Showing papers on "Promyelocytic leukemia protein published in 2002"
TL;DR: The SIRT1 deacetylase is established as a novel negative regulator of p53 function capable of modulating cellular senescence in mammalian cells upon overexpression of either PML or oncogenic Ras.
Abstract: The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.
866 citations
TL;DR: The PML gene, involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL), encodes a protein which localizes to the PML-nuclear body, a subnuclear macromolecular structure, which controls apoptosis, cell proliferation, and senescence.
566 citations
TL;DR: This review focuses on the current theories for molecular and biochemical functions of the PML NB and the supporting evidence for each and the results of these strategies and their inherent limitations are discussed within this review.
Abstract: It has become clear that the nucleus is organized into discrete structures. Although not membrane bound, these structures are considered nuclear organelles. Widespread interest in one such nuclear organelle, the promyelocytic leukemia nuclear body (NB), has emerged because of its link to several human disorders, including acute promyelocytic leukemia and AIDS. Studies of the physiological effects of promyelocytic leukemia NBs and the promyelocytic leukemia protein (PML) indicate that these play roles in growth control, transformation suppression, apoptosis and Ras induced senescence. Unfortunately, the molecular and biochemical bases for physiological phenomena associated with PML are not well understood. PML and, by inference, the PML NB have been ascribed apparently disparate roles in transcription, DNA repair, DNA replication, and RNA transport. Its clear physiological importance means that defining a set of discrete biochemical functions for the PML NB is critical. This review focuses on the current theories for molecular and biochemical functions of the PML NB and the supporting evidence for each.
PML NBs, also known as PML oncogenic domains, nuclear domain 10's, or Kremer bodies, are currently defined by the presence of PML at these nuclear structures. PML and its associated NBs were first described in a series of studies in the early 1990s which showed that PML was fused to the retinoic acid receptor alpha (RARα) in acute promyelocytic leukemia (APL) patients (reviewed in reference 63). These studies demonstrated that PML NBs were similar to those previously observed by electron microscopy in the 1960s (63). Intriguingly, NBs were disrupted in the APL patients but reformed after treatment with all-trans-retinoic acid (ATRA) (36). Reformation of bodies correlated with remission of disease in patients. This was the first evidence that the integrity of these structures may be critically important to the health of the cell (36, 63). These findings sparked widespread interest in the function of these organelles. These early studies also revealed that the PML protein contained a set of novel zinc-binding domains, known as the RING and B-boxes. Early on, it was proposed that PML utilized these zinc fingers to directly bind DNA and thus alter gene expression. However, in the past 10 years, it has become clear that the RING and B-box domains forge protein associations that are critical to the integrity of this multiprotein complex and subsequent physiological function(s) of this organelle (36, 40, 63).
Most reported strategies for assessing PML NB function in essence are designed to answer the following questions: what nuclear structures are the bodies near to, what other macromolecules localize with the body, and what are the effects of disrupting the body? These strategies arise because the discrete biochemical functions of either PML or PML bodies are not known. The results of these strategies and their inherent limitations are discussed within this review. Further, the following considerations should be taken into account in an assessment of PML NB function. First, the expression of the PML gene is not required for viability, since PML−/− mice develop in essence normally and do not get spontaneous cancers at rates higher than do littermate controls (99). Second, the PML gene is not evolutionarily conserved among eukaryotes, being absent in Drosophila melanogaster, Saccharomyces cerevisiae, and Arabidopsis thaliana (see below). Third, unlike other nuclear organelles, there appears to be no PML bodies in Xenopus laevis. However, the disruption of these organelles apparently contributes to human disease. These features and their potential clues to PML NB function are discussed below.
Because of space limitations, many topics are not discussed here. For instance, the study of viral systems has been key to our current understanding of the PML NB. However, an in-depth discussion of these contributions is beyond the scope of this review, but this topic is discussed elsewhere (25, 59, 72). Many excellent and comprehensive reviews have been written on various aspects of the physiological functions of PML and its relationship to APL (36, 40, 59, 63, 82). The present review attempts to describe current understanding of the molecular and biochemical underpinnings of PML NB function.
