Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in <Emphasis Type="Italic">Dictyostelium</Emphasis>

The calmodulin-binding protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar protein. Prior to this study, the dynamics of specific nucleolar proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar protein and the only nucleolar calmodulin-binding protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.     

<Emphasis Type="Italic">Dictyostelium</Emphasis> puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Nucleomorphin (NumA1) is a nucleolar/nucleoplasmic protein linked to cell cycle in Dictyostelium. It interacts with puromycin-sensitive aminopeptidase A (PsaA) which in other organisms is a Zn²⁺-metallopeptidase thought to be involved in cell cycle progression and is involved in several human diseases. Here, we have shown that Dictyostelium PsaA contains domains characteristic of the M1 family of Zn²⁺-metallopeptidases: a GAMEN motif and a Zn²⁺-binding domain. PsaA colocalized with NumA1 in the nucleoplasm in vegetative cells and was also present to a lesser extent in the cytoplasm. The same localization pattern was observed in cells from slugs, however, in fruiting bodies PsaA was only detected in spore nuclei. During mitosis PsaA redistributed mainly throughout the cytoplasm. It possesses a functional nuclear localization signal (⁶⁸⁰RKRF⁶⁸³) necessary for nuclear entry. To our knowledge, this is the first nuclear localization signal identified in a Psa from any organism. Treatment with Ca²⁺ chelators or calmodulin antagonists indicated that neither Ca²⁺ nor calmodulin is involved in PsaA localization. These results are interpreted in terms of the inter-relationship between NumA1 and PsaA in cell function in Dictyostelium.     

Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH₂ terminus of importin-β that is distinct from that used to bind importin-α.     

Rad53 homologue forkhead-associated kinase A (FhkA) and Ca<Superscript>2+</Superscript>-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Background

During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca²⁺-binding protein 4a (CBP4a), which is a binding partner of NumA1.

Methods

Polyclonal antibodies were produced in-house. Cells were fixed and probed with either anti-FhkA or anti-CBP4a in order to determine cellular localization during interphase and throughout the stages of mitosis. Colocalization with DAPI nuclear stain allowed us to determine the location of the nucleus and nucleolus while colocalization with anti-α-tubulin allowed us to determine the cell cycle stage.

Results

Here we verify two novel nucleolar proteins, Rad53 homologue FhkA which localized around the edge of the nucleolus and CBP4a which was detected throughout the entire nucleolus. Treatment with the Ca²⁺ chelator BAPTA (5?mM) showed that the nucleolar localization of CBP4a is Ca²⁺-dependent. In response to actinomycin D (0.05?mg/mL) CBP4a disappeared from the nucleolus while FhkA protruded from the nucleus, eventually pinching off as cytoplasmic circles. FhkA and CBP4a redistributed differently during mitosis. FhkA redistributed throughout the entire cell and at the nuclear envelope region from prometaphase through telophase. In contrast, during prometaphase CBP4a relocated to many large, discrete “CBP4a islands” throughout the nucleoplasm. Two larger “CBP4a islands” were also detected specifically at the metaphase plate region.

Conclusions

FhkA and CBP4a represent the sixth and seventh nucleolar proteins that have been verified to date in Dictyostelium and the third and fourth studied during mitosis. The protein-specific distributions of all of these nucleolar proteins during interphase and mitosis provide unique insight into nucleolar protein dynamics in this model organism setting the stage for future work.

     

Mechanisms controlling subcellular localization of the G(1) cyclins Cln2p and Cln3p in budding yeast

Different G₁ cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G₁ cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated. The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle. In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity. Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein. This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm

The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase) which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2, ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4 showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2, multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus.     

