A cyclin-binding motif within the amino-terminal homology domain of EBNA3C binds cyclin A and modulates cyclin A-dependent kinase activity in Epstein-Barr virus-infected cells

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the alpha1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. Virol. 78:1981-1991, 2004). It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.     

SCFSkp2 complex targeted by Epstein-Barr virus essential nuclear antigen

The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCFSkp2, known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCFSkp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCFSkp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCFSkp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCFSkp2-dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCFSkp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.     

Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1

EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.     

Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.     

Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation.

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.     

EBNA3C Augments Pim-1 Mediated Phosphorylation and Degradation of p21 to Promote B-Cell Proliferation

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr¹⁴⁵ residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers.     

Cyclic AMP induces inhibition of cyclin A expression and growth arrest in human hepatoma cells

Classical cytotoxic therapy has been minimally useful in the treatment of hepatocellular carcinoma. In an effort to develop a new approach to the treatment of this neoplasm, we have investigated the signal transduction pathways regulating the growth of human hepatoma cells. In the data reported here, cyclic AMP (cAMP), a negative growth regulator for many cells of epithelial origin, induced G1 synchronization and apoptosis in the HepG2 human hepatoma cell line. The effects of cAMP on the components of the G1/S transition were analyzed. There was no detectable effect of two different cAMP analogs, 8-bromo cAMP or dibutyryl cAMP on the level of the D-type cyclins, cyclin E, cyclin-dependent kinase 2, cyclin-dependent kinase 4, p53, or the cyclin-dependent kinase inhibitors p21 or p27. In contrast, the cAMP analogs induced a dramatic downregulation of cyclin A protein, cyclin A messenger RNA, and cyclin A-dependent kinase activity. Cyclin A-dependent kinase has been shown to be required for the G1-S transition. Furthermore, cyclin A deregulation has been implicated in the pathogenesis of hepatocellular carcinoma. The data reported here suggest a novel signal transduction-based approach to hepatoma therapy.     

beta3-endonexin as a novel inhibitor of cyclin A-associated kinase

Cyclin A is indispensable for S phase cell cycle progression and is suggested to be a crucial target of cell adhesion signals. In this study, we demonstrate that beta3-endonexin, a molecule known to associate with the integrin beta3 cytoplasmic domain, specifically binds cyclin A. Deletion of the amino-terminal 52-amino-acid residues including the cyclin-binding RxL motif abolishes the ability of beta3-endonexin to interact with cyclin A. In an in vitro kinase assay, beta3-endonexin inhibits pRB kinase activity associated with cyclin A-Cdk2 while leaving its histone H1 kinase activity unaffected. Coexpression of beta3-endonexin in yeast cells overcomes growth suppression caused by an activation of cyclin A-associated kinase. Our results indicate that beta3-endonexin is a novel cyclin A-binding molecule that regulates cyclin A-associated pRB kinase activity.     

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.     

细胞周期抑制蛋白p27的研究进展

p27基因位于人类染色体12p13,其编码的蛋白对cyclinsCDKs具有广泛的抑制活性,是细胞周期调控的抑制蛋白。它以化学剂量的方式调节细胞周期的进程,参与细胞的生长、分化等过程。对p27基因的发现、基因结构、对细胞周期和细胞分化的调控机制以及与肿瘤的关系作一介绍。     

Human papillomavirus 18 E1^E4 protein interacts with cyclin A/CDK 2 through an RXL motif

The human papilloma virus E4 protein is highly expressed in late times of infection. Evidence to date suggests that E4 is essential for amplification of the viral genome and that it can influence cell cycle. Examination of the sequences encoding the E4 proteins from several genotypes of human papillomavirus revealed the presence of RXL-containing motifs reminiscent of the cyclin-binding motifs that have been identified in several cyclin-binding proteins. When baculovirus-produced human cyclin E and cyclin A with cdk2 were incubated in vitro with a GST-E4 fusion protein, both cyclin E and A stably interacted with the GST-E4 protein containing the full E4 sequence from HPV18. The interaction was not dependent on the presence of the kinase subunit but was dependent on the integrity of the RXL motif in E4. When incubated with cell extracts from the C33A human cervical carcinoma cell line or when expressed in C33A cells, the GST-E4 protein formed interactions with cyclin A and cdk2 and kinase activity could be demonstrated in the GST-E4 complex. In contrast to the baculovirus-produced cyclin E, cellular cyclin E failed to detectably interact with GST-E4 suggesting that the HPV18 E4 sequences are capable of interacting only with cyclin A in mammalian cells. These observations suggest that human papillomavirus E4 proteins can interact with cyclin A/cdk2, which may contribute to viral manipulation of the host cell cycle.     

The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms

The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.     

Switching cyclin D-Cdk4 kinase activity on and off

The cyclin-cdks are master regulators of cell proliferation. These serine/threonine kinases are the motors that both start and stop the cell cycle in response to proliferative or antiproliferative signals. They phosphorylate substrates required to trigger orderly cell cycle progression, and thus their activity is tightly regulated in order to prevent inappropriate activation. One of the main interfaces between the extraceullar environment and the cell cycle machinery is the interaction of the cyclin-cdks with two families of stoichiometric cyclin kinase inhibitors (CKIs), the Ink4s and the Cip/Kips. As their name suggests, the CKIs have historically been considered negative regulators of the cyclin-cdks, responsible for rapidly and effectively turning off cyclin-cdk activity. However, the interaction of cyclin D-cdk4 with the Cip/Kip family, and with p27Kip1 in particular, appeared complex. In addition to its ability to inhibit cyclin D-cdk4, p27 appeared to be a required assembly factor for the complex, binding in a non-inhibitory mode at least some of the time. Whether p27 was a cyclin D-cdk4/6 inhibitor or not was controversial, and how it might switch between these two modes was unknown. Arguing for a two state mechanism, we have recently shown that p27 can be both a cdk4 bound-inhibitor and a bound-non-inhibitor, depending on the growth state of the cell. This perspective highlights the significance of this finding in terms of normal cell cycle progression and tumor development.     

Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1)

Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).