324 citations
TL;DR: It is speculated that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing and alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.
312 citations
TL;DR: The hypothesis that PML/RARα can cooperate with FLT3-ITD to induce an APL-like disease in the mouse is tested and activating mutations inFLT3 can functionally substitute for the additional mutations that occur during mouse APL progression.
Abstract: Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor α (RARα). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARα in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARα from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARα can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARα transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARα transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARα and FLT3-ITD in development of the murine APL phenotype.
307 citations
TL;DR: These results provide the first evidence of a functional relationship between PML and a checkpoint kinase in γ irradiation-induced apoptosis and an ataxia telangiectasia-mutated (ATM)–hCds1/Chk2–PML pathway.
Abstract: The promyelocytic leukaemia (PML) gene is translocated in most acute promyelocytic leukaemias and encodes a tumour suppressor protein. PML is involved in multiple apoptotic pathways and is thought to be pivotal in gamma irradiation-induced apoptosis. The DNA damage checkpoint kinase hCds1/Chk2 is necessary for p53-dependent apoptosis after gamma irradiation. In addition, gamma irradiation-induced apoptosis also occurs through p53-independent mechanisms, although the molecular mechanism remains largely unknown. Here, we report that hCds1/Chk2 mediates gamma irradiation-induced apoptosis in a p53-independent manner through an ataxia telangiectasia-mutated (ATM)-hCds1/Chk2-PML pathway. Our results provide the first evidence of a functional relationship between PML and a checkpoint kinase in gamma irradiation-induced apoptosis.
249 citations
TL;DR: It is demonstrated that ionizing radiation induces the stable association of p53 with hMre11 and PML, and this suggests that the PML NBs are involved in the recognition and/or processing of DNA breaks and possibly in the recruitment of proteins required for both checkpoint and DNA-repair responses.
Abstract: PML nuclear bodies (PML NBs) respond to many cellular stresses including viral infection, heat shock, arsenic and oncogenes and have been implicated in the regulation of p53-dependent replicative senescence and apoptosis. Recently, the hMre11/Rad50/NBS1 repair complex, involved in Double Strand Breaks (DSBs) repair, was found to colocalize within PML NBs, suggesting a role for these nuclear sub-domains in the DNA repair signalling pathway. We report here that in normal human fibroblasts, after ionizing radiation (IR), the PML NBs are modified and recognize sites of DNA breaks (ssDNA breaks and DSBs). Eight to 12 h after radiation PML NBs associate with hMre11 Ionizing Radiation-Induced Foci (IRIF), and subsequently with p53 within discrete foci. The PML, hMre11 and p53 colocalizing structures mark sites of DSBs as identified by immunolocalization with anti phosphorylated histone gamma-H2AX. Furthermore, we demonstrate that ionizing radiation induces the stable association of p53 with hMre11 and PML. These results suggest that the PML NBs are involved in the recognition and/or processing of DNA breaks and possibly in the recruitment of proteins (p53 and hMre11) required for both checkpoint and DNA-repair responses.
188 citations
TL;DR: It is shown that three classes of PML nuclear bodies can be distinguished, on the basis of their dynamic properties in living cells, and one class is particularly noteworthy in that it moves by a metabolic-energy-dependent mechanism.
Abstract: Promyelocytic leukaemia (PML) nuclear bodies are present in most mammalian cell nuclei. PML bodies are disrupted by PML retinoic acid receptor alpha (RAR alpha) oncoproteins in acute promyelocytic leukaemia. These bodies contain numerous proteins, including Sp100, SUMO-1, HAUSP(USP7), CBP and BLM, and they have been implicated in aspects of transcriptional regulation or as nuclear storage depots. Here, we show that three classes of PML nuclear bodies can be distinguished, on the basis of their dynamic properties in living cells. One class of PML bodies is particularly noteworthy in that it moves by a metabolic-energy-dependent mechanism. This represents the first example of metabolic-energy-dependent transport of a nuclear body within the mammalian cell nucleus.
188 citations
TL;DR: This review will focus on the novel, nuclear functions of eif4E and how they contribute to eIF4E's growth-activating and oncogenic properties.
