scholarly journals Mask family proteins ANKHD1 and ANKRD17 regulate YAP nuclear import, stability and phase separation

2019 ◽  
Author(s):  
Clara Sidor ◽  
Nerea Borreguero-Munoz ◽  
Georgina C Fletcher ◽  
Ahmed Elbediwy ◽  
Hannah Vanyai ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Tissue Growth ◽  
Ankyrin Repeat ◽  
Liquid Droplets ◽  
Transcriptional Coactivator ◽  
Cytoplasmic Transport ◽  
Nuclear Localisation Sequence ◽  
Kh Domain

AbstractThe Mask family of multiple ankyrin repeat and KH domain proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two Ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. Here we show that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving colloidal phase separation into large liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction. Our results suggest that Mask family proteins promote YAP nuclear import and phase separation to regulate YAP stability and transcriptional activity.

scholarly journals Mask family proteins ANKHD1 and ANKRD17 regulate YAP nuclear import and stability

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Clara Sidor ◽  
Nerea Borreguero-Munoz ◽  
Georgina C Fletcher ◽  
Ahmed Elbediwy ◽  
Oriane Guillermin ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Tissue Growth ◽  
Ankyrin Repeat ◽  
Molecular Function ◽  
Liquid Droplets ◽  
Transcriptional Coactivator ◽  
Nuclear Localisation ◽  
Cytoplasmic Transport ◽  
Nuclear Localisation Sequence

Mask family proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. Here we show that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving formation of liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction.

scholarly journals Destabilization of Ran C-terminus promotes GTP loading and occurs in multiple Ran cancer mutations

2018 ◽  
Author(s):  
Yuqing Zhang ◽  
Jinhan Zhou ◽  
Yuping Tan ◽  
Qiao Zhou ◽  
Aiping Tong ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Mitotic Spindle ◽  
Nuclear Export ◽  
Cellular Transformation ◽  
Nuclear Pore ◽  
Ras Proteins ◽  
Transformation Mechanism ◽  
C Terminus ◽  
Cytoplasmic Transport ◽  
Cancer Mutations

AbstractRan (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other diverse functions in the cytoplasm, as well as in cellular transformation when activated. Unlike other Ras superfamily proteins, Ran contains an auto-inhibitory C-terminal tail, which packs against its G domain and bias Ran towards binding GDP over GTP. The biological importance of this C-terminal tail is not well understood. By disrupting the interaction between the C-terminus and the G domain, we were able to generate Ran mutants that are innately active and potently bind to RanBP1 (Ran Binding Protein 1), nuclear export factor CRM1 and nuclear import factor KPNB1. In contrast to previously reported activated Ran mutants, the C-terminus destabilized mutants are hydrolysis competent in cells, support nuclear transport, and do not form nuclear rim staining. Crystal structures show that one of these C-terminal mutations slightly changes its mode of binding to RanBP1. Finally, a high percentage of Ran C-terminus mutations from cancer patients were found to be destabilizing and hyperactivating, suggesting that Ran C-destabilization might be an unprecedented cellular transformation mechanism in affected cancers. This study also highlights a new drug design strategy towards treating patients with hyperactivated Ras proteins including K-Ras.

scholarly journals Tau conformers in FTLD-MAPT undergo liquid-liquid phase separation and perturb the nuclear envelope

2020 ◽  
Author(s):  
Sang-Gyun Kang ◽  
Zhuang Zhuang Han ◽  
Nathalie Daude ◽  
Emily McNamara ◽  
Serene Wohlgemuth ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Envelope ◽  
Solid Phase ◽  
Critical Mass ◽  
Liquid Phase Separation ◽  
Liquid Droplets ◽  
Cytoplasmic Transport ◽  
End Stage ◽  
Liquid Liquid Phase Separation

