scholarly journals Dual function of the NDR-kinase Dbf2 in the regulation of the F-BAR protein Hof1 during cytokinesis

2013 ◽  
Vol 24 (9) ◽  
pp. 1290-1304 ◽  
Author(s):  
Franz Meitinger ◽  
Saravanan Palani ◽  
Birgit Hub ◽  
Gislene Pereira
Keyword(s):  
Critical Role ◽  
Coiled Coil ◽  
Dual Function ◽  
Serine Residue ◽  
Bar Domain ◽  
Division Site ◽  
Coiled Coil Region ◽  
Ndr Kinase

The conserved NDR-kinase Dbf2 plays a critical role in cytokinesis in budding yeast. Among its cytokinesis-related substrates is the F-BAR protein Hof1. Hof1 colocalizes at the cell division site with the septin complex and, as mitotic exit progresses, moves to the actomyosin ring (AMR). Neither the function of Hof1 at the septin complex nor the mechanism by which Hof1 supports AMR constriction is understood. Here we establish that Dbf2 has a dual function in Hof1 regulation. First, we show that the coiled-coil region, which is adjacent to the conserved F-BAR domain, is required for the binding of Hof1 to septins. The Dbf2-dependent phosphorylation of Hof1 at a single serine residue (serine 313) in this region diminishes the recruitment of Hof1 to septins both in vitro and in vivo. Genetic and functional analysis indicates that the binding of Hof1 to septins is important for septin rearrangement and integrity during cytokinesis. Furthermore, Dbf2 phosphorylation of Hof1 at serines 533 and 563 promotes AMR constriction most likely by inhibiting the SH3-domain–dependent interactions of Hof1. Thus our data show that Dbf2 coordinates septin and AMR functions during cytokinesis through the regulation/control of Hof1.

scholarly journals Eps15 membrane-binding and -bending activity acts redundantly with Fcho1 during clathrin-mediated endocytosis

2016 ◽  
Vol 27 (17) ◽  
pp. 2675-2687 ◽  
Author(s):  
Lei Wang ◽  
Adam Johnson ◽  
Michael Hanna ◽  
Anjon Audhya
Keyword(s):  
Genetic Interaction ◽  
Critical Role ◽  
Membrane Binding ◽  
Accessory Proteins ◽  
Membrane Remodeling ◽  
Bar Domain ◽  
C Elegans ◽  
Accessory Factor

Clathrin coat assembly on membranes requires cytosolic adaptors and accessory proteins, which bridge triskeleons with the lipid bilayer and stabilize lattice architecture throughout the process of vesicle formation. In Caenorhabditis elegans, the prototypical AP-2 adaptor complex, which is activated by the accessory factor Fcho1 at the plasma membrane, is dispensable during embryogenesis, enabling us to define alternative mechanisms that facilitate clathrin-mediated endocytosis. Here we uncover a synthetic genetic interaction between C. elegans Fcho1 (FCHO-1) and Eps15 (EHS-1), suggesting that they function in a parallel and potentially redundant manner. Consistent with this idea, we find that the FCHO-1 EFC/F-BAR domain and the EHS-1 EH domains exhibit highly similar membrane-binding and -bending characteristics in vitro. Furthermore, we demonstrate a critical role for EHS-1 when FCHO-1 membrane-binding and -bending activity is specifically eliminated in vivo. Taken together, our data highlight Eps15 as an important membrane-remodeling factor, which acts in a partially redundant manner with Fcho proteins during the earliest stages of clathrin-mediated endocytosis.

scholarly journals The AMPK-related kinase NUAK1 controls cortical axons branching though a local modulation of mitochondrial metabolic functions

2020 ◽  
Author(s):  
Marine Lanfranchi ◽  
Géraldine Meyer-Dilhet ◽  
Raphael Dos Reis ◽  
Audrey Garcia ◽  
Camille Blondet ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Metabolic Regulation ◽  
Critical Role ◽  
Dual Function ◽  
Axon Branching ◽  
Cellular Mechanisms ◽  
Peripheral Neurons ◽  
Branch Formation