120 citations
TL;DR: Daxx can affect Pax5's roles as an activator or repressor in B cells and a role for Daxx as a transcriptional coactivator is described.
116 citations
TL;DR: PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology, and in vivo observations corroborated results showing more virus production in PML−/− fibroblasts.
Abstract: The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML−/− mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML−/− mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML−/− fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.
TL;DR: In this article, the authors observed that purified RINGs from a variety of functionally unrelated proteins, including promyelocytic leukemia protein, KAP-1/TIF1β, Z, Mel18, and BRCA1-associated RING domain (BARD1), self-assemble into supramolecular structures in vitro that resemble those they form in cells.
Abstract: RING domains act in a variety of unrelated biochemical reactions, with many of these domains forming key parts of supramolecular assemblies in cells. Here, we observe that purified RINGs from a variety of functionally unrelated proteins, including promyelocytic leukemia protein, KAP-1/TIF1β, Z, Mel18, breast cancer susceptibility gene product 1 (BRCA1), and BRCA1-associated RING domain (BARD1), self-assemble into supramolecular structures in vitro that resemble those they form in cells. RING bodies form polyvalent binding surfaces and scaffold multiple partner proteins. Separation of RING bodies from monomers reveals that self-assembly controls and amplifies their specific activities in two unrelated biochemistries: reduction of 5′ mRNA cap affinity of eIF4E by promyelocytic leukemia protein and Z, and E3 ubiquitin conjugation activity of BARD1:BRCA1. Functional significance of self-assembly is underscored by partial restoration of assembly and E3 activity of cancer predisposing BRCA1 mutant by forced oligomerization. RING self-assembly creates bodies that act structurally as polyvalent scaffolds, thermodynamically by amplifying activities of partner proteins, and catalytically by spatiotemporal coupling of enzymatic reactions. These studies reveal a general paradigm of how supramolecular structures may function in cells.
TL;DR: A novel murine model system based on the transduction of purified murine hematopoietic progenitors using high-titer retroviral vectors encoding promyelocytic leukemia-RAR (PML-R AR), and the green fluorescent protein (GFP) as a marker faithfully reproduces the main characteristics of human APL is reported.
TL;DR: It is demonstrated that in vivo activation of cAMP signaling contributes to APL clearance, independently of its RA-sensitivity, thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach.
Abstract: Differentiation therapy for acute myeloid leukemia uses transcriptional modulators to reprogram cancer cells. The most relevant clinical example is acute promyelocytic leukemia (APL), which responds dramatically to either retinoic acid (RA) or arsenic trioxide (As2O3). In many myeloid leukemia cell lines, cyclic adenosine monophosphate (cAMP) triggers growth arrest, cell death, or differentiation, often in synergy with RA. Nevertheless, the toxicity of cAMP derivatives and lack of suitable models has hampered trials designed to assess the in vivo relevance of theses observations. We show that, in an APL cell line, cAMP analogs blocked cell growth and unraveled As2O3-triggered differentiation. Similarly, in RA-sensitive or RA-resistant mouse models of APL, continuous infusions of 8-chloro-cyclic adenosine monophosphate (8-Cl-cAMP) triggered major growth arrest, greatly enhanced both spontaneous and RA- or As2O3-induced differentiation and accelerated the restoration of normal hematopoiesis. Theophylline, a well-tolerated phosphodiesterase inhibitor which stabilizes endogenous cAMP, also impaired APL growth and enhanced spontaneous or As2O3-triggered cell differentiation in vivo. Accordingly, in an APL patient resistant to combined RA–As2O3 therapy, theophylline induced blast clearance and restored normal hematopoiesis. Taken together, these results demonstrate that in vivo activation of cAMP signaling contributes to APL clearance, independently of its RA-sensitivity, thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach.
TL;DR: The effect of As2O3 on histone H3 phosphoacetylation at the CASPASE-10 gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.
TL;DR: It is demonstrated that Daxx interacts in vitro and in vivo with MSP58 via its NH2-terminal segment, which is distinct from the binding region of Fas, apoptosis signal-regulating kinase 1, and promyelocytic leukemia protein, suggesting a unique modulatory role of MSP 58 on DaxX function.