AbstractRecent studies show that a single MAPT gene mutation can promote alternative tau misfolding pathways engendering divergent forms of frontotemporal dementia and that under conditions of molecular crowding, the repertoire of tau forms can include liquid-liquid phase separation (LLPS). We show here that following pathogenic seeding, tau condenses on the nuclear envelope (NE) and disrupts nuclear-cytoplasmic transport (NCT). Interestingly, NE fluorescent tau signals and small fluorescent inclusions behaved as demixed liquid droplets in living cells. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 μm2, indicating that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Our findings indicate that tau undergoing LLPS is more toxic amongst a spectrum of alternative conformers; LLPS droplets on the NE that disrupt NCT serve to trigger cell death and can act as nurseries for fibrillar structures abundantly detected in end-stage disease.

scholarly journals Direct Association and Nuclear Import of the Hepatitis B Virus X Protein with the NF-κB Inhibitor IκBα

1999 ◽  
Vol 19 (9) ◽  
pp. 6345-6354 ◽  
Author(s):  
Robert Weil ◽  
Hüseyin Sirma ◽  
Carlo Giannini ◽  
Dina Kremsdorf ◽  
Christine Bessia ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Critical Role ◽  
Ankyrin Repeat ◽  
Nuclear Accumulation ◽  
X Protein ◽  
Tnf Α ◽  
B Virus

ABSTRACT The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-κB. A recent report indicates that X activates NF-κB by acting on two cytoplasmic inhibitors of this family of transcription factors: IκBα and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IκBα, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IκBα (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-κB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IκBα (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IκBα mediating the interaction with the p50 and p65 subunits of NF-κB and is also close to the nuclear export sequence of IκBα, therefore providing a potential explanation for the nuclear accumulation of IκBα with X. This association can also be observed upon the induction of endogenous IκBα by tumor necrosis factor alpha (TNF-α) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-κB activation following TNF-α treatment, probably by preventing the reassociation of newly synthesized nuclear IκBα with DNA-bound NF-κB complexes.

scholarly journals Promotion of importin α–mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3

2005 ◽  
Vol 169 (3) ◽  
pp. 415-424 ◽  
Author(s):  
Christian Faul ◽  
Stefan Hüttelmaier ◽  
Jun Oh ◽  
Virginie Hachet ◽  
Robert H. Singer ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Protein Targeting ◽  
Underlying Mechanism ◽  
Intracellular Protein ◽  
Regulatory Processes ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Importin Α ◽  
Localization Signal

14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin α binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

scholarly journals The nuclear localization sequence mediates hnRNPA1 amyloid fibril formation revealed by cryoEM structure

2020 ◽  
Author(s):  
Yunpeng Sun ◽  
Kun Zhao ◽  
Wencheng Xia ◽  
Jinge Gu ◽  
Yeyang Ma ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Localization ◽  
Amyloid Fibril ◽  
Low Complexity ◽  
Nuclear Localization Sequence ◽  
Amyloid Aggregation ◽  
Dynamic Phase ◽  
Fibril Structure ◽  
Cytoplasmic Transport ◽  
Localization Sequence

AbstractHuman heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-β2 (Kapβ2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapβ2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminate structural understandings on the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapβ2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

Mechanisms of Receptor-Mediated Nuclear Import and Nuclear Export

Traffic ◽  
2005 ◽  
Vol 6 (3) ◽  
pp. 187-198 ◽  
Author(s):  
Lucy F. Pemberton ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Import ◽  
Nuclear Export

scholarly journals Nuclear Targeting of Adenovirus Type 2 Requires CRM1-mediated Nuclear Export

2005 ◽  
Vol 16 (6) ◽  
pp. 2999-3009 ◽  
Author(s):  
Sten Strunze ◽  
Lloyd C. Trotman ◽  
Karin Boucke ◽  
Urs F. Greber
Keyword(s):  
Nuclear Export ◽  
Adenovirus Type ◽  
Nuclear Pore ◽  
Nuclear Targeting ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Transport ◽  
Viral Particles ◽  
Organizing Center ◽  
Adenovirus Type 2

Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.

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