HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors.

HER2/Neu gene mutations have been identified in lung cancer. Expression of a HER2 mutant containing a G776(YVMA) insertion in exon 20 was more potent than wild-type HER2 in associating with and activating signal transducers, phosphorylating EGFR, and inducing survival, invasiveness, and tumorigenicity. HER2(YVMA) transphosphorylated kinase-dead EGFR(K721R) and EGFR(WT) in the presence of EGFR tyrosine kinase inhibitors (TKIs). Knockdown of mutant HER2 in H1781 lung cancer cells increased apoptosis and restored sensitivity to EGFR TKIs. The HER2 inhibitors lapatinib, trastuzumab, and CI-1033 inhibited growth of H1781 cells and cells expressing exogenous HER2(YVMA). These data suggest that (1) HER2(YVMA) activates cellular substrates more potently than HER2(WT); and (2) cancer cells expressing this mutation remain sensitive to HER2-targeted therapies but insensitive to EGFR TKIs.

A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency.

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We found that beta-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated beta-catenin accumulation; LANA itself upregulated expression of beta-catenin in transfected cells. LANA stabilizes beta-catenin by binding to the negative regulator GSK-3beta, causing a cell cycle-dependent nuclear accumulation of GSK-3beta. The LANA C terminus contains sequences similar to the GSK-3beta-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3beta or stabilize beta-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3beta can therefore be a source of beta-catenin dysregulation in human cancers.

Transforming growth factor {beta} (TGF-{beta})-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-{beta} function.

Transforming growth factor beta (TGF-beta) inhibits proliferation and promotes cell migration. In TGF-beta-treated MCF10A mammary epithelial cells overexpressing HER2 and by chromatin immunoprecipitation, we identified novel Smad targets including protein tyrosine phosphatase receptor type kappa (PTPRK). TGF-beta up-regulated PTPRK mRNA and RPTPkappa (receptor type protein tyrosine phosphatase kappa, the protein product encoded by the PTPRK gene) protein in tumor and nontumor mammary cells; HER2 overexpression down-regulated its expression. RNA interference (RNAi) of PTPRK accelerated cell cycle progression, enhanced response to epidermal growth factor (EGF), and abrogated TGF-beta-mediated antimitogenesis. Endogenous RPTPkappa associated with EGF receptor and HER2, resulting in suppression of basal and ErbB ligand-induced proliferation and receptor phosphorylation. In MCF10A/HER2 cells, TGF-beta enhanced cell motility, FAK phosphorylation, F-actin assembly, and focal adhesion formation and inhibited RhoA activity. These responses were abolished when RPTPkappa was eliminated by RNA interference (RNAi). In cells expressing RPTPkappa RNAi, phosphorylation of Src at Tyr527 was increased and (activating) phosphorylation of Src at Tyr416 was reduced. These data suggest that (i) RPTPkappa positively regulates Src; (ii) HER2 signaling and TGF-beta-induced RPTPkappa converge at Src, providing an adequate input for activation of FAK and increased cell motility and adhesion; and (iii) RPTPkappa is required for both the antiproliferative and the promigratory effects of TGF-beta.

Reduction of cytosolic p27(Kip1) inhibits cancer cell motility, survival, and tumorigenicity.

We generated a p27(Kip1) mutant (p27deltaNLS) that localized exclusively in cell cytosol. Expression of p27deltaNLS in MCF7 breast cancer cells down-regulated RhoA and increased motility, survival, and Akt levels without an effect on cell cycle distribution. RNA interference of p27 in U87 glioma cells, which express p27 predominantly in the cytoplasm, inhibited motility and survival. Conversely, knockdown of p27 in COS7 cells, with >95% nuclear p27 expression, accelerated proliferation but had no effect on motility or survival. U87 cells in which p27 had been eliminated by RNA interference exhibited lower Akt levels, shorter Akt turnover, and markedly impaired tumorigenicity in vivo. These xenografts were less invasive and exhibited increased apoptosis compared with p27-expressing tumors. Expression of cytosolic p27 in primary human breast carcinomas correlated linearly with Akt content as measured by immunohistochemistry. These data suggest that cytoplasmic p27 can exert oncogenic functions by modulating Akt stability, cell survival, and tumorigenicity.