TL;DR: It is shown that the minor capsid protein L2 of human papillomavirus (HPV) type 33 induces a reorganization of ND10-associated proteins, and the differential change in protein composition observed provides further evidence to suggest that the ND10 -associated proteins are an important interface of viral life cycle and host cell.
TL;DR: The findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific, and further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APl cells.
TL;DR: Data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.
Abstract: The adapter protein tumor necrosis factor receptor (TNFR)1–associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.
TL;DR: The human ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB-2) cDNA is identified, as a novel RA-induced gene in APL cells, which strongly enhanced PML-RARα strongly enhanced RA- induced ASB- 2 mRNA expression.
TL;DR: Evidence of a functional interaction between the promyelocytic leukemia zinc finger protein (PLZF) and DRAL/FHL2 is presented, the first reported incidence of a bona fide FHL protein-mediated corepression and supports the notion of these proteins having a role as coregulators of tissue-specific gene expression.
TL;DR: It is suggested that CREB expression could be a useful marker for leukemia in patients with acute disease and a role for CREB in leukemogenesis is suggested.
TL;DR: To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type bacULovirus or a baculovirus expressing the M r 69,000 form of PML.
Abstract: A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the M(r) 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of alpha, beta, or gamma groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the alpha 0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication.
TL;DR: It is reported for the first time that some NDH II/RNA helicase A is a component of promyelocytic leukemia nuclear bodies (PML NBs), and it is suggested thatNDH II recruitment to PML NBs is connected with transcriptional regulation of interferon-α–inducible genes attached to PMl NBs.
Abstract: It is known that nuclear DNA helicase II (NDH II) links CREB-binding protein directly to RNA polymerase II holoenzyme, and that this interaction is essential for gene activation by CREB Here, we report for the first time that some NDH II/RNA helicase A is a component of promyelocytic leukemia nuclear bodies (PML NBs) An autoimmune serum specific for PML NBs was identified and used in immunoprecipitation experiments NDH II was present in the immunoprecipitates as shown by mass spectrometry and by immunoblotting Immunofluorescence and ultrastructural studies showed that NDH II colocalizes with a small subset of PML NBs in control cells, however, colocalizes with practically all bodies in interferon-alpha-stimulated cells After interferon stimulation, more PML NBs were found to contain newly synthesized RNA, as indicated by bromouridine incorporation PML NBs also contain RNA polymerase II The association of NDH II with PML NBs was transcriptionally dependent, and NDH II was present in all bodies with nascent RNA Blocking of mRNA synthesis caused NDH II relocalization from nucleoplasm to nucleoli Based on the data, we suggest that NDH II recruitment to PML NBs is connected with transcriptional regulation of interferon-alpha-inducible genes attached to PML NBs
TL;DR: The present study provides the first evidence that physiological factors modulate the mRNA transport functions of eIF4E and that this regulation is PML dependent.
Abstract: The eukaryotic initiation factor 4E (eIF4E), when dysregulated, transforms cells. A substantial fraction of eIF4E forms nuclear bodies that colocalize with those associated with the promyelocytic leukemia protein PML. Overexpression studies indicate that nuclear eIF4E promotes the transport of cyclin D1 mRNA from the nucleus to the cytoplasm and that PML is a key negative regulator of this function. Since previous studies used overexpression methods, the physiological relevance of eIF4E mRNA transport function or its interaction with PML remained unknown. Therefore, we monitored whether eIF4E-dependent transport could be modulated in response to environmental conditions. Here we report that cadmium treatment, which disperses PML nuclear bodies, leaves eIF4E bodies intact, leading to increased transport of cyclin D1 mRNA and increased cyclin D1 protein levels. Removal of cadmium allows PML to reassociate with eIF4E nuclear bodies, leading to decreased cyclin D1 transport and reduced cyclin D1 protein levels. In contrast, we show that treating cells with interferon increased the levels of PML protein at the PML-eIF4E nuclear body, leading to nuclear retention of cyclin D1 transcripts and reduced cyclin D1 protein levels. Neither interferon nor cadmium treatment altered cyclin D1 levels in PML(-/-) cells. Consistently, overexpression of a series of PML and eIF4E mutant proteins established that PML eIF4E interaction is required for the observed effects of cadmium and interferon treatment. The present study provides the first evidence that physiological factors modulate the mRNA transport functions of eIF4E and that this regulation is PML dependent.
