The Alternative TrkAIII Splice Variant Targets the Centrosome and Promotes Genetic Instability

The hypoxia-regulated alternative TrkAIII splice variant expressed by human neuroblastomas exhibits oncogenic potential, driven by in-frame exon 6 and 7 alternative splicing, leading to omission of the receptor extracellular immunoglobulin C₁ domain and several N-glycosylation sites. Here, we show that the TrkAIII oncogene promotes genetic instability by interacting with and exhibiting catalytic activity at the centrosome. This function depends upon intracellular TrkAIII accumulation and spontaneous interphase-restricted activation, in cytoplasmic tyrosine kinase (tk) domain orientation, predominantly within structures that closely associate with the fully assembled endoplasmic reticulum intermediate compartment and Golgi network. This facilitates TrkAIII tk-mediated binding of γ-tubulin, which is regulated by endogenous protein tyrosine phosphatases and geldanamycin-sensitive interaction with Hsp90, paving the way for TrkAIII recruitment to the centrosome. At the centrosome, TrkAIII differentially phosphorylates several centrosome-associated components, increases centrosome interaction with polo kinase 4, and decreases centrosome interaction with separase, the net results of which are centrosome amplification and increased genetic instability. The data characterize TrkAIII as a novel internal membrane-associated centrosome kinase, unveiling an important alternative mechanism to “classical” cell surface oncogenic receptor tk signaling through which stress-regulated alternative TrkAIII splicing influences the oncogenic process.Alternative splicing is fundamental for differential protein expression from the same gene and not only increases the proteomic complexity of higher organisms (29) but is also involved in cancer pathogenesis, activating several oncogenes and inactivating several oncosuppressors (17).The neurotrophin receptor tropomyosin-related kinase A (TrkA) is among the proto-oncogenes activated by alternative splicing, with a novel hypoxia-regulated oncogenic alternative TrkAIII splice variant recently identified in advanced-stage human neuroblastomas (NB) and primary glioblastomas (44, 45). In contrast to wild-type TrkAI/TrkAII, the expression of which is associated with better prognosis for NB, induces NB cell differentiation, exhibits a tumor suppressor function in NB models in vivo (9, 19, 22, 30, 44, 45), and may regulate both spontaneous and therapy-induced NB regression (30), TrkAIII is expressed by more-advanced-stage NB and exhibits oncogenic activity in NB models (44, 45). This has challenged the hypothesis of an exclusively tumor-suppressing function for TrkA in NB by providing a way through which tumor-suppressing signals from TrkA can be converted to oncogenic signals from TrkAIII during tumor progression.The oncogenic potential of TrkAIII, characterized by NIH 3T3 cell transforming and NB xenograft primary and metastatic tumor-promoting activity (44, 45), is driven by in-frame alternative splicing of exons 6 and 7. This results in the omission of the receptor extracellular immunoglobulin C₁ (Ig C₁) Ig-like domain and several N-glycosylation sites important in regulating TrkA cell surface expression and preventing ligand-independent activation (2, 44, 45, 48). As a consequence, and unlike TrkAI and TrkAII, TrkAIII is not expressed at the cell surface but accumulates in the intracellular membrane compartment, within which it exhibits spontaneous tyrosine kinase (tk) and phosphoinositol-3 kinase (PI3K) activity and induces chronic signaling through PI3K/Akt/NF-κB but not Ras/mitogen-activated protein kinase (MAPK), inducing a more stress-resistant, angiogenic, and tumorigenic NB cell phenotype (44, 45). This differs from ligand-activated cell surface TrkA, which signals transiently through Ras/MAPK in NB cells to induce differentiation and a less angiogenic and tumorigenic NB cell phenotype (9, 19, 22, 30, 44, 45). This difference in signaling provides a potential basis for the opposing tumor-suppressing and oncogenic effects of alternative TrkA splice variants, which may not only depend upon the dislocation of TrkAIII from cell surface caveolae, which are the sites of TrkAI expression and Ras/MAPK signal initiation (45, 48), but also TrkAIII-associated PI3K activity below the Ras/MAPK activation threshold and/or TrkAIII-associated PI3K antagonism of Raf/MEK/extracellular signal-regulated kinase signaling (44). Signal transduction from intracellular TrkAIII bears close resemblance to the transient signaling through PI3K/Akt/NF-κB but not Ras/MAPK induced by A2a adenosine receptor/c-Src-mediated transactivation of immature TrkAI within the Golgi network (GN) (37), suggesting that the intracellular localization of TrkAIII is a critical determinant of both differential signaling and oncogenic potential.Intracellular nonnuclear membranes are separated into the endoplasmic reticulum (ER), ER-GN intermediate compartment (ERGIC), GN, and transport vesicles, which assemble around, integrate, and interact with the centrosome (38). The centrosome, comprised of two centrioles embedded within a pericentriolar matrix of over 100 proteins, including γ-tubulin, acts as the major microtubule-organizing center and orchestrates the assembly, organization, and integration of the ER, ERGIC, GN, and associated vesicles (38). The centrosome also maintains genomic integrity by duplicating once per cell cycle S phase, ensuring bipolar mitotic spindle formation, accurate chromosome segregation, and the inheritance of a single centrosome by each daughter cell (23).Centrosome duplication is tightly regulated by protein kinases Aurora-A and -B, polo kinases 1 and 4 (Plk-1 and Plk-4), Cdk2, PI3K, Zyg-1, Syk, Nek2, regulators Pin-1 and separase, and related phosphatases (11, 12, 18, 31, 42, 47, 53). The deregulation of centrosome duplication leads to centrosome amplification and subsequently to aberrant mitotic spindle formation, which promotes aneuploidy and polyploidy. These manifestations of genetic instability represent hallmarks of malignancy and drive tumor progression by promoting a more malignant phenotype (11, 12, 26, 35, 49). Centrosome amplification and subsequent genetic instability are induced by kinases that target the centrosome, the loss of centrosome-associated kinase inhibitors, altered levels of centrosome-associated regulators, and oncosuppressor inactivation (6, 7, 11, 12, 35, 40, 42, 49, 53).The localization of TrkAIII to internal membranes, as a prerequisite for oncogenic activity (44, 45), makes identification of the membrane context within which TrkAIII exhibits activity and novel substrate interactions within this environment important in elucidating how TrkAIII exerts oncogenic potential. In the present study, we unveil a novel oncogenic mechanism for TrkAIII by demonstrating that TrkAIII activation within membranes that associate closely with the assembled ER/ERGIC/GN facilitates recruitment to the centrosome, results in centrosome amplification, and promotes genetic instability.     

