DOCSLIB.ORG

Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation Published OnlineFirst February 4, 2020; DOI: 10.1158/1541-7786.MCR-19-0052 MOLECULAR CANCER RESEARCH | SIGNAL TRANSDUCTION AND FUNCTIONAL IMAGING Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation Kate Tubbesing1, Jamie Ward1, Raymond Abini-Agbomson1, Aditi Malhotra1, Alena Rudkouskaya1, Janine Warren1, John Lamar1, Nina Martino1, Alejandro P. Adam1,2, and Margarida Barroso1 ABSTRACT ◥ Early sorting endosomes are responsible for the trafficking and exhibited prolonged and increased EGF-induced activation of function of transferrin receptor (TfR) and EGFR. These receptors EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). play important roles in iron uptake and signaling and are critical for Rab4A overexpression in MCF10A cells produced EEA1-positive breast cancer development. However, the role of morphology, enlarged endosomes that displayed prolonged and amplified receptor composition, and signaling of early endosomes in breast EGF-induced EGFR-p1068 activation. Knockdown of Rab4A cancer remains poorly understood. A novel population of enlarged lead to increased endosomal size in MCF10A, but not in early endosomes was identified in breast cancer cells and tumor MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted xenografts but not in noncancerous MCF10A cells. Quantitative in enhanced EGF-induced activation of EGFR-p1068 in MDA- analysis of endosomal morphology, cargo sorting, EGFR activa- MB-231 as well as downstream signaling in MCF10A cells. tion, and Rab GTPase regulation was performed using super- Altogether, this extensive characterization of early endosomes resolution and confocal microscopy followed by 3D rendering. in breast cancer cells has identified a Rab4-modulated enlarged MDA-MB-231 breast cancer cells have fewer, but larger EEA1- early endosomal compartment as the site of prolonged and positive early endosomes compared with MCF10A cells. Live-cell increased EGFR activation. imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but Implications: Enlarged early endosomes play a Rab4-modulated not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes role in regulation of EGFR activation in breast cancer cells. Introduction pathways are initiated from this dynamic organelle (8, 9). Thus, while multiple receptors share an initial endocytosis pathway, their cellular Characterization of the morphology and subcellular localization of fate can differ substantially. For example, Tf bound to its receptor early endosomes (EE) is essential for the understanding of receptor delivers iron into cells via the early and recycling endocytic path- trafficking and signaling in cancer cells. Recently, the endocytic way (10), whereas LDL, which plays a key role in lipid metabolism, is pathway has been shown to be defective in various human can- released from LDLR in EE and delivered to late endosomes/lyso- cers (1, 2). Furthermore, clathrin-dependent and -independent endo- somes (11). Moreover, EGF-induced internalization of EGFR into EEs cytosis show significant alterations in several cancer cell lines (3–5). is required for its signaling function, and EGFR's kinase activity is Importantly, the size and cellular distribution of endosomes have been downregulated via the ubiquitination-dependent late endosomal/lyso- suggested as a potential biomarker in cancer (6). Moreover, peri- somal pathway (12). Therefore, studying the altered endocytic traf- nuclear endosomal signaling complexes have been shown to play a ficking of Tf-TfR, EGF-EGFR, and LDL-LDLR in cancer is crucial to crucial role in the regulation of signaling cascades involved in cancer advance our understanding of the crosstalk between cell signaling and progression (7). However, many aspects of EE function, regulation, metabolism. and morphology in breast cancer cells remain unclear. Endosomal compartments are identified and regulated by one or Membrane-bound receptors involved in nutrient uptake, for exam- more small Rab-GTPases, which modulate essential membrane func- ple, transferrin-receptor (TfR) and low-density lipoprotein (LDL), as tions via interactions with Rab effector proteins (9, 13). Rab5 is an well as in cell signaling, for example, EGFR, play an important role in important regulator of the EEs (9). Two well-characterized Rab5 cancer development and progression. These receptors are endocytosed effectors, EEA1 (EE Antigen 1) and APPL1, can occupy the same or via multiple internalization pathways into EE, which act as a literal separate EEs (14, 15). Altered expression and activity levels of Rab “fork in the road,” as both the endocytic recycling and lysosomal GTPases affect the endocytosis, degradation, and recycling of mem- brane-bound receptors, leading to increased cancer cell proliferation 1 and motility as well as metastatic behavior (1, 2, 16, 17). Department of Molecular and Cellular Physiology, Albany Medical College, fi Albany, New York. 2Department of Ophthalmology, Albany Medical College, Rab4 is the most commonly ampli ed Rab GTPase in invasive Albany, New York. breast carcinoma (4). Rab4 and Rab11 work in a coordinated manner to regulate the fast and slow recycling compartments, respectively Note: Supplementary data for this article are available at Molecular Cancer – fl Research Online (http://mcr.aacrjournals.org/). (18 20). Moreover, Rab4 was shown to in uence Tf-mediated cellular iron accumulation (21). Rab4 inactivation, through expres- Corresponding Author: Margarida Barroso, Albany Medical College, 47 New Scotland Av, Albany, NY 12208. Phone: 518-262-6435; Fax: 518-262-8101; E-mail: sion of either a dominant-negative Rab4-S22N mutation or a Rab4 [email protected] GTPase activating protein (TBC1D16), increases EGFR degrada- tion (22, 23) and weakens the invasive behavior of breast cancer Mol Cancer Res 2020;18:757–73 cells in vivo (4). As the regulation of EGFR trafficking and signaling doi: 10.1158/1541-7786.MCR-19-0052 involves multiple Rab proteins, the role of Rab4 in EGFR regulation Ó2020 American Association for Cancer Research. remains unclear (24). AACRJournals.org | 757 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 4, 2020; DOI: 10.1158/1541-7786.MCR-19-0052 Tubbesing et al. EEs have been characterized as tubulovesicular compartments with Tumor xenografts an average diameter between 100 and 500 nm (9, 25). In this study, All animal procedures were conducted with the approval of the super-resolution (SR) microscopy, time-lapse live-cell imaging, and Institutional Animal Care and Use Committee at Albany Medical 3D whole-cell quantitative microscopy approaches, as well as immu- College, which is American Association for Laboratory Animal Care nostaining and immunoblotting signaling assays were used to char- accredited. Human tumor xenografts were generated as described in acterize an enlarged endosomal population that is also the site of ref. 27. increased and prolonged EGF-induced EGFR activation upon phos- phorylation at tyrosine 1068 (EGFR-p1068), in aggressive triple- 3D dSTORM microscopy negative MDA-MB-231 cells but not in nontransformed mammary Cells were preincubated for 30 minutes with CI medium and epithelial MCF10A cells. Furthermore, Rab4A overexpression led to an incubated with 20 mg/mL AF647-Tf in CI medium for 1 hour, prior increase in EEA1-positive endosomal size as well as an extended and to PBS washes and fixation with 4% PFA. 3D dSTORM microscopy elevated EGF-induced EGFR-p1068 activation in those enlarged and postprocessing image analysis was performed as described in endosomes in MCF10A cells. Unexpectedly, Rab4A knockdown led ref. 28 and in Supplementary Methods. to an increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A-depleted MDA-MB-231 cells displayed an Confocal and Airyscan microscopy fi elevated EGF-induced activation of EGFR-p1068. These ndings Zeiss LSM880 with Fast Airyscan detector or LSM510 (Zeiss) were establish enlarged EEs in breast cancer cells as central players in the used to collect z-stack images. Fast Airyscan was performed at Nyquist fi regulation of cargo traf cking and cell signaling, Here, we suggest a sampling and using SR settings. For