 promoter were then each cloned at a HindIII site in pLC-18URR, a derivative of pLJd containing the 1.1 kbHPV-18 URR (2) . In pLC clones, the bacterial origin of replication was replaced with that from pBluescript SK+ for improved plasmid yields in E. coli. For protein expression in COS-7 cells, the wild-type and mutated E7 open reading frame were recloned into pMTX (68) . For expression of GST-E7 proteins in E. coli, intermediate clones were first generated with a small deleted fragment of PstI-NsiI in pBS-E7-SV40P, and then the BamHI-SalI fragment of E7 gene was cloned in-frame into BamHI-SalI of pGEX2T/M (69) . These constructions produced a fusion protein with three extra residues GLL between GST and E7. All mutations were verified by DNA sequencing, and protein synthesis was demonstrated by SDS-PAGE (data not shown).

Cells Lines and Epithelial Raft Cultures of Neonatal Foreskin Keratinocytes.
Cell lines COS-7, C33A, and K562 were cultured in DMEM or RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD). For E7 protein phosphorylation, COS-7 cells were electroporated with pMTX-E7 or the E7 mutations. Cells were labeled with ortho-32P-phosphoric acid (8 µCi/ml; Amersham Pharmacia Biotech, Piscataway, NJ) for 4 h after starvation for phosphate for 4 h. Cell extracts were made as described.5

PHKs were recovered from neonatal foreskins collected from a local hospital as described (38) . Only the second passage of PHKs was used. Amphotropic recombinant retroviruses were prepared, infection of PHKs was conducted, and raft cultures developed as described (238) . In some experiments, the cultures were exposed to 50 µg/ml of BrdUrd for the final 12 h of culturing prior to fixation with 10% buffered formalin and paraffin embedding. Four-µm sections were cut, deparaffinized, and stained with H&E for histology or used for immunohistochemical staining or in situ hybridization.

Immunohistochemistry and in Situ Hybridization.
Immunohistochemical staining was performed with monoclonal antibodies against PCNA (PC-10 at 1:100; Dako, Carpinteria, CA), cyclin E (HE-12 at 1:25; PharMingen, San Diego, CA), p21cip1 (AB-1 at 1:20; Calbiochem, La Jolla, CA), or BrdUrd (at 1:100; Zymed, South San Francisco, CA). The color development was performed with the Histostain kit (Zymed). A 1.2-kb HindIII-ClaI fragment from the coding region of the human DNA pol {alpha} p180 catalytic subunit cDNA in the pGEM1 vector was a gift from Dr. Teresa S-F. Wang. In vitro transcription to synthesize 35S-UTP-labeled antisense and sense riboprobes (>1000 Ci/mmol, 10 mCi/ml; Amersham Pharmacia Biotech) was made as described with T7 and SP6 RNA polymerase, respectively (Life Technologies). In situ hybridization was conducted with the probe applied at 25% saturation as described (2) .

Protein Binding Assays.
Plasmids pMTX-E7 and pMTX-E7 mutations were electroporated into COS cells as described previously (70) . For immunoblots, cells were harvested 48 h after transfection. Protein concentrations were quantified by Bradford assays, and 100 µg of protein from each lysate were separated on a 14% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), and probed with a 1:2000 dilution of polyclonal antibody against HPV-18 E7 protein (71) . For pull-down assays, cell extracts were prepared from K562 or C33A cells as follows. Trypsinized cells were washed once with buffer A [10 mM HEPES (pH 8), 150 mM NaCl, 1 mM EDTA, 0.5% NP40, and 1 mM DTT], suspended in buffer B [20 mM HEPES (pH 8.0), 250 mM NaCl, 1 mM EDTA, 0.5% NP40, and 1 mM DTT] for 30 min on ice, and cleared by centrifugation in a minicentrifuge at 4°C for 10 min. Glutathione-Sepharose beads with bound bacterial extracts containing GST-E7 or GST-E7 mutations were added to K562 or C33A extracts and incubated at 4°C for 1 h. The beads were washed three times with buffer B and boiled in SDS sample buffer. Proteins released from the beads were separated by electrophoresis in 10% SDS-PAGE and immunoblotted with the polyclonal antibody against pRb (C-15 at 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), p107 (C-18 at 1:1000; Santa Cruz Biotechnology), or HPV-18 E7. Alternatively, GST-pRb pocket protein expressed in E. coli (72) and bound to glutathione-Sepharose beads were used to pull-down wild-type or mutated E7 protein from extracts of transfected COS cells. The immunoprecipitates were analyzed by immunoblots with E7 antibody by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

Immunoprecipitation.
For immunoprecipitation, COS-7 cell extracts were prepared as described.5 Briefly, after 1-h incubation with 10 µl of rabbit anti-HPV-18 E7 anti-serum at 4°C, 30 µl of protein A-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) were added to each cell extract at 4°C for another 1 h. The immunoprecipitates were washed five times with buffer B, and proteins were solubilized in sample buffer for separation by SDS-PAGE. Gels were either transferred to PVDF membranes for immunoblots by ECL or dried to detect 32P-labeled proteins using a PhosphorImager.


    Acknowledgments
 
We thank Dr. Brian J. Wiatrak of the Alabama Children’s Hospital for providing laryngeal papilloma specimens, and we thank the nurses at Cooper Green Hospital for collecting neonatal foreskins. We thank Ge Jin and Dolores Madden for embedding and sectioning of patient biopsies and raft cultures. We also thank Dr. Teresa Wang for supplying the plasmid for preparing human DNA polymerase {alpha} p180 riboprobe.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by USPHS Research Grant CA36200. Back

2 Present address: Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294-0005. Back

3 To whom requests for reprints should be addressed. Phone: (205) 975-8300; Fax: (205) 975-6075; E-mail: LTChow{at}uab.edu Back

4 The abbreviations used are: HPV, human papillomavirus; PCNA, proliferating cell nuclear antigen; cdk, cyclin-dependent kinase; CR, conserved region; URR, upstream regulatory region; PHK, primary human keratinocyte; pRb, the retinoblastoma susceptibility protein; CKII, casein kinase II; pol {alpha}, human DNA polymerase {alpha}; BrdUrd, bromodeoxyuridine. Back

5 F. Noya, W-M. Chien, T. R. Broker, and L. T. Chow, unpublished results. Back

Received for publication 3/29/00. Revision received 6/12/00. Accepted for publication 6/19/00.


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 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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