Cdk5-Dependent Regulation of Rho Activity,Cytoskeletal Contraction,and Epithelial Cell Migration via Suppression of Src and p190RhoGAP

Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.Cell migration is essential for morphogenesis during embryonic development and for epithelial homeostasis and wound healing throughout life. As myosin II is involved in all aspects of cell migration, from cell polarization and adhesion to protrusion and tail retraction (34, 48), the signaling pathways regulating myosin-dependent cytoskeletal contraction are of particular interest. Myosin contraction is regulated by phosphorylation of myosin regulatory light chain (MRLC) at Thr18/Ser19. Although a number of kinases have been identified which phosphorylate these sites, the principal kinases in most cells are myosin light chain kinase (MLCK), a calcium/calmodulin-regulated enzyme, and Rho kinase (ROCK), a downstream effector of the Rho family GTPase RhoA. To provide the stringent control of cytoskeletal contraction needed for migration, RhoA is subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1 (4, 21), and negative regulation by GTPase-activating proteins (GAPs), such as the Src-regulated protein p190RhoGAP (1, 3, 10, 13). An additional level of regulation is provided by guanine nucleotide dissociation inhibitors, which bind to inactive RhoA and other Rho family GTPases, sequestering them in the cytosol (3). Two major downstream effectors of RhoA with regard to the cytoskeleton are the mammalian homologue of diaphanous, involved in actin polymerization (43), and ROCK, which phosphorylates MRLC and myosin phosphatase (20).Cdk5, a serine/threonine kinase, is an atypical member of the well-known family of cyclin-dependent kinases (Cdks). Unlike the other Cdks, it has no known function in cell cycle regulation and is activated by one of two noncyclin proteins, p35 or p39 (16, 41). Phosphorylation of Cdk5 at Y15 increases its activity severalfold (36, 49). Although Cdk5 is most abundant in neuronal cells, where it regulates migration, cytoskeletal dynamics, and membrane trafficking (37, 38, 45), a growing body of evidence indicates that Cdk5 has similar functions in nonneuronal cells (35). In particular, Cdk5 has been shown to strengthen cell-to-matrix adhesion and regulate migration in lens epithelial cells (28), corneal epithelial cells (11, 12, 40), keratinocytes (27), and CHO-K1 cells (15). The effects of Cdk5 on adhesion and migration have been linked, at least in part, to Cdk5-dependent phosphorylation of talin, which strengthens adhesion by slowing the rate of focal adhesion turnover (15). However, we have observed that Cdk5 not only binds to focal adhesions, where talin is located, but also to stress fibers (33). Moreover, in spreading cells, Cdk5 exerts its greatest effect on adhesion 1 to 2 h after plating (28), when stress fiber contraction is pronounced and Cdk5 activity is maximum (33). Therefore, we hypothesized that Cdk5 might regulate the MRLC phosphorylation necessary for stress fiber contraction and stability. To test this possibility, we examined the relationship of Cdk5 activity to MRLC phosphorylation and cytoskeletal contraction in spreading human lens epithelial cells.     