whole-cell analysis, z-stack images complex role for Rab4 in the regulation of endosomal size as well as of were collected to visualize cells from top to bottom, and for live-cell intensity and duration of EGFR activation. imaging, 3–5 images per z-stack were collected per time point. Air- yscan processing (pixel reassignment) was performed in Zen Black Materials and Methods software under default settings. Live-cell imaging is described in Supplementary Methods. Cell culture Cells were purchased from ATCC and tested routinely for Myco- Colocalization analysis plasma. MCF10A cells were cultured in DMEM/F12 with 5% horse Pearson correlation coefficient in z-stacks was determined using serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera “coloc” module from Imaris software (Bitplane, Inc.). For statistical toxin, 10 mg/mL bovine insulin, and penicillin/streptomycin. Human analysis, at least 3 z-stack whole images per condition were analyzed. breast cancer cells were cultured in DMEM with 10% FBS, 4 mmol/L Extended description of the colocalization analysis is found in Sup- L-glutamine, and 10 mmol/L HEPES, pH 7.4. Cells were passaged less plementary Methods and Supplementary Fig. S1. than 15 times. No further cell line authentication was
Load more

Recommended publications
  • Redefining the Specificity of Phosphoinositide-Binding by Human
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.163253; this version posted June 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins Nilmani Singh1†, Adriana Reyes-Ordoñez1†, Michael A. Compagnone1, Jesus F. Moreno Castillo1, Benjamin J. Leslie2, Taekjip Ha2,3,4,5, Jie Chen1* 1Department of Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; 2Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; 3Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218; 4Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205; 5Howard Hughes Medical Institute, Baltimore, MD 21205, USA †These authors contributed equally to this work. *Correspondence: [email protected]. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.163253; this version posted June 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. ABSTRACT Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employed a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates.
    [Show full text]
  • Lowe Syndrome-Linked Endocytic Adaptors Direct Membrane Cycling
    bioRxiv preprint doi: https://doi.org/10.1101/616664; this version posted April 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Lowe Syndrome-linked endocytic adaptors direct membrane 2 cycling kinetics with OCRL in Dictyostelium discoideum. 3 Running Title: F&H motifs and OCRL in Dictyostelium discoideum 4 Alexandre Luscher5+, Florian Fröhlich1,7+, Caroline Barisch5, Clare Littlewood 4, Joe Metcalfe4, 5 Florence Leuba5, Anita Palma6, Michelle Pirruccello1,2, Gianni Cesareni6, Massimiliano Stagi4, Tobias 6 C. Walther7, Thierry Soldati5*, Pietro De Camilli1,2,3*, Laura E. Swan1,2,4* 7 1. Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA 8 2. Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration, and 9 Repair, Yale University School of Medicine, New Haven, CT 06510, USA. 10 3. Department of Neuroscience and Kavli Institute for Neuroscience, Yale University School of 11 Medicine, New Haven, CT 06510, USA. 12 4. Department of Cellular and Molecular Physiology, University of Liverpool, Crown St, Liverpool, 13 L69 3BX, UK 14 5.Department of Biochemistry, Faculty of Science, University of Geneva, Science II, 30 quai Ernest- 15 Ansermet, 1211 Geneva-4, Switzerland 16 6.Department of Biology, University of Rome, Tor Vergata, Rome Italy 17 7. Department of Genetics and Complex Diseases, Harvard School of Public Health, Department of 18 Cell Biology, Harvard Medical School, Howard Hughes Medical Institute, Boston, MA 02115, USA 19 + : contributed equally to this manuscript 20 *: Correspondence : [email protected]; [email protected]; [email protected] 21 Lead correspondence: [email protected] 22 1 bioRxiv preprint doi: https://doi.org/10.1101/616664; this version posted April 24, 2019.