Refractory lympho-epithelial carcinoma of the nasopharynx: a case report illustrating a protracted clinical course.

Nasopharyngeal carcinoma is an uncommon cancer in North America. Its clinical course is typified by locally advanced disease at diagnosis and has a high propensity for both regional and distant spread. It is, therefore, typically treated with a combination of radiation and chemotherapy. This report describes our 10-year clinical and radiological findings in a 48-year-old Vietnamese male patient with locally-advanced T4N1M0 lympho-epithelial carcinoma of the nasopharynx. Despite a long remission period after his initial course of aggressive chemoradiation, his tumor recurred locally after 4 years. Thereafter, throughout a period of over 10 years, he has been treated with multiple courses of re-irradiation and three different trials of chemotherapy. He was ultimately provided with over 30 months of progression-free tumor control with the epidermal growth factor receptor (EGFR)-inhibitor cetuximab. This case illustrates the commonly protracted course of this disease and its responsiveness to multiple treatment modalities.

Convergence of p53 and transforming growth factor beta (TGFbeta) signaling on activating expression of the tumor suppressor gene maspin in mammary epithelial cells.

Using two-dimensional difference gel electrophoresis, we identified the tumor suppressor gene maspin as a transforming growth factor beta (TGFbeta) target gene in human mammary epithelial cells. TGFbeta up-regulatesMaspin expression both at the RNA and protein levels. This up-regulation required Smad2/3 function and intact p53-binding elements in the Maspin promoter. DNA affinity immunoblot and chromatin immunoprecipitation revealed the presence of both Smads and p53 at the Maspin promoter in TGFbeta-treated cells, suggesting that both transcription factors cooperate to induce Maspin transcription. TGFbeta did not activate Maspin-luciferase reporter in p53-mutant MDA-MB-231 breast cancer cells, which exhibit methylation of the endogenous Maspin promoter. Expression of ectopic p53, however, restored ligand-induced association of Smad2/3 with a transfected Maspin promoter. Stable transfection of Maspin inhibited basal and TGFbeta-stimulated MDA-MB-231 cell motility. Finally, knockdown of endogenous Maspin in p53 wild-type MCF10A/HER2 cells enhanced basal and TGFbeta-stimulated motility. Taken together, these data support cooperation between the p53 and TGFbeta tumor suppressor pathways in the induction of Maspin expression, thus leading to inhibition of cell migration.

HER2/Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by transforming growth factor beta.

In HER2 (ErbB2)-overexpressing cells, transforming growth factor beta (TGF-beta), via activation of phosphoinositide-3 kinase (PI3K), recruits actin and actinin to HER2, which then colocalizes with Vav2, activated Rac1, and Pak1 at cell protrusions. This results in prolonged Rac1 activation, enhanced motility and invasiveness, Bad phosphorylation, uncoupling of Bad/Bcl-2, and enhanced cell survival. The recruitment of the HER2/Vav2/Rac1/Pak1/actin/actinin complex to lamellipodia was abrogated by actinin siRNAs, dominant-negative (dn) p85, gefitinib, and dn-Rac1 or dn-Pak1, suggesting that the reciprocal interplay of PI3K, HER2 kinase, and Rac GTPases with the actin cytoskeleton is necessary for TGF-beta action in oncogene-overexpressing cells. Thus, by recruiting the actin skeleton, TGF-beta "cross-links" this signaling complex at cell lamellipodia; this prolongs Rac1 activation and increases metastatic properties and survival of HER2-overexpressing cells.

Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells.

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.

Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry.

We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.

Regulation of expression of the Epstein-Barr virus BamHI-A rightward transcripts.