Dictyostelium discoideum CenB Is a Bona Fide Centrin Essential for Nuclear Architecture and Centrosome Stability

Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.Centrins (also known as caltractins) are small calcium-binding proteins of the EF-hand superfamily and are thought to have diversified by gene duplication (37). The first centrin was discovered in the unicellular green algae Tetraselmis striata more than 20 years ago (45). Since then, members of this family of proteins have been found in groups as diverse as yeasts, insects, plants, and humans, making these proteins essentially ubiquitous among eukaryotic cells (55). Furthermore, centrins have been included within the 347 “eukaryotic signature proteins” that are thought to be indispensable for the eukaryotic cell and share no similarities with prokaryotic proteins (21). Many lower eukaryotes have a single centrin gene (e.g., Saccharomyces cerevisiae and Chlamydomonas reinhardtii); however, up to three or four centrin paralogs have been found in higher eukaryotes (e.g., Xenopus laevis, Mus musculus, and Homo sapiens). Centrins have a high level of structural resemblance to calmodulin, exhibiting the characteristic two globular domains interconnected by a linker loop. Each globular domain in turn contains two helix-loop-helix motifs that, in calmodulins, bind calcium ions. However, in many centrins these motifs are slightly modified, and not all four of them have affinity for calcium in the normal range associated with signal transduction (33).Throughout the course of evolution, centrins have acquired multiple functionalities in addition to the archetypical role at the microtubule organizing center (MTOC). For example, the centrin of the flagellated green algae C. reinhardtii (CrCen) localizes to the basal bodies, to the fibers that interconnect the basal bodies and the nucleus, and to the axoneme. CrCen is required for normal basal body replication, segregation, and maturation (26). In addition, it plays an active role in the contraction of MTOC-related fibers (47, 57) and regulates the activity of the inner dynein arm in a calcium-regulated fashion (30).In the budding yeast Saccharomyces cerevisiae, centrin (ScCDC31) localizes primarily to a specialized region of the nuclear envelope (NE) called the half bridge (49), which is in close proximity to the MTOC (known as the spindle pole body [SPB]). Conditional mutants of ScCDC31 show cell cycle arrest and failure to duplicate the SPB (23, 49). CDC31 also binds the NEF2 complex and is required for efficient nucleotide excision repair. CDC31 mutants unable to bind to the complex showed an increased sensitivity to UV (1). In addition, CDC31 is involved in mRNA export through its interaction with SAC3 at the nuclear pore (11). Mammalian cells typically have four centrin paralogs; however, human cells express only three (HsCen1 to -3) and the fourth is a pseudogene (gene ID, 729338) (9, 13, 31, 36). All human centrins show partial localization at the centrioles, in a tissue-specific fashion (HsCen1) or ubiquitously (HsCen2 and -3) (29, 56). Knockdown of HsCen2 inhibits centriole duplication and induces cell division arrest in HeLa cells (46). Additionally, HsCen2 was shown to play a role similar to that of CDC31 in stimulating nucleotide excision repair by binding to xeroderma pigmentosum group C protein (38).The social amoeba Dictyostelium discoideum has emerged as a powerful model organism, in part because it is haploid, it is easy to propagate, and its genome has been recently completed (4, 8, 25). D. discoideum cells undergo a closed mitosis during which the NE remains intact. They also have multiple modes of cytokinesis (53), making them a very useful model for studying the cell division machinery. These cells lack basal bodies and have acentriolar centrosomes that are similar in their trilaminar core structure to yeast SPBs (17). However, D. discoideum interphase centrosomes are not embedded in the NE but are attached to it, and they are surrounded by a centrosomal corona analogous to the pericentriolar material of animal cell centrosomes (4). Centrosomal duplication in D. discoideum involves extensive structural changes and is synchronized with mitosis. It begins at early prophase, by increasing its size to about twice that of an interphase centrosome. At the prophase-prometaphase transition, the corona and the fibrous link to the nucleus are disassembled. This is followed by the insertion of the core into the NE. By metaphase, the two outer layers have come apart and migrated to opposite ends of the cell nucleus, where they organize the spindle. The anaphase-telophase transition marks the beginning of centrosomal maturation. The outer layers fold back into themselves, inducing the formation of a middle layer and a corona, and returning to the size of an interphase centrosome. Finally, the two maturing centrosomes transition out of the NE at the end of mitosis and reform the fibrous link that connects them to the NE (17, 52). It has recently been shown that the D. discoideum Sun1 protein is a key component of the fibrous link that bridges and anchors the centrosome to the cell nucleus (58). DdSun1 predominantly localizes to the nuclear membrane and links chromatin to other components of the fibrous link. Truncation or knockdown of DdSun1 promotes separation of the inner and outer NE membranes, inducing aberrant nuclear morphology and loss of the nucleus-centrosome connection (observed as protrusions of the outer NE membrane). Additionally, cells develop supernumerary centrosomes and aberrant spindles, leading to poor chromosome segregation. All this suggests that the centrosome-nucleus link is of extreme importance in maintaining the genetic stability of the cell.D. discoideum has two known centrin proteins, DdCenA (originally named DdCrp) and DdCenB. The initial characterization of DdCenA describes a very divergent centrin that localizes to the centrosomal corona and to the nucleus (5). The second centrin protein, known as DdCenB, was originally identified as a putative member of the centrin family based on sequence similarity by the Dictyostelium Genome Consortium and remained uncharacterized until now. In this work, we report the initial characterization of DdCenB, including molecular cloning, sequence analysis, cellular localization, and analysis of functional roles.     