    [Show full text]
  • APPL1 Associates with Trka and GIPC1 and Is Required for Nerve Growth Factor-Mediated Signal Transduction
    University of Montana ScholarWorks at University of Montana Biomedical and Pharmaceutical Sciences Faculty Publications Biomedical and Pharmaceutical Sciences 12-2006 APPL1 Associates with TrkA and GIPC1 and is Required for Nerve Growth Factor-Mediated Signal Transduction D. C. Lin C. Quevedo N. E. Brewer A. Bell J. R. Testa See next page for additional authors Follow this and additional works at: https://scholarworks.umt.edu/biopharm_pubs Part of the Medical Sciences Commons, and the Pharmacy and Pharmaceutical Sciences Commons Let us know how access to this document benefits ou.y Recommended Citation Lin, D. C.; Quevedo, C.; Brewer, N. E.; Bell, A.; Testa, J. R.; Grimes, Mark L.; Miller, F. D.; and Kaplan, D. R., "APPL1 Associates with TrkA and GIPC1 and is Required for Nerve Growth Factor-Mediated Signal Transduction" (2006). Biomedical and Pharmaceutical Sciences Faculty Publications. 13. https://scholarworks.umt.edu/biopharm_pubs/13 This Article is brought to you for free and open access by the Biomedical and Pharmaceutical Sciences at ScholarWorks at University of Montana. It has been accepted for inclusion in Biomedical and Pharmaceutical Sciences Faculty Publications by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. Authors D. C. Lin, C. Quevedo, N. E. Brewer, A. Bell, J. R. Testa, Mark L. Grimes, F. D. Miller, and D. R. Kaplan This article is available at ScholarWorks at University of Montana: https://scholarworks.umt.edu/biopharm_pubs/13 MOLECULAR AND CELLULAR BIOLOGY, Dec. 2006, p. 8928–8941 Vol. 26, No. 23 0270-7306/06/$08.00ϩ0 doi:10.1128/MCB.00228-06 Copyright © 2006, American Society for Microbiology.
    [Show full text]
  • Structure of the APPL1 BAR-PH Domain and EMBO Characterization of Its Interaction with Rab5 Open
    The EMBO Journal (2007) 26, 3484–3493 | & 2007 European Molecular Biology Organization | Some Rights Reserved 0261-4189/07 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL Structure of the APPL1 BAR-PH domain and EMBO characterization of its interaction with Rab5 open This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproductioninany medium, provided the original authorand source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission. Guangyu Zhu1, Jia Chen2, Jay Liu3, Introduction Joseph S Brunzelle4, Bo Huang2, Endocytosis induced by ligandÀreceptor interaction has been Nancy Wakeham1, Simon Terzyan1, 2 2 3 directly linked to signal transduction mediated by Rab5 and Xuemei Li , Zihe Rao , Guangpu Li its effector APPL1 (Adaptor protein containing PH domain 1,2, and Xuejun C Zhang * PTB domain and Leucine zipper motif; Miaczynska et al, 1Crystallography Research Program, Oklahoma Medical Research 2004; Mao et al, 2006). The small GTPase Rab5 is a generally Foundation, Oklahoma City, OK, USA, 2National Laboratory of acknowledged prominent regulator of vesicle trafficking en- Biomacromolecules, Institute of Biophysics, Chinese Academy of route from the plasma membrane to early endosomes (Li, 3 Sciences, Beijing, China, Department of Biochemistry and Molecular 1996), whereas APPL1 (also called DIP13a) is identified with Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA and 4Department of Molecular Pharmacology and signaling pathways of adiponectin, insulin, EGF, follicle Biological Chemistry, Feinberg Medical School, Northwestern stimulating hormone receptor, neurotrophin receptor University, Chicago, IL, USA (TrkA), oxidative stress, and DCC-mediated apoptosis (Liu et al, 2002; Miaczynska et al, 2004; Lin et al, 2006; Mao et al, APPL1 is an effector of the small GTPase Rab5.
    [Show full text]
  • Adaptor Protein APPL1 Couples Synaptic NMDA Receptor with Neuronal Prosurvival Phosphatidylinositol 3-Kinase/Akt Pathway
    The Journal of Neuroscience, August 29, 2012 • 32(35):11919–11929 • 11919 Cellular/Molecular Adaptor Protein APPL1 Couples Synaptic NMDA Receptor with Neuronal Prosurvival Phosphatidylinositol 3-Kinase/Akt Pathway Yu-bin Wang, Jie-jie Wang, Shao-hua Wang, Shuang-Shuang Liu, Jing-yuan Cao, Xiao-ming Li, Shuang Qiu, and Jian-hong Luo Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circum- stances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR- dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we exploredtheroleofanadaptorprotein—adaptorproteincontainingpHdomain,PTBdomain,andleucinezippermotif(APPL1)—inthis signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs.
