[en] Recent data suggest that K-Ras4B (hereafter K-Ras) can drive cancer cell stemness via calmodulin (CaM)-dependent, non-canonical Wnt-signalling. Here we examined whether another Ca2+-binding protein, the CaM-related centrin1, binds to K-Ras and could mediate some K-Ras functions that were previously ascribed to CaM. While CaM and centrin1 appear to distinguish between peptides that were derived from their classical targets, they both bind to K-Ras in cells. Cellular BRET- and immunoprecipitation data suggest that CaM engages more with K-Ras than centrin1 and that the interaction with the C-terminal membrane anchor of K-Ras is sufficient for this. Surprisingly, binding of neither K-Ras nor its membrane anchor alone to CaM or centrin1 is sensitive to inhibition of prenylation. In support of an involvement of the G-domain of K-Ras in cellular complexes with these Ca2+-binding proteins, we find that oncogenic K-RasG12V displays increased engagement with both CaM and centrin1. This is abrogated by addition of the D38A effector-site mutation, suggesting that K-RasG12V is held together with CaM or centrin1 in complexes with effectors. When treated with CaM inhibitors, the BRET-interaction of K-RasG12V with centrin1 was also disrupted in the low micromolar range, comparable to that with CaM. While CaM predominates in regulating functional membrane anchorage of K-Ras, it has a very similar co-distribution with centrin1 on mitotic organelles. Given these results, a significant overlap of the CaM- and centrin1-dependent functions of K-Ras is suggested.
Disciplines :
Biochimie, biophysique & biologie moléculaire
Auteur, co-auteur :
Manoharan, Ganesh babu; Department of Life Sciences and Medicine, University of Luxembourg, Luxembourg
LAURINI, Christina ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
BOTTONE, Sara ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
BEN FREDJ, Nesrine ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
ABANKWA, Daniel ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Co-auteurs externes :
no
Langue du document :
Anglais
Titre :
K-Ras Binds Calmodulin-Related Centrin1 with Potential Implications for K-Ras Driven Cancer Cell Stemness
Date de publication/diffusion :
2023
Titre du périodique :
Cancers
eISSN :
2072-6694
Maison d'édition :
Multidisciplinary Digital Publishing Institute (MDPI), Basel, Suisse
Peer reviewed :
Peer reviewed vérifié par ORBi
Projet FnR :
FNR13673303 - Investigation Of Nanoscale Ras Polarizations In Instructing Cancer Cell Stemness, 2019 (01/09/2020-31/08/2023) - Daniel Abankwa
Prior I.A. Hood F.E. Hartley J.L. The Frequency of Ras Mutations in Cancer Cancer Res. 2020 80 2969 2974 10.1158/0008-5472.CAN-19-3682 32209560
Castel P. Rauen K.A. McCormick F. The duality of human oncoproteins: Drivers of cancer and congenital disorders Nat. Rev. Cancer 2020 20 383 397 10.1038/s41568-020-0256-z 32341551
Abankwa D. Gorfe A.A. Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again Biomolecules 2020 10 1522 10.3390/biom10111522 33172116
Schmick M. Kraemer A. Bastiaens P.I. Ras moves to stay in place Trends Cell Biol. 2015 25 190 197 10.1016/j.tcb.2015.02.004
Newlaczyl A.U. Coulson J.M. Prior I.A. Quantification of spatiotemporal patterns of Ras isoform expression during development Sci. Rep. 2017 7 41297 10.1038/srep41297
Wang M.T. Holderfield M. Galeas J. Delrosario R. To M.D. Balmain A. McCormick F. K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling Cell 2015 163 1237 1251 10.1016/j.cell.2015.10.041