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expressed in all EBV latency programs, including EBV-infected B cells in healthy carriers. Despite their ubiquitous expression, the regulation and biological function of BARTs are still unclear. In this study, the BART 5' termini were characterized by using a procedure that selects capped, full-length mRNAs. Two TATA-less promoter regions, designated P1 and P2, were mapped. P1 had relatively high basal activity in both epithelial and B cells, whereas P2 exhibited higher activity in epithelial cells. Upon EBV infection of B cells, transcription from P1 was detected soon after infection, while expression from P2 was delayed. Promoter-reporter assays in transiently transfected cells revealed that P1 and P2 were differentially regulated. Interferon regulatory factor 7 (IRF7) and IRF5 negatively regulated P1 activity. c-Myc and C/EBP family members positively regulated P2. Regulation of P2 by C/EBPs was characterized by electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter assays. More-abundant BART expression in epithelial cells correlated with the relative expression of positive and negative regulators in these cells.

CCAAT/enhancer-binding protein-alpha is induced during the early stages of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic cycle reactivation and together with the KSHV replication and transcription activator (RTA) cooperatively stimulates the viral RTA, MTA, and PAN promoters.

During the immediate-early (IE) phase of reactivation from latency, the Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator protein (RTA) (or ORF50) is thought to be the most critical trigger that upregulates expression of many downstream viral lytic cycle genes, including the delayed-early (DE) gene encoding the replication-associated protein (RAP) (or K8). RAP physically interacts with and stabilizes the cellular transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha), leading to upregulated expression of the cellular C/EBPalpha and p21(CIP-1) proteins followed by G(0)/G(1) cell cycle arrest. Furthermore, RTA also interacts with C/EBPalpha, and both RAP and RTA cooperate with C/EBPalpha to activate the RAP promoter through binding to a strong proximal C/EBP binding site that also serves as an RTA-responsive element (RRE). Here we show that C/EBPalpha also activates the IE RTA promoter in transient-cotransfection reporter gene assays and that addition of either RTA or RAP enhances the effect. Electrophoretic mobility shift assay and deletion analysis revealed three C/EBP binding sites that mediate cooperative transactivation of the RTA promoter by C/EBPalpha and RTA. Furthermore, chromatin immunoprecipitation assay results showed that the endogenous C/EBPalpha, RTA, and RAP proteins all associate with RTA promoter sequences in tetradecanoyl phorbol acetate-induced primary effusion lymphoma (PEL) cells. Induction of endogenous KSHV RTA mRNA in PEL cells by exogenously introduced C/EBPalpha was confirmed by reverse transcription-PCR analysis and by double-label indirect immunofluorescence assays. Reciprocally, expression of exogenous RTA also led to an increase of endogenous C/EBPalpha expression that could be detected by Western immunoblot assays even in KSHV-negative DG75 cells. Cotransfected RTA also increased positive C/EBPalpha autoregulation of the C/EBPalpha promoter in transient-cotransfection reporter gene assays. Finally, C/EBPalpha proved to strongly activate the promoters of two other KSHV DE genes encoding PAN (polyadenylated nuclear) RNA and MTA (ORF57), which was again mediated by C/EBP binding sites that also contribute to RTA activation. Overall, these results support a model in which the cellular transcription factor C/EBPalpha and RTA:C/EBPalpha interactions play important roles both upstream and downstream of the two major KSHV regulatory proteins RTA and RAP during the early stages of lytic cycle reactivation.

CCAAT/enhancer binding protein alpha binds to the Epstein-Barr virus (EBV) ZTA protein through oligomeric interactions and contributes to cooperative transcriptional activation of the ZTA promoter through direct binding to the ZII and ZIIIB motifs during induction of the EBV lytic cycle.