    [Show full text]
  • The Endosomal Protein Appl1 Mediates Akt Substrate Specificity and Cell Survival in Vertebrate Development
    The Endosomal Protein Appl1 Mediates Akt Substrate Specificity and Cell Survival in Vertebrate Development Annette Schenck,1,4 Livia Goto-Silva,1 Claudio Collinet,1 Muriel Rhinn,2,5 Angelika Giner,1 Bianca Habermann,1,3 Michael Brand,2 and Marino Zerial1,* 1Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany 2Biotechnology Center and Center for Regenerative Therapies, University of Technology Dresden, Tatzberg 47-51, 01307 Dresden, Germany 3Scionics Computer Innovation GmbH, Tatzberg 47, 01307 Dresden, Germany 4Present address: Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands. 5Present address: Institut de Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire, CNRS/INSERM/ULP, Boite Postal 10142, 67404 Illkirch Cedex, France. *Correspondence: [email protected] DOI 10.1016/j.cell.2008.02.044 SUMMARY grown in the past decade, uncovering central signaling hubs such protein kinase B (Akt) and mitogen-activated protein During development of multicellular organisms, cells kinases (MAPK) (Dhillon et al., 2007; Manning and Cantley, respond to extracellular cues through nonlinear sig- 2007). However, our understanding of the mechanisms underly- nal transduction cascades whose principal compo- ing the specificity of signal transmission and processing requires nents have been identified. Nevertheless, the molec- new conceptual advances. Signaling components such as ular mechanisms underlying specificity of cellular kinases often possess various potential substrates, leading to responses remain poorly understood. Spatial distri- highly branched signaling networks rather than linear cascades. This poses the problem of how a physiological response can be bution of signaling proteins may contribute to signal- elicited with high specificity, with information flowing through ing specificity.
    [Show full text]
  • Mutations and Protein Interaction Landscape Reveal Key Cellular Events Perturbed in Upper Motor Neurons with HSP and PLS
    brain sciences Article Mutations and Protein Interaction Landscape Reveal Key Cellular Events Perturbed in Upper Motor Neurons with HSP and PLS Oge Gozutok 1, Benjamin Ryan Helmold 1 and P. Hande Ozdinler 1,2,3,4,* 1 Department of Neurology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave, Chicago, IL 60611, USA; [email protected] (O.G.); [email protected] (B.R.H.) 2 Center for Molecular Innovation and Drug Discovery, Center for Developmental Therapeutics, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60611, USA 3 Mesulam Center for Cognitive Neurology and Alzheimer’s Disease, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA 4 Feinberg School of Medicine, Les Turner ALS Center at Northwestern University, Chicago, IL 60611, USA * Correspondence: [email protected]; Tel.: +1-(312)-503-2774 Abstract: Hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are rare motor neuron diseases, which affect mostly the upper motor neurons (UMNs) in patients. The UMNs display early vulnerability and progressive degeneration, while other cortical neurons mostly remain functional. Identification of numerous mutations either directly linked or associated with HSP and PLS begins to reveal the genetic component of UMN diseases. Since each of these mutations are identified on genes that code for a protein, and because cellular functions mostly depend on protein- protein interactions, we hypothesized that the mutations detected in patients and the alterations in Citation: Gozutok, O.; Helmold, B.R.; protein interaction domains would hold the key to unravel the underlying causes of their vulnerability. Ozdinler, P.H. Mutations and Protein In an effort to bring a mechanistic insight, we utilized computational analyses to identify interaction Interaction Landscape Reveal Key Cellular Events Perturbed in Upper partners of proteins and developed the protein-protein interaction landscape with respect to HSP Motor Neurons with HSP and PLS.