TL;DR: The cloning and characterization of a full-length IE1 transcript and protein (IE1B) from human herpesvirus 6 (HHV-6) variant B highlights the similarity and divergence between IE1 from both variants and provides useful information pertaining to the early phase of infection.
TL;DR: It is demonstrated that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1), which has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression.
Abstract: Human cytomegalovirus (HCMV) is a member of the herpesvirus family, exhibiting a narrow host range and a characteristic temporal cascade of gene expression in permissive cells. While HCMV poses a low threat to healthy individuals, it is life threatening to the immunocompromised, including prenatally infected newborns and AIDS patients (3).
Primary transcripts from the major immediate-early region undergo alternative splicing to yield two major gene products. The 1.95-kb IE1 transcript is comprised of exons 1 to 4 and gives rise to the abundant IE72 gene product. This 491-amino-acid protein is present throughout HCMV infection (42). The IE2 transcript is comprised of exons 1, 2, 3, and 5 and encodes IE86, which is a promiscuous transactivator of both viral and cellular promoters. During infection, IE72 and IE86 are the first and most abundantly expressed proteins and are required for the subsequent induction of the early and late genes. IE72 and IE86 contain a common transactivation domain encoded within exon 3, which encodes amino acids 25 to 85 of both proteins (37). IE86 and IE72 independently and synergistically activate heterologous promoters (8, 9, 16, 48). Cellular permissiveness for HCMV infection requires IE72 transactivation of the major immediate-early protein enhancer through the NF-κB site (43). Thus, both IE86 and IE72 are major gene regulatory factors that play essential roles in HCMV infection.
We have discovered that IE72 is a viral kinase capable of phosphorylating itself, as well as E2F-1, -2, and -3 and the pocket proteins p107 and p130, but not E2F-4 or -5 or pRb (36). The important role that IE72 plays in HCMV lytic growth is underscored by the fact that a recombinant virus bearing a deletion of exon 4 in the major immediate-early region is severely impaired for replication at a low multiplicity of infection (MOI) (30). This block in DNA replication correlates with a defect in the accumulation of ppUL44, an early gene product required for viral DNA polymerase, and failure to form DNA replication compartments, which may be related to a failure to disrupt the nuclear structures referred to as PML oncogenic domains (PODs) (2), nuclear domain 10, or nuclear dots; these defects can be corrected when IE72 is supplied in trans (2, 15, 50).
IE72 is involved in viral effects on numerous cellular processes including gene regulation, cell cycle progression, signal transduction, POD dispersal, and apoptosis (2, 25, 27, 32, 50, 52). Posttranslational modifications are common mechanisms for the regulation of multifunctional proteins. Our studies have determined that IE72 is autophosphorylated (36) and is also phosphorylated at distinct sites by a cellular kinase(s) (C. Himmelheber and J. Azizkhan-Clifford, unpublished data). The present investigation demonstrates that IE72 exhibits a novel posttranslational modification in which the small ubiquitin-like modifier (SUMO-1) is covalently attached to lysine 450 of IE72.
