A CRM1-Mediated Nuclear Export Signal Is Essential for Cytoplasmic Localization of Neurogenin 3 in Neurons

Neurogenin 3 (Ngn3), a proneural gene, regulates dendritogenesis and synaptogenesis in mouse hippocampal neurons. Ngn3 is transiently exported from the cell nucleus to the cytoplasm when neuronal polarity is initiated, suggesting that the nucleo-cytoplasmic transport of the protein is important for its action on neuronal development. In this study, we identified for the first time a functional nuclear export sequence (NES2; ¹³¹YIWALTQTLRIA¹⁴²) in Ngn3. The green fluorescent protein (EGFP)-NES2 fusion protein was localized in the cytoplasm and its nucleo-cytoplasmic shuttling was blocked by the CRM1 specific export inhibitor leptomycin B. Mutation of a leucine residue to alanine (L135A) in the NES2 motif resulted in both cytoplasmic and nuclear localization of the EGFP-NES2 fusion protein and in the nuclear accumulation of ectopic full-length myc-Ngn3. In addition, point mutation of the leucine 135 counteracted the effects of Ngn3 on neuronal morphology and synaptic inputs indicating that the cytoplasmic localization of Ngn3 is important for neuronal development. Pharmacological perturbation of the cytoskeleton revealed that cytoplasmic Ngn3 is associated with microtubules.     

Sigma1 Receptor Inhibits TRPC1-Mediated Ca2+ Entry That Promotes Dopaminergic Cell Death

Cellular and Molecular Neurobiology - Regulation of Ca2+ homeostasis is essential for neuronal function and its survival. Recent data suggest that TRPC1 function as the endogenous store-mediated...     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

A Nuclear Localization Signal in Herpesvirus Protein VP1-2 Is Essential for Infection via Capsid Routing to the Nuclear Pore

To initiate infection, herpesviruses must navigate to and transport their genomes across the nuclear pore. VP1-2 is a large structural protein of the virion that is conserved in all herpesviruses and plays multiple essential roles in virus replication, including roles in early entry. VP1-2 contains an N-terminal basic motif which functions as an efficient nuclear localization signal (NLS). In this study, we constructed a mutant HSV strain, K.VP1-2ΔNLS, which contains a 7-residue deletion of the core NLS at position 475. This mutant fails to spread in normal cells but can be propagated in complementing cell lines. Electron microscopy (EM) analysis of infection in noncomplementing cells demonstrated capsid assembly, cytoplasmic envelopment, and the formation of extracellular enveloped virions. Furthermore, extracellular virions isolated from noncomplementing cells had similar profiles and abundances of structural proteins. Virions containing VP1-2ΔNLS were able to enter and be transported within cells. However, further progress of infection was prevented, with at least a 500- to 1,000-fold reduction in the efficiency of initiating gene expression compared to that in the revertant. Ultrastructural and immunofluorescence analyses revealed that the K.VP1-2ΔNLS mutant was blocked at the microtubule organizing center or immediately upstream of nuclear pore docking and prior to gene expression. These results indicate that the VP1-2 NLS is not required for the known assembly functions of the protein but is a key requirement for the early routing to the nuclear pore that is necessary for successful infection. Given its conservation, we propose that this motif may also be critical for entry of other classes of herpesviruses.     

Molecular Control of Nuclear and Subnuclear Targeting of the Plant CDK Inhibitor ICK1 and ICK1-Mediated Nuclear Transport of CDKA

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS^ICK1 could transport the much larger GUS (β-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS^ICK1 is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1^1–162 that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.     

LIM and SH3 Protein -1 Modulates CXCR2-Mediated Cell Migration

Background

The chemokine receptor CXCR2 plays a pivotal role in migration of neutrophils, macrophages and endothelial cells, modulating several biological responses such as angiogenesis, wound healing and acute inflammation. CXCR2 is also involved in pathogenesis of chronic inflammation, sepsis and atherosclerosis. The ability of CXCR2 to associate with a variety of proteins dynamically is responsible for its effects on directed cell migration or chemotaxis. The dynamic network of such CXCR2 binding proteins is termed as “CXCR2 chemosynapse”. Proteomic analysis of proteins that co-immunoprecipitated with CXCR2 in neutrophil-like dHL-60 cells revealed a novel protein, LIM and SH3 protein 1 (LASP-1), binds CXCR2 under both basal and ligand activated conditions. LASP-1 is an actin binding cytoskeletal protein, involved in the cell migration.

Methodology/Principal Findings

We demonstrate that CXCR2 and LASP-1 co-immunoprecipitate and co-localize at the leading edge of migrating cells. The LIM domain of LASP-1 directly binds to the carboxy-terminal domain (CTD) of CXCR2. Moreover, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4. Using a site-directed and deletion mutagenesis approach, Iso323-Leu324 of the conserved LKIL motif on CXCR2-CTD was identified as the binding site for LASP-1. Interruption of the interaction between CXCR2-CTD and LIM domain of LASP-1 by dominant negative and knock down approaches inhibited CXCR2-mediated chemotaxis. Analysis for the mechanism for inhibition of CXCR2-mediated chemotaxis indicated that LASP-1/CXCR2 interaction is essential for cell motility and focal adhesion turnover involving activation of Src, paxillin, PAK1, p130CAS and ERK1/2.

Conclusions/Significance

We demonstrate here for the first time that LASP-1 is a key component of the “CXCR2 chemosynapse” and LASP-1 interaction with CXCR2 is critical for CXCR2-mediated chemotaxis. Furthermore, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated chemotaxis. Thus, LASP-1 may serve as a new link coordinating the flow of information between chemokine receptors and nascent focal adhesions, especially at the leading edge. Thus the association between the chemokine receptors and LASP-1 suggests to the presence of a CXC chemokine receptor-LASP-1 pro-migratory module in cells governing the cell migration.     

Discrimination between mammalian RNases H-1 and H-2.

The two principal RNases H in mammalian cells, H-1 and H-2, differ in their responses to sale, divalent metal, and sulfhydryl inhibition. Specific reaction conditions that provide unambiguous discrimination between RNases H-1 and H-2 with only two assays are described. The assays were used for identification in a new purification procedure for RNases H-1 and H-2.     

Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.     

Loss of TBL1XR1 Disrupts Glucocorticoid Receptor Recruitment to Chromatin and Results in Glucocorticoid Resistance in a B-Lymphoblastic Leukemia Model

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.     

死亡受体5介导细胞凋亡的研究及应用

死亡受体DR5(death receptor 5)属于肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)超家族的成员,其胞质区部分含有死亡结构域(death domain,DD),广泛分布于各种肿瘤细胞和正常组织细胞的膜上.配体TRAIL与肿瘤细胞表面的DR5结合,可诱导大多数肿瘤凋亡,而对正常的组织几乎没有作用.近年来死亡受体DR5与细胞凋亡的关系已成为研究热点之一,对DR5介导细胞凋亡的机制和应用进展作一综述.