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G₂/M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G₂/M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.Despite broad progress in studies of cell cycle control in eukaryotes, advanced models are lacking for the regulation of mitotic entry in human cells. This regulation is pivotal in cell cycle control, and a better understanding of it may be crucial to improving cytotoxic cancer chemotherapy, the mainstay of cancer treatment. Models of mitotic entry in higher eukaryotes revolve around activation of the cyclin B/Cdk1 (cyclin-dependent kinase 1 or Cdc2) complex, which drives the major events of mitosis. A rise in the cyclin B level triggers mitotic entry in Xenopus egg extracts but not in mammalian cells (15, 47). Inhibitory phosphorylation of Cdk1 on the ATP-binding site residue tyrosine 15 (Y15) has been recognized as a key constraint throughout eukaryotes (29, 42). Wee1 and Myt kinases perform this phosphorylation in vertebrate cells, where Wee1 appears to be dominant (34). Kim and Ferrell and others have recently developed an elegant model for ultrasensitive, switch-like inactivation of Wee1 by cyclin B/Cdk1 in a positive feedback loop that contributes to mitotic entry in Xenopus egg extracts (27).Although cyclin A(A2)/Cdk2 is traditionally omitted from models of mitotic entry, accumulating evidence from several different approaches suggests that cyclin A/Cdk complexes play roles. Cyclin A levels rise during S phase and peak in G₂ before falling abruptly in prometaphase of mitosis (60). Microinjection of cyclin A/Cdk2 complexes in human G₂ phase cells was observed to drive mitotic entry (14). Conversely, microinjection of antibodies directed against cyclin A in S-phase cells inhibited mitotic entry without an apparent effect on bulk DNA synthesis (45). In complementary approaches that supported biochemical analyses, cyclin A RNA interference (RNAi) or induction of a dominant negative mutant of Cdk2 (Cdk2-dn), the major cyclin A binding partner, inhibited mitotic entry (13, 15, 21, 37). In these settings, cyclin B/Cdk1 complexes accumulated in inactive, Y15-phosphorylated forms (13, 21, 37). Cdc25 phosphatases, which can reverse this phosphorylation, show reduced activity in this context (37), but increased Cdc25 activity could not readily overcome the arrest (13). RNAi-mediated knockdown of Wee1 was found capable of overriding the arrest mediated by cyclin A RNAi, suggesting that Wee1 is a key rate-limiting factor (13). However, whether and by what mechanisms cyclin A complexes might regulate Wee1 and drive Cdk1 dephosphorylation and mitotic entry have remained unclear.Recently, genetic studies in mice have reinforced these observations while providing evidence for some cell type differences (24). Although Cdk2 is not essential, in its absence Cdk1 binds more cyclin A and E and provides redundant functions (4, 25, 44). Deletion of the cyclin A gene is lethal for embryos and adults (24). Gene deletion in fibroblasts in vitro did not completely abrogate their proliferation but caused S and G₂/M delays. In this setting cyclin E was upregulated, and combined deletion of cyclin E yielded arrest in G₁, S, and G₂/M phases. Cyclin A gene deletion was alone sufficient to block proliferation of hematopoietic stem cells, suggesting that cyclin A is essential for their proliferation.Wee1 is regulated on multiple levels, including inhibitory phosphorylation in the amino-terminal regulatory domain (NRD), residues 1 to 292. This region is predicted to be intrinsically disordered (56), and few functional elements have been identified in it. The cyclin B/Cdk1 complex has been thought to be the principal or exclusive kinase responsible for NRD phosphorylation (18, 27, 28). Two sites in the Xenopus embryonic Wee1 NRD, Thr 104 and Thr 150 (referred to here by the homologous residue, T239, in human somatic Wee1), have been identified as Cdk phosphorylation sites that inhibit Wee1 activity (28). Recent studies of Xenopus somatic Wee1 suggest that T239 phosphorylation may antagonize the function of a surrounding motif, dubbed the Wee box (43). This small, conserved region appears to augment the activity of the carboxy-terminal kinase domain.We show here that cyclin A/Cdk2 complexes directly bind Wee1 as a substrate in human cells. In particular, a conserved cyclin A/Cdk binding RXL motif in the Wee1 NRD is required for efficient T239 phosphorylation. Further analysis revealed that RXL1 is located within a Crm1 binding site that mediates Wee1 export during S and G₂ phases. Cyclin A/Cdk2 activity appears to foster Wee1 export, but this export is not essential for mitotic entry. These findings further define roles of cyclin A/Cdk complexes in regulating Wee1 and mitotic entry in human cells and dissect the mechanisms and consequences of Wee1 redistribution during the run-up to mitosis.     

Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Mitotic centromere-associated kinesin (MCAK) plays an essential role in spindle formation and in correction of improper microtubule-kinetochore attachments. The localization and activity of MCAK at the centromere/kinetochore are controlled by Aurora B kinase. However, MCAK is also abundant in the cytosol and at centrosomes during mitosis, and its regulatory mechanism at these sites is unknown. We show here that cyclin-dependent kinase 1 (Cdk1) phosphorylates T537 in the core domain of MCAK and attenuates its microtubule-destabilizing activity in vitro and in vivo. Phosphorylation of MCAK by Cdk1 promotes the release of MCAK from centrosomes and is required for proper spindle formation. Interfering with the regulation of MCAK by Cdk1 causes dramatic defects in spindle formation and in chromosome positioning. This is the first study demonstrating that Cdk1 regulates the localization and activity of MCAK in mitosis by directly phosphorylating the catalytic core domain of MCAK.Chromosomes are properly attached to the mitotic spindles, and chromosome movement is tightly linked to the structure and dynamics of spindle microtubules during mitosis. Important regulators of microtubule dynamics are the kinesin-13 proteins (37). This kinesin superfamily is defined by the localization of the conserved kinesin core motor domain in the middle of the polypeptide (19). Kinesin-13 proteins induce microtubule depolymerization by disassembling tubulin subunits from the polymer end (6). Among them, mitotic centromere-associated kinesin (MCAK) is the best-characterized member of the family. It depolymerizes microtubules in vitro and in vivo, regulates microtubule dynamics, and has been implicated in correcting misaligned chromosomes (12, 14, 16, 24). In agreement with these observations, both overexpression and inhibition of MCAK result in a disruption of microtubule dynamics, leading further to improper spindle assembly and errors in chromosome alignment and segregation (7, 11, 15, 22, 33). The importance of MCAK in ensuring the faithful segregation of chromosomes is consistent with the observation that MCAK is highly expressed in several types of cancer and thus is likely to be involved in causing aneuploidy (25, 32).While MCAK is found both in the cytoplasm and at the centromeres throughout the cell cycle, it is highly enriched on centrosomes, the centromeres/kinetochores, and the spindle midzone during mitosis (18, 21, 36, 38). In accordance with its localizations, MCAK affects many aspects throughout mitosis, from spindle assembly and maintenance (3, 10, 36) to chromosome positioning and segregation (14, 21, 35). Thus, the precise control of the localization and activity of MCAK is crucial for maintaining genetic integrity during mitosis. Regulation of MCAK on the centromeres/kinetochores by Aurora B kinase in mitosis has been intensively investigated (1, 28, 29, 43). The data reveal that MCAK is phosphorylated on several serine/threonine residues by Aurora B, which inhibits the microtubule-destabilizing activity of MCAK and regulates its localization on chromosome arms/centromeres/kinetochores during mitosis (1, 18, 28). Moreover, in concert with Aurora B, ICIS (inner centromere KinI stimulator), a protein targeting the inner centromeres in an MCAK-dependent manner, may regulate MCAK at the inner centromeres and prevent kinetochore-microtubule attachment errors in mitosis by stimulating the activity of MCAK (27). Interestingly, hSgo2, a recently discovered inner centromere protein essential for centromere cohesion, has been reported to be important in localizing MCAK to the centromere and in spatially regulating its mitotic activity (13). These data highlight that the activity and localization of MCAK on the centromeres/kinetochores during mitosis are tightly controlled by Aurora B and its cofactors. Remarkably, MCAK concentrates at spindle poles from prophase to telophase during mitosis (18); however, only a few studies have been done to deal with that issue. Aurora A-depleted prometaphase cells delocalize MCAK from spindle poles but accumulate the microtubule-stabilizing protein ch-TOG at poles (5), implying that Aurora A might influence the centrosomal localization of MCAK in mitosis. Aurora A is also found to be important for focusing microtubules at aster centers and for facilitating the transition from asters to bipolar spindles in Xenopus egg extracts (42). In addition, it has been revealed that Ca²⁺/calmodulin-dependent protein kinase II gamma (CaMKII gamma) suppresses MCAK''s activity, which is essential for bipolar spindle formation in mitosis (11). More work is required to gain insight into the regulatory mechanisms of MCAK at spindle poles during mitosis.Deregulated cyclin-dependent kinases (Cdks) are very often linked to genomic and chromosomal instability (20). Cyclin B1, the regulatory subunit of Cdk1, is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment (2, 4). We observed that knockdown of cyclin B1 induces defects in chromosome alignment and mitotic spindle formation (N.-N. Kreis, M. Sanhaji, A. Krämer, K. Sommor, F. Rödel, K. Strebhardt, and J. Yuan, submitted for publication). Yet, how Cdk1/cyclin B1 carries out these functions is not very well understood. In this context, it is extremely interesting to investigate the relationship between the essential mitotic kinase Cdk1 and the microtubule depolymerase MCAK in human cells.     

Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1

Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G₁/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.Human cytomegalovirus (HCMV), a highly prevalent β-herpesvirus, can cause serious birth defects and disease in immunocompromised individuals, and it may be associated with cancer and cardiovascular disease (53). Viral gene expression is temporally regulated and is dependent on many cellular factors for a productive infection. Immediate early (IE) genes are expressed by 2 h postinfection (p.i.) and transactivate the early genes required for viral DNA replication. The expression of the late genes, which encode proteins involved in virion maturation and egress, is dependent on viral DNA replication.The virus has adopted different strategies for altering the cellular environment to make it more conducive to productive infection, including the stimulation of host cell DNA replication pathways, cell cycle deregulation and arrest, immune evasion, and inhibition of apoptosis (53). Although HCMV encodes its own DNA polymerase, it is dependent on other cellular resources for DNA replication. Infection of quiescent cells induces passage toward S phase such that the host cell is stimulated to generate proteins and DNA precursors necessary for genome replication; however, entry into S phase and cellular DNA replication are subsequently blocked and the cell arrests in G₁/S (1, 10, 11, 14, 30, 45). Cellular resources are thereby presumably free to be efficiently utilized for viral replication. Cell cycle arrest by HCMV is achieved in part through the misregulation of several cell cycle proteins, including the phosphorylation and accumulation of the Rb family pocket proteins, upregulation of cyclins E and B and their associated kinase activities, inhibition of cyclin A expression, stabilization of p53, and accumulation of Cdc6 and geminin, which inhibits licensing of the cellular origins of DNA replication (8, 17, 30, 49, 54, 65). Some of these cell cycle defects can be attributed to a deregulation of the anaphase-promoting complex (APC) (8, 72, 79, 80), an E3 ubiquitin ligase that is responsible for the timely degradation of cell cycle proteins and mitotic cyclins to promote cycle progression from mitosis through G₁ to S phase (58, 74). As the APC also appears to be a common target among other viruses, including the chicken anemia virus, adenoviruses, and poxviruses (23, 36, 52, 70), understanding the mechanisms leading to its inactivation during viral infection has been of great interest.As we have previously reported, multiple mechanisms may be involved in disabling the APC during HCMV infection (72), which is not surprising given the complexity of its structure and regulation (for a review, see references 58 and 74). The APC is a large multisubunit complex consisting of at least 11 conserved core subunits, as well as other species-specific subunits. In metazoans, the APC2 and APC11 subunits form the catalytic core, and along with APC10, provide the platform for binding the E2 ubiquitin-conjugating enzyme. Each of the APC3, APC8, APC6, and APC7 subunits contain multiple copies of the tetratricopeptide repeat (TPR) motif and together make up the TPR subcomplex, which provides a platform of protein interaction surfaces for binding the coactivators (i.e., Cdh1 and Cdc20) and various substrates. These two subcomplexes are bridged by the large scaffolding subunit APC1, with the TPR subcomplex tethered to APC1 through APC4 and APC5. The binding between APC1, APC4, APC5, and APC8 is also interdependent, such that the loss of one subunit decreases the association of the other three (71).The APC is activated by either of its coactivators, Cdh1 or Cdc20, which also function in recruiting specific substrates to the APC during different phases of the cell cycle. The phosphorylation of several APC subunits at the onset of mitosis, including APC1 and the TPR subunits, by cyclin B/cyclin-dependent kinase 1 (Cdk1) and Plk1 allows the binding of Cdc20 and subsequent activation of the APC (APC^Cdc20) (19, 37), whereas the binding and activation of the complex by Cdh1 is inhibited through its phosphorylation by cyclin B/Cdk1 (9, 29, 38, 83). As cells pass the spindle assembly checkpoint, APC^Cdc20 ubiquitinates securin (to allow for sister chromatid separation) and cyclin B for degradation by the proteasome (42, 67). The subsequent inactivation of Cdk1 and activation of mitotic phosphatases during late anaphase relieves the inhibitory phosphorylation on Cdh1, presumably by Cdc14 (6, 38, 44), which then allows Cdh1 to bind and activate the APC (APC^Cdh1). APC^Cdh1 ubiquitinates Cdc20 and mitotic cyclins for degradation to facilitate mitotic exit and maintains their low levels, along with S-phase regulators (e.g., Cdc6, geminin, etc.), during G₁ (16, 50, 59, 63). The inactivation of APC^Cdh1 as cells enter S phase may be mediated in part through the phosphorylation of Cdh1 by cyclin A/Cdk2 (46) and Cdh1 binding to the inhibitor Emi1 (25). The inactivation of Cdh1 by phosphorylation has been shown in all organisms studied thus far (e.g., yeast, Drosophila, plants, mammals, etc.), and mutants mimicking constitutively phosphorylated Cdh1 on Cdk consensus sites can neither bind nor activate the APC in vivo or in vitro (9, 29, 38, 69, 83).During HCMV infection of fibroblasts in G₀/G₁, however, Cdh1 becomes prematurely phosphorylated in a Cdk-independent manner and no longer associates with the APC (72). This dissociation does not appear to be due to an overexpression of Emi1 (79). Cdc20 also can no longer associate with the APC (79), suggesting a defect in the APC core. We have further shown that the APC core complex disassembles during the infection, with the TPR subunits (i.e., APC3, APC7, and APC8) and APC10 localizing to the cytosol, while APC1 remains nuclear (72). Interestingly, both the phosphorylation of Cdh1 and the dissociation of the APC occur at similar times during HCMV infection. Although either of these mechanisms could render the APC inactive, it was unclear whether these processes are linked or represent independent (or redundant) pathways. The causative factor(s) in mediating these events and the question of whether such a factor(s) was of cellular or viral origin also remained unresolved.On the basis of the results of several recent studies (26, 32, 62), the viral protein kinase UL97 emerged as a likely candidate for involvement in the phosphorylation of Cdh1. Conserved among herpesviruses, UL97 functions in viral genome replication (7, 32, 81) and in nuclear egress of viral capsids (21, 39, 48). UL97 is present in the tegument of the virus particle (76) and is also expressed de novo with early kinetics (i.e., detectable by 5 h p.i. by Western blot assay), with increased expression at later times of the infection (51, 76, 77). UL97 is a serine/threonine (S/T) protein kinase (22), and recent studies have further characterized it as a Cdkl mimic, with predicted structural similarity to Cdk2 (64) and common substrates. UL97 has been shown to phosphorylate in vitro nuclear lamin A/C (21), the carboxyl-terminal domain of RNA polymerase II (5), the translation elongation factor 1δ (EF1δ) (33), and Rb (26, 