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RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency

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Abstract

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.

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References

  1. Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136:731–745

    Article  PubMed  CAS  Google Scholar 

  2. Kondrashov N, Pusic A, Stumpf CR et al (2011) Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell 145:383–397

    Article  PubMed  CAS  Google Scholar 

  3. Ron D, Chen CH, Caldwell J, Jamieson L, Orr E, Mochly-Rosen D (1994) Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc Natl Acad Sci U S A 91:839–843

    Article  PubMed  CAS  Google Scholar 

  4. Ron D, Luo J, Mochly-Rosen D (1995) C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo. J Biol Chem 270:24180–24187

    Article  PubMed  CAS  Google Scholar 

  5. Besson A, Wilson TL, Yong VW (2002) The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J Biol Chem 277:22073–22084

    Article  PubMed  CAS  Google Scholar 

  6. Gibson TJ (2012) RACK1 research – ships passing in the night? FEBS Lett 586(17):2787–2789

    Article  PubMed  CAS  Google Scholar 

  7. Chang BY, Chiang M, Cartwright CA (2001) The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1. J Biol Chem 276:20346–20356

    Article  PubMed  CAS  Google Scholar 

  8. Mamidipudi V, Zhang J, Lee KC, Cartwright CA (2004) RACK1 regulates G1/S progression by suppressing Src kinase activity. Mol Cell Biol 24:6788–6798

    Article  PubMed  CAS  Google Scholar 

  9. Lopez-Bergami P, Habelhah H, Bhoumik A, Zhang W, Wang LH, Ronai Z (2005) RACK1 mediates activation of JNK by protein kinase C [corrected]. Mol Cell 19:309–320

    Article  PubMed  CAS  Google Scholar 

  10. Lopez-Bergami P, Huang C, Goydos JS et al (2007) Rewired ERK-JNK signaling pathways in melanoma. Cancer Cell 11:447–460

    Article  PubMed  CAS  Google Scholar 

  11. Yaka R, Thornton C, Vagts AJ, Phamluong K, Bonci A, Ron D (2002) NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1. Proc Natl Acad Sci USA 99:5710–5715

    Article  PubMed  CAS  Google Scholar 

  12. Reiner CL, McCullar JS, Kow RL, Le JH, Goodlett DR, Nathanson NM (2010) RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking. PLoS One 5:e13517

    Article  PubMed  Google Scholar 

  13. Angenstein F, Evans AM, Settlage RE et al (2002) A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons. J Neurosci 22:8827–8837

    PubMed  CAS  Google Scholar 

  14. Buensuceso CS, Woodside D, Huff JL, Plopper GE, O’Toole TE (2001) The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration. J Cell Sci 114:1691–1698

    PubMed  CAS  Google Scholar 

  15. Serrels B, Sandilands E, Serrels A et al (2010) A complex between FAK, RACK1, and PDE4D5 controls spreading initiation and cancer cell polarity. Curr Biol 20:1086–1092

    Article  PubMed  CAS  Google Scholar 

  16. Smith KJ, Baillie GS, Hyde EI et al (2007) 1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin and RACK1. Cell Signal 19:2612–2624

    Article  PubMed  CAS  Google Scholar 

  17. Li X, Baillie GS, Houslay MD (2009) Mdm2 directs the ubiquitination of beta-arrestin-sequestered cAMP phosphodiesterase-4D5. J Biol Chem 284:16170–16182

    Article  PubMed  CAS  Google Scholar 

  18. Serrels B, Sandilands E, Frame MC (2011) Signaling of the direction-sensing FAK/RACK1/PDE4D5 complex to the small GTPase Rap1. Small GTPases 2:54–61

    Article  PubMed  Google Scholar 

  19. Chang BY, Conroy KB, Machleder EM, Cartwright CA (1998) RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells. Mol Cell Biol 18:3245–3256