    [Show full text]
  • Expression of APPL1 Is Correlated with Clinicopathologic Characteristics and Poor Prognosis in Patients with Gastric Cancer
    APPL1 EXPRESSION ORIGINALIN GASTRIC CANCER, ARTICLE Zhai et al. Expression of APPL1 is correlated with clinicopathologic characteristics and poor prognosis in patients with gastric cancer † † † † † † J.S. Zhai MD,* J.G. Song MD, C.H. Zhu MD, K. Wu PhD, Y. Yao PhD, and N. Li MD* ABSTRACT Background Although appl1 is overexpressed in many cancers, its status in gastric cancer (gc) is not known. In the present study, we used relevant pathologic and clinical data to investigate appl1 expression in patients with gc. Methods In 47 gc and 27 non-gc surgical specimens, immunohistochemistry was used to detect the expression of appl1, and reverse-transcriptase polymerase chain reaction (rt-pcr) was used to detect messenger rna (mrna). A scatterplot visualized the relationship between survival time and mrna expression in gc patients. The log-rank test and other survival statistics were used to determine the association of appl1 expression with the pathologic features of the cancer and clinical outcomes. Results In gc, appl1 was expressed in 28 of 47 specimens (59.6%), and in non-gc, it was expressed in 7 of 23 specimens (30.4%, p < 0.05). The expression of mrna in gc was 0.82 [95% confidence interval (ci): 0.78 to 0.86], and in non-gc, it was 0.73 (95% ci: 0.69 to 0.77; p < 0.05). Immunohistochemistry demonstrated that, in gc, appl1 expression was correlated with depth of infiltration (p = 0.005), lymph node metastasis (p = 0.017), and TNM stage (p = 0.022), but not with pathologic type (p = 0.41).
    [Show full text]
  • APPL1 Gates Long-Term Potentiation Through Its Plekstrin Homology Domain Mónica Fernández-Monreal*, Carla Sánchez-Castillo and Joséa
    © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2793-2803 doi:10.1242/jcs.183475 RESEARCH ARTICLE APPL1 gates long-term potentiation through its plekstrin homology domain Mónica Fernández-Monreal*, Carla Sánchez-Castillo and JoséA. Esteban* ABSTRACT mechanistic connection between activity-dependent signaling Hippocampal synaptic plasticity involves both membrane trafficking pathways and the membrane trafficking machinery is far from events and intracellular signaling, but how these are coordinated is far clear. One potential link might be provided by phosphorylated from clear. The endosomal transport of glutamate receptors in and out derivatives of phosphatidylinositol or phosphoinositides. Different of the postsynaptic membrane responds to multiple signaling phosphoinositides are specifically distributed at the plasma cascades triggered by synaptic activity. In this work, we have membrane and distinct intracellular compartments, where they can identified adaptor protein containing a plekstrin homology domain, function both as second messengers and as key spatial cues for the phosphotyrosine-binding domain and leucine zipper motif 1 (APPL1) recruitment of additional molecules (Di Paolo and De Camilli, 2006). as a crucial element linking trafficking and signaling during synaptic Alterations in phosphoinositide metabolism are involved in several – plasticity. We show that APPL1 knockdown specifically impairs PI3K- neurological disorders such as Charcot Marie Tooth syndrome type dependent forms of synaptic plasticity, such as long-term potentiation 4J (Chow et al., 2007), amyotrophic lateral sclerosis (ALS) and (LTP) and metabotropic-glutamate-receptor-dependent long-term primary lateral sclerosis (Chow et al., 2009), Down syndrome depression (mGluR-LTD). Indeed, we demonstrate that APPL1 is (Voronov et al., 2008), Lowe syndrome (Olivos-Glander et al., 1995) required for the activation of the phosphatidylinositol triphosphate and Alzheimer disease (Berman et al., 2008).
    [Show full text]
  • Membrane Curvature Protein Exhibits Interdomain Flexibility and Binds A
    THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 49, pp. 40996–41006, November 30, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Membrane Curvature Protein Exhibits Interdomain Flexibility and Binds a Small GTPase* Received for publication, August 27, 2012, and in revised form, September 24, 2012 Published, JBC Papers in Press, October 10, 2012, DOI 10.1074/jbc.M112.349803 Gordon J. King‡, Jacqueline Stöckli§, Shu-Hong Hu‡, Brit Winnen‡, Wilko G. A. Duprez‡, Christopher C. Meoli§, Jagath R. Junutula¶, Russell J. Jarrott‡, David E. James§ʈ1, Andrew E. Whitten‡2, and Jennifer L. Martin‡3 From the ‡Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia, the §Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, New South Wales 2010, Australia, ¶Genentech, Inc., South San Francisco, California 94080, and the ʈSchool of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2050, Australia Background: APPL2 is an endosomal Rab effector forming part of a signaling pathway linking cell surface and nucleus. Results: Crystal and solution structures of APPL2 were solved, and Rab partners were identified. Conclusion: APPL2 interacts tightly with Rab31, and APPL2 structures reveal unexpected domain motion that could have functional implications. Significance: APPL2 dynamics and interactions may be crucial for its cell signaling role. Downloaded from The APPL1 and APPL2 proteins (APPL (adaptor protein, BAR domain proteins are crescent- or banana-shaped mole- phosphotyrosine interaction, pleckstrin homology (PH) do- cules that generate, sense, or maintain membrane curvature (1).