ABSTRACTThe precise regulation of the cellular mechanisms underlying axonal morphogenesis is essential to the formation of functional neuronal networks. We previously identified the autism-candidate kinase NUAK1 as a central regulator of axon branching in mouse cortical neurons through the control of mitochondria trafficking. How does local mitochondrial position or function regulate axon branching during development? Here, we characterized the metabolic regulation in the developing axon and report a marked metabolic decorrelation between axon elongation and collateral branching. We next solved the cascade of event leading to presynaptic clustering and mitochondria recruitment during spontaneous branch formation. Interestingly and contrary to peripheral neurons, mitochondria are recruited after but not prior to branch formation in cortical neurons. Using flux metabolomics and fluorescent biosensors, we observed that NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration. Upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Altogether, our results indicate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondria distribution and activity, and suggest that a mitochondrial-dependent remodeling of local metabolic homeostasis plays a critical role during axon morphogenesis.

scholarly journals MICAL-L1 Links EHD1 to Tubular Recycling Endosomes and Regulates Receptor Recycling

2009 ◽  
Vol 20 (24) ◽  
pp. 5181-5194 ◽  
Author(s):  
Mahak Sharma ◽  
Sai Srinivas Panapakkam Giridharan ◽  
Juliati Rahajeng ◽  
Naava Naslavsky ◽  
Steve Caplan
Keyword(s):  
Coiled Coil ◽  
Live Cells ◽  
Endocytic Recycling ◽  
Interacting Protein ◽  
Recycling Endosomes ◽  
Coiled Coil Region ◽  
Membrane Tubules ◽  
Tubular Membranes

Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.

scholarly journals VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis

2021 ◽  
Author(s):  
Cheng-Hsiang Kuo ◽  
Yi-Hsun Huang ◽  
Po-Ku Chen ◽  
Gang-Hui Lee ◽  
Ming-Jer Tang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Coiled Coil ◽  
Angiogenic Activity ◽  
Sprouting Angiogenesis ◽  
Membrane Microdomain

Objective: VEGF (vascular endothelial growth factor) plays a critical role in physiological and pathological angiogenesis. Endothelial 3D podosomes (3DPs) are a type of F-actin-rich membrane microdomain, predominantly found in endothelial tip cells controlled by VEGF signaling during sprouting angiogenesis, such as occurs in retinal vasculature development. The molecular mechanisms governing 3DP formation have not been completely elucidated. Approach and Results: By using in vitro cell models and in vivo mouse models, we study the role of TM (thrombomodulin) in VEGF-induced endothelial 3DPs. Here, we report that VEGF can induce the expression of TM via ROCK2 (Rho-associated coiled-coil kinase 2). Furthermore, ROCK2 can catalyze the phosphorylated activation of ezrin to promote the association of the cytoplasmic domain of TM with F-actin in 3DPs and thereby promote the formation of 3DPs. We used endothelial cells transfected with different TM mutants as models to verify the role of TM domains in 3DPs and angiogenic activity. TM expression in endothelial cells augments angiogenic activity, a response that is dependent on the interaction of the cytoplasmic tail of TM with ezrin, and the integrity of the lectin-like domain of TM. Thus, as compared with wild-type counterparts, mice lacking the lectin-like domain of TM exhibit reduced neovascularization of granulation tissues during cutaneous wound healing and less retinal neovascularization in a model of oxygen-induced retinopathy. Conclusions: VEGF-ROCK2-ezrin-TM-F-actin axis promotes the formation of the lipid raft membrane-associated complex configuration, 3DP, which plays a critical role in mediating tube formation and cell migration of endothelial cells in sprouting angiogenesis.

scholarly journals Phosphorylation of the Mitotic Regulator Protein Hec1 by Nek2 Kinase Is Essential for Faithful Chromosome Segregation

2002 ◽  
Vol 277 (51) ◽  
pp. 49408-49416 ◽  
Author(s):  
Yumay Chen ◽  
Daniel J. Riley ◽  
Lei Zheng ◽  
Phang-Lang Chen ◽  
Wen-Hwa Lee
Keyword(s):  
Chromosome Segregation ◽  
Critical Role ◽  
Coiled Coil ◽  
Serine Phosphorylation ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Chromosomal Segregation ◽  
M Phase

Hec1 (highlyexpressed incancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G2/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G2/M. Nek2 phosphorylates Hec1 on serine residue 165, bothin vitroandin vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitivenimamutation inAspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 inSaccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser165to Ala or yeast Hec1 mutant changing Ser201to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.