    PubMed  CAS  Google Scholar 

  20. Mamidipudi V, Cartwright CA (2009) A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways. Oncogene 28:4421–4433

    Article  PubMed  CAS  Google Scholar 

  21. Wehner P, Shnitsar I, Urlaub H, Borchers A (2011) RACK1 is a novel interaction partner of PTK7 that is required for neural tube closure. Development 138:1321–1327

    Article  PubMed  CAS  Google Scholar 

  22. Arimoto K, Fukuda H, Imajoh-Ohmi S, Saito H, Takekawa M (2008) Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nat Cell Biol 10:1324–1332

    Article  PubMed  CAS  Google Scholar 

  23. Ohn T, Kedersha N, Hickman T, Tisdale S, Anderson P (2008) A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly. Nat Cell Biol 10:1224–1231

    Article  PubMed  CAS  Google Scholar 

  24. McGough NN, He DY, Logrip ML et al (2004) RACK1 and brain-derived neurotrophic factor: a homeostatic pathway that regulates alcohol addiction. J Neurosci 24:10542–10552

    Article  PubMed  CAS  Google Scholar 

  25. Robles MS, Boyault C, Knutti D, Padmanabhan K, Weitz CJ (2010) Identification of RACK1 and protein kinase Calpha as integral components of the mammalian circadian clock. Science 327:463–466

    Article  PubMed  CAS  Google Scholar 

  26. Rachfall N, Schmitt K, Bandau S, Smolinski N, Ehrenreich A, Valerius O, Braus GH (2012) RACK1/Asc1p, a ribosomal node in cellular signaling. Mol Cell Proteomics. doi:10.1074/mcp.M112.017277

  27. Baum S, Bittins M, Frey S, Seedorf M (2004) Asc1p, a WD40-domain containing adaptor protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes. Biochem J 380:823–830

    Article  PubMed  CAS  Google Scholar 

  28. Chantrel Y, Gaisne M, Lions C, Verdiere J (1998) The transcriptional regulator Hap1p (Cyp1p) is essential for anaerobic or heme-deficient growth of Saccharomyces cerevisiae: genetic and molecular characterization of an extragenic suppressor that encodes a WD repeat protein. Genetics 148:559–569

    PubMed  CAS  Google Scholar 

  29. Gerbasi VR, Weaver CM, Hill S, Friedman DB, Link AJ (2004) Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression. Mol Cell Biol 24:8276–8287

    Article  PubMed  CAS  Google Scholar 

  30. Nunez A, Franco A, Madrid M, Soto T, Vicente J, Gacto M, Cansado J (2009) Role for RACK1 orthologue Cpc2 in the modulation of stress response in fission yeast. Mol Biol Cell 20:3996–4009

    Article  PubMed  CAS  Google Scholar 

  31. Zeller CE, Parnell SC, Dohlman HG (2007) The RACK1 ortholog Asc1 functions as a G-protein beta subunit coupled to glucose responsiveness in yeast. J Biol Chem 282:25168–25176

    Article  PubMed  CAS  Google Scholar 

  32. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR 3rd (1999) Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17:676–682

    Article  PubMed  CAS  Google Scholar 

  33. Sengupta J, Nilsson J, Gursky R, Spahn CM, Nissen P, Frank J (2004) Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nat Struct Mol Biol 11:957–962

    Article  PubMed  CAS  Google Scholar 

  34. Rabl J, Leibundgut M, Ataide SF, Haag A, Ban N (2011) Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331:730–736

    Article  PubMed  CAS  Google Scholar 

  35. Jannot G, Bajan S, Giguere NJ, Bouasker S, Banville IH, Piquet S, Hutvagner G, Simard MJ (2011) The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans. EMBO Rep 12:581–586

    Article  PubMed  CAS  Google Scholar 

  36. Kuroha K, Akamatsu M, Dimitrova L, Ito T, Kato Y, Shirahige K, Inada T (2010) Receptor for activated C kinase 1 stimulates nascent polypeptide-dependent translation arrest. EMBO Rep 11:956–961

    Article  PubMed  CAS  Google Scholar 

  37. Ceci M, Gaviraghi C, Gorrini C, Sala LA, Offenhauser N, Marchisio PC, Biffo S (2003) Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature 426:579–584