SUMO-1 (also known as sentrin, GMP1, PIC1, and Ubl1, or in Saccharomyces cerevisiae as SMt3), a ubiquitin-like protein sharing 48% homology with ubiquitin (5), functions as an important reversible protein modifier. Since the discovery of SUMO-1 in 1996, the list of proteins that have been reported to be SUMO-1 modified has been expanding (see reference 51 for a review); many SUMO-modified proteins are associated with PODs. The major POD structural component, promyelocytic leukemia protein (PML), was first discovered in acute promyelocytic leukemia patients exhibiting a chromosomal translocation, t(15;17), that fuses the retinoic acid receptor to PML (7, 11, 14, 22). PODs have more recently been identified as sites of cellular and viral replication processes (26). While POD organization has been linked to cell transformation, PODs are the target for a variety of viral proteins, and the loss of POD organization is thought to be important in the viral infection cycle (see references 1 and 18 for reviews). SUMO-1 conjugation accounts for specific effects, including altered stability, gene regulation, subcellular localization, and protein-protein interactions (10, 19, 24, 39). For example, the SUMO-1 conjugation of PML was reported to function as a POD targeting signal (34), whereas SUMO-1 modification of RanGAP1 enables its interaction at the nuclear pore complex and SUMO-1 modification of p53 increases its transactivation activity. In this study, we have mapped the site of SUMO modification on IE72 to lysine 450 and have demonstrated that IE72 sumoylation is not involved in the ability of IE72 to target or disrupt PODs, nor does it appear to significantly alter IE72 stability, transactivation activity, or ability to complement IE72-deficient HCMV. The precise role of IE72 sumoylation remains to be determined.
TL;DR: The human cytomegalovirus tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0.
Abstract: The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E. coli lacZ gene controlled by various promoters. In this system, pp71 stimulated β-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing β-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML−/−) as in control animals, and in PML−/− fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.
TL;DR: The structure, composition, and dynamics that may provide insights into the function of PML bodies are discussed and the changes that occur are interpreted as evidence for a role of these structures in gene transcription.
Abstract: The promyelocytic leukemia (PML) nuclear body is one of many subnuclear domains in the eukaryotic cell nucleus. It has received much attention in the past few years because it accumulates the promyelocytic leukemia protein called PML. This protein is implicated in many nuclear events and is found as a fusion with the retinoic acid receptor RARα in leukemic cells. The importance of PML bodies in cell differentiation and growth is implicated in acute promyelocitic leukemia cells, which do not contain PML bodies. Treatment of patients with drugs that reverse the disease phenotype also causes PML bodies to reform. In this review, we discuss the structure, composition, and dynamics that may provide insights into the function of PML bodies. We also discuss the repsonse of PML bodies to cellular stresses, such as virus infection and heat shock. We interpret the changes that occur as evidence for a role of these structures in gene transcription. We also examine the role of the posttranslational modification, SUMO...
TL;DR: Structural analysis revealed a role for the unique N-terminal domain of RGS12TS-S in its transcriptional repressor and cell cycle-regulating activities and showed that the RGS domain was dispensable for these functions.
Abstract: RGS12TS-S, an 1,157-amino-acid RGS protein (regulator of G protein signaling), is a nuclear protein that exhibits a unique pattern of subnuclear organization into nuclear foci or dots when expressed endogenously or ectopically. We now report that RGS12TS-S is a nuclear matrix protein and identify structural determinants that target this protein to the nuclear matrix and to discrete subnuclear sites. We also determine the relationship between RGS12TS-S-decorated nuclear dots and known subnuclear domains involved in control of gene expression and provide the first evidence that RGS12TS-S is functionally involved in the regulation of transcription and cell cycle events. A novel nuclear matrix-targeting sequence was identified that is distinct from a second novel motif needed for targeting RGS12TS-S to nuclear dots. RGS12TS-S nuclear dots were distinct from Cajal bodies, SC-35 domains, promyelocytic leukemia protein nuclear bodies, Polycomb group domains, and DNA replication sites. However, RGS12TS-S inhibited S-phase DNA synthesis in various tumor cell lines independently of Rb and p53 proteins, and its prolonged expression promoted formation of multinucleated cells. Expression of RGS12TS-S dramatically reduced bromo-UTP incorporation into sites of transcription. RGS12TS-S, when tethered to a Gal4 DNA binding domain, dramatically inhibited basal transcription from a Gal4-E1b TATA promoter in a histone deacetylase-independent manner. Structural analysis revealed a role for the unique N-terminal domain of RGS12TS-S in its transcriptional repressor and cell cycle-regulating activities and showed that the RGS domain was dispensable for these functions. These results provide novel insights into the structure and function of RGS12TS-S in the nucleus and demonstrate that RGS12TS-S possesses biological activities distinct from those of other members of the RGS protein family.