Quinlan M.P. Quatela S.E. Philips M.R. Settleman J. Activated Kras, but not Hras or Nras, may initiate tumors of endodermal origin via stem cell expansion Mol. Cell. Biol. 2008 28 2659 2674 10.1128/MCB.01661-07
Najumudeen A.K. Jaiswal A. Lectez B. Oetken-Lindholm C. Guzman C. Siljamaki E. Posada I.M. Lacey E. Aittokallio T. Abankwa D. Cancer stem cell drugs target K-ras signaling in a stemness context Oncogene 2016 35 5248 5262 10.1038/onc.2016.59
Okutachi S. Manoharan G.B. Kiriazis A. Laurini C. Catillon M. McCormick F. Yli-Kauhaluoma J. Abankwa D. A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness Front. Cell Dev. Biol. 2021 9 665673 10.3389/fcell.2021.665673
Alvarez-Moya B. Lopez-Alcala C. Drosten M. Bachs O. Agell N. K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function Oncogene 2010 29 5911 5922 10.1038/onc.2010.298
Dharmaiah S. Bindu L. Tran T.H. Gillette W.K. Frank P.H. Ghirlando R. Nissley D.V. Esposito D. McCormick F. Stephen A.G. et al. Structural basis of recognition of farnesylated and methylated KRAS4b by PDEdelta Proc. Natl. Acad. Sci. USA 2016 113 E6766 E6775 10.1073/pnas.1615316113
Chippalkatti R. Abankwa D. Promotion of cancer cell stemness by Ras Biochem. Soc. Trans. 2021 49 467 476 10.1042/BST20200964
Tidow H. Nissen P. Structural diversity of calmodulin binding to its target sites FEBS J. 2013 280 5551 5565 10.1111/febs.12296
Grant B.M.M. Enomoto M. Ikura M. Marshall C.B. A Non-Canonical Calmodulin Target Motif Comprising a Polybasic Region and Lipidated Terminal Residue Regulates Localization Int. J. Mol. Sci. 2020 21 2751 10.3390/ijms21082751
Grant B.M.M. Enomoto M. Back S.I. Lee K.Y. Gebregiworgis T. Ishiyama N. Ikura M. Marshall C.B. Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety Sci. Signal. 2020 13 eaaz0344 10.1126/scisignal.aaz0344
Ismail S.A. Chen Y.X. Rusinova A. Chandra A. Bierbaum M. Gremer L. Triola G. Waldmann H. Bastiaens P.I. Wittinghofer A. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo Nat. Chem. Biol. 2011 7 942 949 10.1038/nchembio.686
Villalonga P. Lopez-Alcala C. Bosch M. Chiloeches A. Rocamora N. Gil J. Marais R. Marshall C.J. Bachs O. Agell N. Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling Mol. Cell. Biol. 2001 21 7345 7354 10.1128/MCB.21.21.7345-7354.2001
Chandra A. Grecco H.E. Pisupati V. Perera D. Cassidy L. Skoulidis F. Ismail S.A. Hedberg C. Hanzal-Bayer M. Venkitaraman A.R. et al. The GDI-like solubilizing factor PDEdelta sustains the spatial organization and signalling of Ras family proteins Nat. Cell Biol. 2011 14 148 158 10.1038/ncb2394
Li C.J. Heim R. Lu P. Pu Y. Tsien R.Y. Chang D.C. Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique J. Cell Sci. 1999 112 1567 1577 10.1242/jcs.112.10.1567
Yu Y.Y. Chen Y. Dai G. Chen J. Sun X.M. Wen C.J. Zhao D.H. Chang D.C. Li C.J. The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells Int. J. Biochem. Cell Biol. 2004 36 1562 1572 10.1016/j.biocel.2003.12.016
Abraham S.J. Nolet R.P. Calvert R.J. Anderson L.M. Gaponenko V. The hypervariable region of K-Ras4B is responsible for its specific interactions with calmodulin Biochemistry 2009 48 7575 7583 10.1021/bi900769j 19583261
Liao J. Planchon S.M. Wolfman J.C. Wolfman A. Growth factor-dependent AKT activation and cell migration requires the function of c-K(B)-Ras versus other cellular ras isoforms J. Biol. Chem. 2006 281 29730 29738 10.1074/jbc.M600668200 16908523
Fischer R. Julsgart J. Berchtold M.W. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase FEBS Lett. 1998 425 175 177 10.1016/S0014-5793(98)00225-7 9541031