62) on sites targeted by Cdks, and there is considerable evidence that UL97 phosphorylates lamin A/C, EF1δ, and Rb on these sites in infected cells as well (21, 26, 33, 62). Given that cyclin A/Cdk2 and cyclin B/Cdk1 complexes normally phosphorylate Cdh1, thus preventing its association with the APC, we hypothesized that UL97 phosphorylates Cdh1 during HCMV infection.In the present study, we provide further mechanistic details of the events and players involved in inactivating the APC during HCMV infection. Evidence that UL97 is the viral factor mediating the phosphorylation of Cdh1 was obtained. However, APC disassembly still occurred at similar times in ΔUL97 and wild-type virus infections, indicating that UL97-mediated phosphorylation of Cdh1 is not required for this event. The inactivation of the APC core complex is further attributed to the loss of the APC5 and APC4 subunits early during the infection. The degradation of these subunits is proteasome dependent and requires de novo synthesis of viral early or cellular proteins. While the primary mechanism of inactivation appears to be the dissociation of the complex and the targeted loss of APC5 and APC4, phosphorylation of Cdh1 may provide a small kinetic advantage and backup mechanism for disabling the APC.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Microtubule-Nucleus Interactions in Dictyostelium discoideum Mediated by Central Motor Kinesins

Kinesins are a diverse superfamily of motor proteins that drive organelles and other microtubule-based movements in eukaryotic cells. These motors play important roles in multiple events during both interphase and cell division. Dictyostelium discoideum contains 13 kinesin motors, 12 of which are grouped into nine families, plus one orphan. Functions for 11 of the 13 motors have been previously investigated; we address here the activities of the two remaining kinesins, both isoforms with central motor domains. Kif6 (of the kinesin-13 family) appears to be essential for cell viability. The partial knockdown of Kif6 with RNA interference generates mitotic defects (lagging chromosomes and aberrant spindle assemblies) that are consistent with kinesin-13 disruptions in other organisms. However, the orphan motor Kif9 participates in a completely novel kinesin activity, one that maintains a connection between the microtubule-organizing center (MTOC) and nucleus during interphase. kif9 null cell growth is impaired, and the MTOC appears to disconnect from its normally tight nuclear linkage. Mitotic spindles elongate in a normal fashion in kif9⁻ cells, but we hypothesize that this kinesin is important for positioning the MTOC into the nuclear envelope during prophase. This function would be significant for the early steps of cell division and also may play a role in regulating centrosome replication.Directed cell migration, organelle transport, and cell division involve fundamental motilities that are necessary for eukaryotic cell viability and function. Much of the force required for these motilities is generated through the cyclical interactions of motor proteins with the cell cytoskeleton. Microtubules (MTs) and actin filaments provide structural support and directional guides, and all eukaryotic organisms have diverse, often extensive families of motors that carry out different tasks. Functional studies have revealed that many of the motors work in combination with others, and that the individual deletion of a single motor activity often is insufficient to produce a defect that substantially impairs cell growth or function. The latter phenomenon is particularly evident in some organisms with simple motor families (14, 42). By contrasting homologous motor functions between simple and complex systems, we hope to learn the details of how each motor is custom-tuned for specific tasks.Dictyostelium discoideum is a compact amoeba that exhibits robust forms of motility common to nearly all animal cells, with speeds that frequently exceed corresponding rates in vertebrate cell models (25, 33, 54). Since Dictyostelium possesses a relatively small number of motor proteins (13 kinesin, 1 dynein, and 13 myosin isoforms [23, 24, 26]), it combines advantages of terrific cytology with straightforward molecular genetics and thus represents an excellent model to investigate individual and combined motor protein actions. To date, 11 of the 13 kinesin motors have been analyzed functionally (5, 17, 18, 30, 42, 46, 51, 60). Only 1 of these 11 motors, Kif3, a member of the kinesin-1 family of organelle transporters, appears to be essential for organism viability (51). Individual disruptions of three kinesin genes (kif1, kif4, and kif12) produce distinctive defects in cell growth or organelle transport (30, 42, 46). Analyses of six of the seven other kinesins reveal important phenotypes but only when combined with other motor disruptions or cell stresses. We address here the roles of the remaining two Dictyostelium MT-based motors.kif6 and kif9 encode two central motor kinesins in the Dictyostelium genome (24). The best-studied isoforms of this motor type are represented by the kinesin-13 family, and they largely function to regulate MT length during cell division (13, 16, 40, 41). In some organisms, kinesin-13 motors also have been shown to operate during interphase and to mediate MT and flagellar length control (3, 4, 15) and perhaps even organelle transport (32, 43, 56). kif6 encodes the kinesin-13 family member in Dictyostelium. We demonstrate that Kif6 activity is essential for viability, and that it plays a primary, conserved role in chromosome segregation during cell division.The second of the central motor kinesins, Kif9, does not group with an existing family (24, 38). The gene disruption of this motor reveals a completely novel function for a kinesin in maintaining a connection between the MT-organizing center (MTOC) and the nucleus. By electron microscopy (EM), the MTOC of Dictyostelium appears as a cytoplasmic cube-shaped structure surrounded by amorphous dense material (39, 44). EM, biochemical analyses, antibody labeling, and live-cell imaging studies have demonstrated that during interphase, the cytoplasmic MTOC is firmly and closely attached to the nucleus (28, 29, 44, 48, 49, 63). Upon entry into mitosis, the MTOC duplicates during prophase and is brought to or into a fenestration of the nuclear envelope, and then it establishes an intranuclear bipolar spindle for division (39, 53, 64). While MTOCs can be purified from Dictyostelium, the methods rely heavily on reagents that actively disrupt the attached nuclei (10, 59). A recent study has identified at least one component of this connection, the nuclear envelope protein Sun-1 (67). The perturbation of Sun-1 affects nuclear shape and results in centrosome detachment, hyperamplification, and aneuploidy. We demonstrate in the current work that the disruption of the Kif9 kinesin also perturbs the MTOC-nucleus linkage. Our results suggest that an MT-mediated mechanism plays a significant role in maintaining an MTOC-nucleus connection during interphase, and we discuss how this connection could be important to regulate centrosome replication and ensure proper chromosome segregation during cell division.     

Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect keratinocytes of the differentiating epithelium of the skin or mucosa (reviewed in references 4 and 63). Of more than 150 different HPV types identified thus far, about 25 infect the anogenital region (9). The low-risk types, such as HPV11 and HPV6, are associated with the development of genital warts, while the high-risk types, such as HPV16, -18, and -31, cause high-grade lesions that can progress to invasive cervical carcinoma (17, 38, 61).The HPV life cycle is coupled with the differentiation program that keratinocytes undergo in the epithelium. After infection of the basal cell layer of the epithelium, the virus establishes and maintains its genome as an extrachromosomal element (episome) in the nucleus of infected cells. While the viral episome is maintained at low levels in basal cells, its amplification to a high copy number is trigged in the upper layers of the epithelium by the action of the viral oncogenes E6 and E7 and the differentiation of the infected keratinocytes (reviewed in reference 21). Replication of the HPV genome relies on the viral proteins E1 and E2 and the host DNA replication machinery. Viral DNA replication is initiated by the binding of E2 to specific sites on the viral origin where it facilitates the recruitment and assembly of E1 into a double hexamer that is required to unwind DNA ahead of the bidirectional replication fork (3, 14, 15, 31, 33, 36, 43-45, 52, 60). In addition to its helicase activity, E1 interacts with several cellular replication factors, including polymerase α-primase, replication protein A (RPA), and topoisomerase I, to replicate the viral episome (5, 6, 19, 32, 35, 39).E1, which belongs to helicase superfamily III (SF3) (22, 26), can be divided into three functional regions. Its C-terminal domain has ATPase and helicase activity and can self-assemble into hexamers. It is also this domain that is contacted by E2 to recruit E1 at the origin (50, 57, 58). The middle portion of E1 encompasses the origin-binding domain (OBD) that binds and dimerizes on specific sequences in the origin (55, 56). We and others previously found that a fragment of E1 containing only the C-terminal enzymatic domain and the OBD is capable of supporting viral DNA replication in vitro but is inactive in vivo (2, 51). This suggested that the N-terminal region of E1 plays an essential regulatory function in vivo. As such, it has been shown for HPV11 E1 that this region contains a cyclin E/A-Cdk2 (cyclin-dependent kinase 2) binding motif (CBM), a bipartite nuclear localization signal (NLS) and an CRM1-dependent nuclear export signal (NES), which together regulate the nucleocytoplasmic shuttling of the protein (10, 30, 34). Specifically, it has been shown that phosphorylation of HPV11 E1 on three serine residues within its N-terminal region inhibits its nuclear export (10, 62). Interestingly, bovine papillomavirus (BPV) E1 was also shown to shuttle between the nucleus and the cytoplasm in a phosphorylation-dependent manner. In this case, however, Cdk2 phosphorylation was found to promote, rather than inhibit, the export of the viral helicase (24). This apparent discrepancy between HPV11 and BPV E1 prompted us to examine the regulation of a third E1 protein, specifically that of the high-risk HPV31.We report here that HPV31 E1 also shuttles between the nucleus and the cytoplasm through its conserved NLS and NES. We determined that nuclear export of HPV31 E1 is dependent on the CRM1 export pathway and is inhibited by Cdk2 phosphorylation of serines 92 and 106. We also found that nuclear export of E1 is not required for transient viral DNA replication and thus investigated its role in viral genome maintenance and amplification in immortalized keratinocytes. In contrast to the wild type (WT), a mutant genome carrying a defective E1 NES was poorly maintained and progressively lost upon cell division, indicating that nuclear export of E1 is required for long-term maintenance of the viral episome. Because nuclear export of E1 is not required for viral DNA replication per se but needed for episomal maintenance over several cell divisions, we investigated the possibility that continuous accumulation of E1 into the nucleus is detrimental to cellular proliferation. In support of this possibility, we found that the accumulation of E1 at high levels in the nucleus impedes cellular proliferation by delaying cell cycle progression in the S phase. In addition, we found that this delay was alleviated when nuclear export of E1 was increased. Altogether, these results suggest that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.