scholarly journals TRIM25 Identification in the Chinese Goose: Gene Structure, Tissue Expression Profiles, and Antiviral Immune Responses In Vivo and In Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Yunan Wei ◽  
Hao Zhou ◽  
Anqi Wang ◽  
Lipei Sun ◽  
Mingshu Wang ◽  
...  
Keyword(s):  
Rna Virus ◽  
Expression Profiles ◽  
Critical Role ◽  
Coiled Coil ◽  
Poly I:C ◽  
Positive Role ◽  
Antiviral Immune Response ◽  
Polycytidylic Acid

The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein play a critical role in the interferon (IFN) response during RNA virus infection. The tripartite motif containing 25 proteins (TRIM25) was reported to modify caspase activation and RIG-I recruitment domains (CARDs) via ubiquitin. These modifications allow TRIM25 to interact with mitochondrial antiviral signaling molecules (MAVs) and form CARD-CARD tetramers. Goose TRIM25 was cloned from gosling lungs, which possess a 1662 bp open reading flame (ORF). This ORF encodes a predicted 554 amino acid protein consisting of a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence has 89.25% sequence identity withAnas platyrhynchosTRIM25, 78.57% withGallus gallusTRIM25, and 46.92% withHomo sapiensTRIM25. TRIM25 is expressed in all gosling and adult goose tissues examined. QRT-PCR revealed that goose TRIM25 transcription could be induced by goose IFN-α, goose IFN-γ, and goose IFN-λ, as well as a35 s polyinosinic-polycytidylic acid (poly(I:C)), oligodeoxynucleotides 2006 (ODN 2006), and resiquimod (R848) in vitro; however, it is inhibited in H9N2 infected goslings for unknown reasons. These data suggest that goose TRIM25 might play a positive role in the regulation of the antiviral immune response.

scholarly journals Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ70 Binding

2002 ◽  
Vol 184 (10) ◽  
pp. 2634-2641 ◽  
Author(s):  
Larry C. Anthony ◽  
Alan A. Dombkowski ◽  
Richard R. Burgess
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Disulfide Bond ◽  
Conformational Changes ◽  
Coiled Coil ◽  
Mrna Synthesis ◽  
Promoter Sequences ◽  
Coiled Coil Region

ABSTRACT RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ70. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ70. We also show that the locked coiled-coil is capable of inducing a conformational change within σ70 that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ70 or upon recognition of the −10 nontemplate strand of DNA.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ai-Ling Tian ◽  
Qi Wu ◽  
Peng Liu ◽  
Liwei Zhao ◽  
Isabelle Martins ◽  
...  
Keyword(s):  
Stress Response ◽  
Immune Responses ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Serine Residue ◽  
Eif2α Phosphorylation ◽  
Lysosomotropic Agents ◽  
Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

scholarly journals Emerging Roles for Ion Channels in Ovarian Cancer: Pathomechanisms and Pharmacological Treatment

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 668
Author(s):  
Concetta Altamura ◽  
Maria Raffaella Greco ◽  
Maria Rosaria Carratù ◽  
Rosa Angela Cardone ◽  
Jean-François Desaphy
Keyword(s):  
Ovarian Cancer ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Critical Role ◽  
Gynecologic Cancer ◽  
Receptor Potential ◽  
Transient Receptor Potential Channels ◽  
Platinum Resistance

Ovarian cancer (OC) is the deadliest gynecologic cancer, due to late diagnosis, development of platinum resistance, and inadequate alternative therapy. It has been demonstrated that membrane ion channels play important roles in cancer processes, including cell proliferation, apoptosis, motility, and invasion. Here, we review the contribution of ion channels in the development and progression of OC, evaluating their potential in clinical management. Increased expression of voltage-gated and epithelial sodium channels has been detected in OC cells and tissues and shown to be involved in cancer proliferation and invasion. Potassium and calcium channels have been found to play a critical role in the control of cell cycle and in the resistance to apoptosis, promoting tumor growth and recurrence. Overexpression of chloride and transient receptor potential channels was found both in vitro and in vivo, supporting their contribution to OC. Furthermore, ion channels have been shown to influence the sensitivity of OC cells to neoplastic drugs, suggesting a critical role in chemotherapy resistance. The study of ion channels expression and function in OC can improve our understanding of pathophysiology and pave the way for identifying ion channels as potential targets for tumor diagnosis and treatment.

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