The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein alpha (C/EBPalpha), leading to the induction of p21-mediated G(1) cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPalpha subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated (35)S-labeled C/EBPalpha and (35)S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPalpha binding but not with ZTA binding in electrophoretic mobility shift assays (EMSAs). Glutathione S-transferase affinity assays with mutant ZTA proteins revealed that the basic DNA binding domain and the key leucine zipper residues required for homodimerization are all required for the interaction with C/EBPalpha. ZTA is known to bind to two ZRE sites within the ZTA promoter and to positively autoregulate its own expression in transient cotransfection assays, but there is conflicting evidence about whether it does so in vivo. Examination of the proximal ZTA upstream promoter region by in vitro EMSA analysis revealed two high-affinity C/EBP binding sites (C-2 and C-3), which overlap the ZII and ZIIIB motifs, implicated as playing a key role in lytic cycle induction. A chromatin immunoprecipitation assay confirmed the in vivo binding of both endogenous C/EBPalpha and ZTA protein to the ZTA promoter after lytic cycle induction but not during the latent state in EBV-infected Akata cells. Reporter assays revealed that cotransfected C/EBPalpha activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPalpha were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIIIB sites in the ZTA promoter strongly reduced C/EBPalpha transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPalpha into EBV-infected HeLa-BX1 cells induced endogenous ZTA mRNA and protein expression, as demonstrated by both reverse transcription-PCR and immunoblotting assays. Finally, double-label immunofluorescence assays suggested that EAD protein expression was activated even better than ZTA expression in latently infected C/EBPalpha-transfected Akata cells, perhaps because of the presence of a strong B-cell-specific repressed chromatin conformation on the ZTA promoter itself during EBV latency.

Early activation of the Kaposi's sarcoma-associated herpesvirus RTA, RAP, and MTA promoters by the tetradecanoyl phorbol acetate-induced AP1 pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma (PEL) cells, but treatment with tetradecanoyl phorbol acetate (TPA) can trigger the full lytic-cycle replication in some of these cells. During lytic-cycle replication, the KSHV-encoded replication and transcription activator (RTA or ORF50), the mRNA transport and accumulation protein (MTA), and the replication-associated protein (RAP) all play crucial roles in expression of downstream viral genes as well as in mediation of viral DNA replication. The cellular CCAAT/enhancer-binding protein alpha (C/EBP alpha) is induced in TPA-treated PEL cells and contributes to transactivation of the promoters for all of these genes through both direct binding and cooperative interactions with RTA and RAP targeted to upstream C/EBP sites. However, little is known about how RTA expression is triggered initially at the earliest stages after TPA induction when the C/EBP alpha levels are still limited. Treatment with TPA proved to significantly induce both AP1 DNA-binding activity and levels of activated phosphorylated cJUN in PEL cells and ectopic expression of cJUN-plus-cFOS-induced RTA protein expression in PEL cells. Cotransfected cJUN plus cFOS or TPA treatment transactivated the KSHV RTA, RAP, and MTA promoters in an AP1-binding site-dependent manner in all three promoters. Chromatin immunoprecipitation assays confirmed that cJUN associates with these KSHV target promoters in PEL cells as early as 4 h after TPA treatment. Furthermore, the KSHV RTA and RAP proteins both interact with cJUN or both cJUN and cFOS in vitro or by coimmunoprecipitation from induced PEL cells and enhance cJUN-plus-cFOS-mediated transactivation of these viral promoters. Both increased phosphorylated cJUN and AP1 DNA-binding activity was detected as early as 1 h after TPA treatment in PEL cells, suggesting that AP1 activity may be crucial for very early activation of the RAP, MTA, and RTA promoters during the KSHV lytic cycle. Finally, expression of RTA alone increased cJUN protein levels severalfold in DG75 cells but did not induce cJUN phosphorylation. Therefore, we suggest that the initiating effects of TPA via the AP1 pathway in PEL cells need to be amplified by RTA for full lytic-cycle induction.