Wu L.J. Xu L.R. Liao J.M. Chen J. Liang Y. Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin PLoS ONE 2011 6 e21929 10.1371/journal.pone.0021929 21750741
Agamasu C. Ghirlando R. Taylor T. Messing S. Tran T.H. Bindu L. Tonelli M. Nissley D.V. McCormick F. Stephen A.G. KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner Biophys. J. 2019 116 1049 1063 10.1016/j.bpj.2019.02.004 30846362
Manoharan G.B. Kopra K. Eskonen V. Härmä H. Abankwa D. High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors Anal. Biochem. 2019 572 25 32 10.1016/j.ab.2019.02.015
Berchtold M.W. Villalobo A. The many faces of calmodulin in cell proliferation, programmed cell death, autophagy, and cancer Biochim. Biophys. Acta 2014 1843 398 435 10.1016/j.bbamcr.2013.10.021
Faust F.M. Slisz M. Jarrett H.W. Calmodulin is labeled at lysine 148 by a chemically reactive phenothiazine J. Biol. Chem. 1987 262 1938 1941 10.1016/S0021-9258(18)61599-6
Yang A. Miron S. Mouawad L. Duchambon P. Blouquit Y. Craescu C.T. Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair Biochemistry 2006 45 3653 3663 10.1021/bi0524868
Friedberg F. Centrin isoforms in mammals. Relation to calmodulin Mol. Biol. Rep. 2006 33 243 252 10.1007/s11033-006-9004-z
Dantas T.J. Daly O.M. Morrison C.G. Such small hands: The roles of centrins/caltractins in the centriole and in genome maintenance Cell. Mol. Life Sci. 2012 69 2979 2997 10.1007/s00018-012-0961-1
Puumalainen M.R. Ruthemann P. Min J.H. Naegeli H. Xeroderma pigmentosum group C sensor: Unprecedented recognition strategy and tight spatiotemporal regulation Cell. Mol. Life Sci. 2016 73 547 566 10.1007/s00018-015-2075-z
Sanz J.M. Grecu D. Assairi L. Ca2+ signaling and Target Binding Regulations: Calmodulin and Centrin In Vitro and In Vivo Bioenergetics 2016 5 1000144 10.4172/2167-7662.1000144
Klein U.R. Nigg E.A. SUMO-dependent regulation of centrin-2 J. Cell Sci. 2009 122 3312 3321 10.1242/jcs.050245
Paoletti A. Moudjou M. Paintrand M. Salisbury J.L. Bornens M. Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles J. Cell Sci. 1996 109 3089 3102 10.1242/jcs.109.13.3089
Hodges M.E. Scheumann N. Wickstead B. Langdale J.A. Gull K. Reconstructing the evolutionary history of the centriole from protein components J. Cell Sci. 2010 123 1407 1413 10.1242/jcs.064873
Graser S. Stierhof Y.D. Lavoie S.B. Gassner O.S. Lamla S. Le Clech M. Nigg E.A. Cep164, a novel centriole appendage protein required for primary cilium formation J. Cell Biol. 2007 179 321 330 10.1083/jcb.200707181
Wall V.E. Garvey L.A. Mehalko J.L. Procter L.V. Esposito D. Combinatorial assembly of clone libraries using site-specific recombination Methods Mol. Biol. 2014 1116 193 208 10.1007/978-1-62703-764-8_14
Zimmermann G. Papke B. Ismail S. Vartak N. Chandra A. Hoffmann M. Hahn S.A. Triola G. Wittinghofer A. Bastiaens P.I. et al. Small molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS signalling Nature 2013 497 638 642 10.1038/nature12205
Vaasa A. Viil I. Enkvist E. Viht K. Raidaru G. Lavogina D. Uri A. High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK Anal. Biochem. 2009 385 85 93 10.1016/j.ab.2008.10.030
Sinijarv H. Wu S. Ivan T. Laasfeld T. Viht K. Uri A. Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity Anal. Biochem. 2017 531 67 77 10.1016/j.ab.2017.05.017 28527909
Manoharan G.B. Okutachi S. Abankwa D. Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells PLoS ONE 2022 17 e0268635 10.1371/journal.pone.0268635 35617282