    [Show full text]
  • APPL1 Promotes the Migration of Gastric Cancer Cells by Regulating Akt2 Phosphorylation
    INTERNATIONAL JOURNAL OF ONCOLOGY 51: 1343-1351, 2017 APPL1 promotes the migration of gastric cancer cells by regulating Akt2 phosphorylation YINGXUN LIU1*, CHUNLI ZHANG1*, LINGYU ZHAO1, NING DU3, NI HOU1,2, TUSHENG SONG1 and CHEN HUANG1,2 1Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061; 2Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi 710061; 3Department of Oncology Surgery, The First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China Received May 22, 2017; Accepted August 25, 2017 DOI: 10.3892/ijo.2017.4121 Abstract. As a multifunctional adaptor protein, APPL1 results demonstrate an important new function of APPL1 in (adaptor protein containing pleckstrin homology domain, regulating cell migration through a mechanism that depends phosphotyrosine binding domain and a leucine zipper motif 1) on Akt2 phosphorylation. is overexpressed in many cancers, and has been implicated in tumorigenesis and tumor progression. The present study Introduction investigated the expression of APPL1 in gastric carcinoma and the function in regulating cell migration. We investigated Gastric cancer (GC) is one of the most common human the expression of APPL1 in gastric carcinoma based upon malignant diseases and the second leading cause of cancer The Cancer Genome Atlas (TCGA) database. The expression mortality worldwide (1). The reason for the high fatality of APPL1 in collected gastric carcinoma tissues and cultured rate associated with this disease is that most cases of GC cells was measured by qRT-PCR and western blot analysis.
    [Show full text]
  • Supplemental Solier
    Supplementary Figure 1. Importance of Exon numbers for transcript downregulation by CPT Numbers of down-regulated genes for four groups of comparable size genes, differing only by the number of exons. Supplementary Figure 2. CPT up-regulates the p53 signaling pathway genes A, List of the GO categories for the up-regulated genes in CPT-treated HCT116 cells (p<0.05). In bold: GO category also present for the genes that are up-regulated in CPT- treated MCF7 cells. B, List of the up-regulated genes in both CPT-treated HCT116 cells and CPT-treated MCF7 cells (CPT 4 h). C, RT-PCR showing the effect of CPT on JUN and H2AFJ transcripts. Control cells were exposed to DMSO. β2 microglobulin (β2) mRNA was used as control. Supplementary Figure 3. Down-regulation of RNA degradation-related genes after CPT treatment A, “RNA degradation” pathway from KEGG. The genes with “red stars” were down- regulated genes after CPT treatment. B, Affy Exon array data for the “CNOT” genes. The log2 difference for the “CNOT” genes expression depending on CPT treatment was normalized to the untreated controls. C, RT-PCR showing the effect of CPT on “CNOT” genes down-regulation. HCT116 cells were treated with CPT (10 µM, 20 h) and CNOT6L, CNOT2, CNOT4 and CNOT6 mRNA were analysed by RT-PCR. Control cells were exposed to DMSO. β2 microglobulin (β2) mRNA was used as control. D, CNOT6L down-regulation after CPT treatment. CNOT6L transcript was analysed by Q- PCR. Supplementary Figure 4. Down-regulation of ubiquitin-related genes after CPT treatment A, “Ubiquitin-mediated proteolysis” pathway from KEGG.
    [Show full text]