    Article  PubMed  CAS  Google Scholar 

  38. Ruan Y, Sun L, Hao Y et al (2012) Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma. J Clin Invest 122:2554–2566

    Article  PubMed  CAS  Google Scholar 

  39. Grosso S, Volta V, Sala LA, Vietri M, Marchisio PC, Ron D, Biffo S (2008) PKCbetaII modulates translation independently from mTOR and through RACK1. Biochem J 415:77–85

    Article  PubMed  CAS  Google Scholar 

  40. Biffo S, Sanvito F, Costa S, Preve L, Pignatelli R, Spinardi L, Marchisio PC (1997) Isolation of a novel beta4 integrin-binding protein (p27(BBP)) highly expressed in epithelial cells. J Biol Chem 272:30314–30321

    Article  PubMed  CAS  Google Scholar 

  41. Strezoska Z, Pestov DG, Lau LF (2002) Functional inactivation of the mouse nucleolar protein Bop1 inhibits multiple steps in pre-rRNA processing and blocks cell cycle progression. J Biol Chem 277:29617–29625

    Article  PubMed  CAS  Google Scholar 

  42. Gandin V, Miluzio A, Barbieri AM, Beugnet A, Kiyokawa H, Marchisio PC, Biffo S (2008) Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation. Nature 455:684–688

    Article  PubMed  CAS  Google Scholar 

  43. Miluzio A, Beugnet A, Grosso S et al (2011) Impairment of cytoplasmic eIF6 activity restricts lymphomagenesis and tumor progression without affecting normal growth. Cancer Cell 19:765–775

    Article  PubMed  CAS  Google Scholar 

  44. Biffo S, Verhaagen J, Schrama LH, Schotman P, Danho W, Margolis FL (1990) B-50/GAP43 expression correlates with process outgrowth in the embryonic mouse nervous system. Eur J Neurosci 2:487–499

    Article  PubMed  Google Scholar 

  45. Biffo S, DeLucia R, Mulatero B, Margolis F, Fasolo A (1990) Carnosine-, calcitonin gene-related peptide- and tyrosine hydroxylase-immunoreactivity in the mouse olfactory bulb following peripheral denervation. Brain Res 528:353–357

    Article  PubMed  CAS  Google Scholar 

  46. Sanvito F, Piatti S, Villa A, Bossi M, Lucchini G, Marchisio PC, Biffo S (1999) The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly. J Cell Biol 144:823–837

    Article  PubMed  CAS  Google Scholar 

  47. Oliver ER, Saunders TL, Tarle SA, Glaser T (2004) Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute. Development 131:3907–3920

    Article  PubMed  CAS  Google Scholar 

  48. Guo J, Wang S, Valerius O et al (2011) Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid. Plant Physiol 155:370–383

    Article  PubMed  CAS  Google Scholar 

  49. Caldarola S, De Stefano MC, Amaldi F, Loreni F (2009) Synthesis and function of ribosomal proteins—fading models and new perspectives. FEBS J 276:3199–3210

    Article  PubMed  CAS  Google Scholar 

  50. Kiss T, Fayet-Lebaron E, Jady BE (2010) Box H/ACA small ribonucleoproteins. Mol Cell 37:597–606

    Article  PubMed  Google Scholar 

  51. Adams DR, Ron D, Kiely PA (2011) RACK1, a multifaceted scaffolding protein: structure and function. Cell Commun Signal 9:22

    Article  PubMed  CAS  Google Scholar 

  52. Brejning J, Arneborg N, Jespersen L (2005) Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts. J Appl Microbiol 98:261–271

    Article  PubMed  CAS  Google Scholar 

  53. Kleinschmidt M, Schulz R, Braus GH (2006) The yeast CPC2/ASC1 gene is regulated by the transcription factors Fhl1p and Ifh1p. Curr Genet 49:218–228