Liyanage M.R. Zaidi A. Johnson C.K. Fluorescence polarization assay for calmodulin binding to plasma membrane Ca2+-ATPase: Dependence on enzyme and Ca2+ concentrations Anal. Biochem. 2009 385 1 6 10.1016/j.ab.2008.10.022
Waxham M.N. Tsai A.L. Putkey J.A. A mechanism for calmodulin (CaM) trapping by CaM-kinase II defined by a family of CaM-binding peptides J. Biol. Chem. 1998 273 17579 17584 10.1074/jbc.273.28.17579 9651352
Zhao Y. Guo X. Yang B. Calcium-induced human centrin 1 self-assembly and double-regulating the binding with peptide R18-Sfi1p Int. J. Biol. Macromol. 2019 128 314 323 10.1016/j.ijbiomac.2019.01.096
Bodle J.C. Loboa E.G. Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies Stem Cells 2016 34 1445 1454 10.1002/stem.2341
Pfleger K.D. Eidne K.A. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET) Nat. Methods 2006 3 165 174 10.1038/nmeth841
Halling D.B. Liebeskind B.J. Hall A.W. Aldrich R.W. Conserved properties of individual Ca2+-binding sites in calmodulin Proc. Natl. Acad. Sci. USA 2016 113 E1216 E1225 10.1073/pnas.1600385113
Herrmann C. Horn G. Spaargaren M. Wittinghofer A. Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor J. Biol. Chem. 1996 271 6794 6800 10.1074/jbc.271.12.6794
Vetter I.R. Linnemann T. Wohlgemuth S. Geyer M. Kalbitzer H.R. Herrmann C. Wittinghofer A. Structural and biochemical analysis of Ras-effector signaling via RalGDS FEBS Lett. 1999 451 175 180 10.1016/S0014-5793(99)00555-4
Thurnher M. Gruenbacher G. Nussbaumer O. Regulation of mevalonate metabolism in cancer and immune cells Biochim. Biophys. Acta (BBA) Mol. Cell Res. 2013 1831 1009 1015 10.1016/j.bbalip.2013.03.003
Abdelkarim H. Leschinsky N. Jang H. Banerjee A. Nussinov R. Gaponenko V. The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations Curr. Opin. Struct. Biol. 2021 71 164 170 10.1016/j.sbi.2021.06.008
Parkkola H. Siddiqui F.A. Oetken-Lindholm C. Abankwa D. FLIM-FRET Analysis of Ras Nanoclustering and Membrane-Anchorage Methods Mol. Biol. 2021 2262 233 250 10.1007/978-1-0716-1190-6_13
Siddiqui F.A. Alam C. Rosenqvist P. Ora M. Sabt A. Manoharan G.B. Bindu L. Okutachi S. Catillon M. Taylor T. et al. PDE6D Inhibitors with a New Design Principle Selectively Block K-Ras Activity ACS Omega 2020 5 832 842 10.1021/acsomega.9b03639
Kohnke M. Schmitt S. Ariotti N. Piggott A.M. Parton R.G. Lacey E. Capon R.J. Alexandrov K. Abankwa D. Design and application of in vivo FRET biosensors to identify protein prenylation and nanoclustering inhibitors Chem. Biol. 2012 19 866 874 10.1016/j.chembiol.2012.05.019
Dontu G. Abdallah W.M. Foley J.M. Jackson K.W. Clarke M.F. Kawamura M.J. Wicha M.S. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells Genes Dev. 2003 17 1253 1270 10.1101/gad.1061803
Santoro A. Vlachou T. Carminati M. Pelicci P.G. Mapelli M. Molecular mechanisms of asymmetric divisions in mammary stem cells EMBO Rep. 2016 17 1700 1720 10.15252/embr.201643021
Cicalese A. Bonizzi G. Pasi C.E. Faretta M. Ronzoni S. Giulini B. Brisken C. Minucci S. Di Fiore P.P. Pelicci P.G. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells Cell 2009 138 1083 1095 10.1016/j.cell.2009.06.048
Chen C. Yamashita Y.M. Centrosome-centric view of asymmetric stem cell division Open Biol. 2021 11 200314 10.1098/rsob.200314
Nussinov R. Zhang M. Tsai C.J. Jang H. Calmodulin and IQGAP1 activation of PI3Kalpha and Akt in KRAS, HRAS and NRAS-driven cancers Biochim. Biophys. Acta (BBA) Mol. Basis Dis. 2018 1864 2304 2314 10.1016/j.bbadis.2017.10.032 29097261