Identification of nuclear import mechanisms for the neuronal Cdk5 activator

The activation of Cdk5 by p35 plays a pivotal role in a multitude of nervous system activities ranging from neuronal differentiation to degeneration. A fraction of Cdk5 and p35 localizes in the nucleus where Cdk5-p35 exerts its functions via protein phosphorylation, and p35 displays a dynamic localization between the cytoplasm and the nucleus. Here, we examined the nuclear import properties of p35. In nuclear import assays, p35 was actively transported into the nuclei of digitonin-permeabilized HeLa cells and cortical neurons by cytoplasmic carrier-mediated mechanisms. Importin-beta, importin-5, and importin-7 were identified to import p35 into the nuclei via a direct interaction with it. An N-terminal region of p35 was defined to interact with the above importins, serving as a nuclear localization signal. Finally, we show that the nuclear localization of p35 does not require the association of Cdk5. Furthermore, Cdk5 and importin-beta/5/7 are mutually exclusive in binding to p35. These results suggest that p35 employs pathways distinct from that used by Cdk5 for transport to the nucleus.     

The cleidocranial dysplasia‐related R131G mutation in the Runt‐related transcription factor RUNX2 disrupts binding to DNA but not CBF‐β

Cleidocranial dysplasia (CCD) is caused by haploinsufficiency in RUNX2 function. We have previously identified a series of RUNX2 mutations in Korean CCD patients, including a novel R131G missense mutation in the Runt‐homology domain. Here, we examine the functional consequences of the RUNX2^R131G mutation, which could potentially affect DNA binding, nuclear localization signal, and/or heterodimerization with core‐binding factor‐β (CBF‐β). Immunofluorescence microscopy and western blot analysis with subcellular fractions show that RUNX2^R131G is localized in the nucleus. Immunoprecipitation analysis reveals that heterodimerization with CBF‐β is retained. However, precipitation assays with biotinylated oligonucleotides and reporter gene assays with RUNX2 responsive promoters together reveal that DNA‐binding activity and consequently the transactivation of potential of RUNX2^R131G is abrogated. We conclude that loss of DNA binding, but not nuclear localization or CBF‐β heterodimerization, causes RUNX2 haploinsufficiency in patients with the RUNX2^R131G mutation. Retention of specific functions including nuclear localization and binding to CBF‐β of the RUNX2^R131G mutation may render the mutant protein an effective competitor that interferes with wild‐type function. J. Cell. Biochem. 110: 97–103, 2010. © 2010 Wiley‐Liss, Inc.     

A Ras subfamily GTPase shows cell cycle-dependent nuclear localization

Previously characterized Ras subfamily proteins have been found to be predominantly associated with the plasma membrane where they function in signal transduction pathways to convey extracellular signals to intracellular targets. Here, we provide evidence that the Dictyostelium Ras subfamily protein RasB has a novel subcellular localization and function. The protein is predominantly localized in the nucleus during most of the cell cycle. Furthermore, during mitosis and cytokinesis RasB assumes a diffuse cellular localization despite the fact that the nuclear membrane stays intact. The linkage between the position of RasB in the cell and division suggests that it may have a role in nuclear division. Consistent with this idea, rasB^– cells exhibit severe growth defects and cells overexpressing an activated version of RasB are multinucleate.     

Nuclear trafficking of the anti‐apoptotic Coxiella burnetii effector protein AnkG requires binding to p32 and Importin‐α1

The obligate intracellular bacterium Coxiella burnetii causes the zoonotic disease Q‐fever. Coxiella pathogenesis depends on a functional type IV secretion system (T4SS). The T4SS effector AnkG inhibits pathogen‐induced host cell apoptosis, which is believed to be important for the establishment of a persistent infection. However, the mode of action of AnkG is not fully understood. We have previously demonstrated that binding of AnkG to p32 is crucial for migration of AnkG into the nucleus and that nuclear localization of AnkG is essential for its anti‐apoptotic activity. Here, we compared the activity of AnkG from the C. burnetii strains Nine Mile and Dugway. Although there is only a single amino acid exchange at residue 11, we observed a difference in anti‐apoptotic activity and nuclear migration. Mutation of amino acid 11 to glutamic acid, threonine or valine results in AnkG mutants that had lost the anti‐apoptotic activity and the ability to migrate into the nucleus. We identified Importin‐α1 to bind to AnkG, but not to the mutants and concluded that binding of AnkG to p32 and Importin‐α1 is essential for migration into the nucleus. Also during Coxiella infection binding of AnkG to p32 and Importin‐α1 is crucial for nuclear localization of AnkG.     

Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression

Cell division requires the coordination of critical protein kinases and phosphatases. Greatwall (Gwl) kinase activity inactivates PP2A-B55 at mitotic entry to promote the phosphorylation of cyclin B–Cdk1 substrates, but how Gwl is regulated is poorly understood. We found that the subcellular localization of Gwl changed dramatically during the cell cycle in Drosophila. Gwl translocated from the nucleus to the cytoplasm in prophase. We identified two critical nuclear localization signals in the central, poorly characterized region of Gwl, which are required for its function. The Polo kinase associated with and phosphorylated Gwl in this region, promoting its binding to 14-3-3ε and its localization to the cytoplasm in prophase. Our results suggest that cyclin B–Cdk1 phosphorylation of Gwl is also required for its nuclear exclusion by a distinct mechanism. We show that the nucleo-cytoplasmic regulation of Gwl is essential for its functions in vivo and propose that the spatial regulation of Gwl at mitotic entry contributes to the mitotic switch.     

Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells

Summary. The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.Correspondence and reprints: Department of Biology, 221 Morrill Science Center II, University of Massachusetts, Amherst, MA 01003, U.S.A.Received September 30, 2002; accepted February 12, 2003 Published online September 23, 2003     

Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.     

Retrospective assessment of cyclin-dependent kinase 5 mRNA and protein expression and its association with patient survival in breast cancer

Cyclin-dependent kinase 5 (Cdk5) is an atypical member of the cyclin-dependent kinase family and functions as a serine/threonine kinase that can be activated by non-cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early-stage invasive breast cancer tumours (n = 1110) with long-term follow-up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer-specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418-0.896, P = .011 and HR = 0.507, 95% CI = 0.318-0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.     

A non-structural protein encoded by Rice Dwarf Virus targets to the nucleus and chloroplast and inhibits local RNA silencing

RNA silencing is a potent antiviral mechanism in plants and animals. As a counter-defense, many viruses studied to date encode one or more viral suppressors of RNA silencing (VSR). In the latter case, how different VSRs encoded by a virus function in silencing remains to be fully understood. We previously showed that the nonstructural protein Pns10 of a Phytoreovirus, Rice dwarf virus (RDV), functions as a VSR. Here we present evidence that another nonstructural protein, Pns11, also functions as a VSR. While Pns10 was localized in the cytoplasm, Pns11 was localized both in the nucleus and chloroplasts. Pns11 has two bipartite nuclear localization signals (NLSs), which were required for nuclear as well as chloroplastic localization. The NLSs were also required for the silencing activities of Pns11. This is the first report that multiple VSRs encoded by a virus are localized in different subcellular compartments, and that a viral protein can be targeted to both the nucleus and chloroplast. These findings may have broad significance in studying the subcellular targeting of VSRs and other viral proteins in viral-host interactions.

     

Nucleocytoplasmic Cdk5 is involved in neuronal cell cycle and death in post-mitotic neurons

In a variety of neurodegenerative disease, despite the frequent correlation of neuronal cell cycle and cell death in the same neuronal populations, the mechanistic pathway linking the two remains undefined. One possible link is the atypical cyclin dependent kinase, Cdk5. Cdk5 exerts a double protective function in neurons, first by suppressing the cell cycle in the nucleus and second by suppressing cell death in the cytoplasm. Cdk5 transport between nucleus and cytoplasm serves to regulate the balance between these two events. Cdk5 nuclear localization relies on its interaction with p27, and its cell cycle suppression activity is achieved by direct binding to E2F1, disrupting the DP1-E2F1 dimer and its DNA binding ability. To bind to E2F1, Cdk5 does not need to be catalytically active but it does require a physical association with both p27 and its cyclin-like activator, p35. Because of this requirement, the proper levels and locations of p27 and p35 are characteristics that endow a neuron a unique form of cell cycle regulation that uses Cdk5 in a non-catalytic role. The findings offer cautionary notes to any strategy aimed at blocking Cdk5 activity as a means of combating neurodegenerative disease. To the extent that these approaches either directly or indirectly influence Cdk5 levels or location, they may produce unexpected and possibly unwanted consequences.     

Characterization of the <Emphasis Type="Italic">Bm61</Emphasis> of the <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.