    Article  PubMed  CAS  Google Scholar 

  54. Mabuchi T, Ichimura Y, Takeda M, Douglas MG (2000) ASC1/RAS2 suppresses the growth defect on glycerol caused by the atp1-2 mutation in the yeast Saccharomyces cerevisiae. J Biol Chem 275:10492–10497

    Article  PubMed  CAS  Google Scholar 

  55. Valerius O, Kleinschmidt M, Rachfall N et al (2007) The Saccharomyces homolog of mammalian RACK1, Cpc2/Asc1p, is required for FLO11-dependent adhesive growth and dimorphism. Mol Cell Proteomics 6:1968–1979

    Article  PubMed  CAS  Google Scholar 

  56. Nilsson J, Sengupta J, Frank J, Nissen P (2004) Regulation of eukaryotic translation by the RACK1 protein: a platform for signalling molecules on the ribosome. EMBO Rep 5:1137–1141

    Article  PubMed  CAS  Google Scholar 

  57. Matsson H, Davey EJ, Draptchinskaia N et al (2004) Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation. Mol Cell Biol 24:4032–4037

    Article  PubMed  CAS  Google Scholar 

  58. Ellis SR, Gleizes PE (2011) Diamond Blackfan anemia: ribosomal proteins going rogue. Semin Hematol 48:89–96

    Article  PubMed  CAS  Google Scholar 

  59. Barkic M, Crnomarković S, Grabusić K et al (2009) The p53 tumor suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival. Mol Cell Biol 29:2489–2504

    Article  PubMed  CAS  Google Scholar 

  60. Egidy G, Julé S, Bossé P et al (2008) Transcription analysis in the MeLiM swine model identifies RACK1 as a potential marker of malignancy for human melanocytic proliferation. Mol Cancer 7:34

    Article  PubMed  Google Scholar 

  61. Lee JH, Cho B, Jun HJ, Seo WD, Kim DW, Cho KJ, Lee SJ (2012) Momilactione B inhibits protein kinase A signaling and reduces tyrosinase-related proteins 1 and 2 expression in melanocytes. Biotechnol Lett 34:805–812

    Article  PubMed  CAS  Google Scholar 

  62. Bolger GB, Baillie GS, Li X et al (2006) Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5. Biochem J 398:23–36

    Article  PubMed  CAS  Google Scholar 

  63. Lynch MJ, Baillie GS, Mohamed A, Li X, Maisonneuve C, Klussmann E, van Heeke G, Houslay MD (2005) RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells. J Biol Chem 280:33178–33189

    Article  PubMed  CAS  Google Scholar 

  64. Levitt JM, Baldwin A, Papadakis A, Puri S, Xylas J, Munger K, Georgakoudi I (2006) Intrinsic fluorescence and redox changes associated with apoptosis of primary human epithelial cells. J Biomed Opt 11:064012

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by grants AIRC IG11390 (Associazione Italiana Ricerca sul Cancro), AICR (Association for International Cancer Research, UK) 09-0061, and TELETHON GGP05043 to S.B. We thank Dr. Dorit Ron (UCSF, San Francisco, USA) for helpful suggestions. Requests concerning reagents or mice should be addressed to S.B. or V.V.

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Authors and Affiliations

  1. Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy

    Viviana Volta, Anne Beugnet, Simone Gallo, Laura Magri, Daniela Brina, Elisa Pesce, Piera Calamita & Stefano Biffo

  2. Environmental and Life Science Department (DISAV), University of Eastern Piedmont, Alessandria, Italy

    Elisa Pesce & Stefano Biffo

  3. Department of Pathology, San Raffaele Scientific Institute, Milan, Italy

    Francesca Sanvito

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  1. Viviana Volta
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  2. Anne Beugnet
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  3. Simone Gallo
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  9. Stefano Biffo
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Correspondence to Stefano Biffo.

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V. Volta and A. Beugnet contributed equally to this work.

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Volta, V., Beugnet, A., Gallo, S. et al. RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency. Cell. Mol. Life Sci. 70, 1439–1450 (2013). https://doi.org/10.1007/s00018-012-1215-y

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