Role of CCAAT/enhancer-binding protein alpha (C/EBPalpha) in activation of the Kaposi's sarcoma-associated herpesvirus (KSHV) lytic-cycle replication-associated protein (RAP) promoter in cooperation with the KSHV replication and transcription activator (RTA) and RAP.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBPalpha) and p21(CIP-1) expression, resulting in G(0)/G(1) cell cycle arrest during the lytic cycle. RAP and C/EBPalpha are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBPalpha; this analysis showed that one of these sites has a very high affinity for C/EBPalpha. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPalpha can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBPalpha together resulted in a threefold increase in activity over that obtained with C/EBPalpha alone. Importantly, the introduction of exogenous Flag-tagged C/EBPalpha triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBPalpha and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBPalpha and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPalpha, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPalpha or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPalpha-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBPalpha and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBPalpha in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBPalpha was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBPalpha and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPalpha occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBPalpha and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPalpha. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPalpha but that full activity requires more than just the core C/EBP binding site.

Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-alpha.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G(1) cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12-24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPalpha and p21(CIP-1) proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPalpha in vitro and stimulates C/EBPalpha-induced expression from both the C/EBPalpha and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPalpha and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G(1) to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPalpha or by deletion of the basic/leucine zipper region in RAP that interacts with C/EBPalpha. Therefore, C/EBPalpha is essential for the p21-mediated inhibition of G(1) to S-phase progression by RAP in KSHV-infected host cells.

Cell cycle arrest by Kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to CCAAT/enhancer-binding protein alpha and p21(CIP-1).

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G(1) cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPalpha and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPalpha and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPalpha. Cotransfection of RAP with C/EBPalpha cooperatively stimulated both the C/EBPalpha and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPalpha, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPalpha in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPalpha sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPalpha promoter in vivo, but only in a C/EBPalpha-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPalpha. Expression of exogenous RAP (Ad-RAP) caused G(1)/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPalpha and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPalpha was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPalpha binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPalpha were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G(1) phase or inducing p21 in a C/EBPalpha-independent process.

CCAAT/enhancer binding protein alpha interacts with ZTA and mediates ZTA-induced p21(CIP-1) accumulation and G(1) cell cycle arrest during the Epstein-Barr virus lytic cycle.

Cellular CCAAT/enhancer binding protein alpha (C/EBPalpha) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G(1)/S through stabilization of p21(CIP-1)/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G(1)/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G(1)/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPalpha and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPalpha and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPalpha by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPalpha in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPalpha from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPalpha and ZTA were found to specifically associate with the C/EBPalpha promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPalpha because it was abolished by clearing with anti-C/EBPalpha antibody. ZTA did not bind to or activate the C/EBPalpha promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPalpha promoter by cotransfected C/EBPalpha in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPalpha, ZTA enhanced the level of C/EBPalpha activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPalpha binding sites. Finally, in C/EBPalpha-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G(1) arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPalpha is essential for at least one pathway of ZTA-induced G(1) arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPalpha leading to greatly enhanced C/EBPalpha and p21 levels through both transcriptional and posttranslational mechanisms.

Phospholipase C gamma 1 is a physiological guanine nucleotide exchange factor for the nuclear GTPase PIKE.

Phospholipase C gamma 1 (PLC-gamma 1) hydrolyses phosphatidylinositol-4,5-bisphosphate to the second messengers inositol-1,4,5-trisphosphate and diacylglycerol. PLC-gamma 1 also has mitogenic activity upon growth-factor-dependent tyrosine phosphorylation; however, this activity is not dependent on the phospholipase activity of PLC-gamma 1, but requires an SH3 domain. Here, we demonstrate that PLC-gamma 1 acts as a guanine nucleotide exchange factor (GEF) for PIKE (phosphatidylinositol-3-OH kinase (PI(3)K) enhancer). PIKE is a nuclear GTPase that activates nuclear PI(3)K activity, and mediates the physiological activation by nerve growth factor (NGF) of nuclear PI(3)K activity. This enzymatic activity accounts for the mitogenic properties of PLC-gamma 1.