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HuR thermal stability is dependent on domain binding and upon phosphorylation

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Abstract

Human antigen R (HuR) is a multitasking RNA binding protein involved in posttranscriptional regulation by recognizing adenine- and uracile-rich elements placed at the 3′-untranslated regions of messenger RNAs (mRNAs). The modular architecture of the protein, which consists of two N-terminal RNA recognition motifs (RRMs) in tandem spaced from a third one by a nuclear-cytoplasmic shuttling sequence, controls the stability of many mRNA targets, as well as their translation rates. A higher level of regulation comes from the fact that both localization and function of HuR are strictly regulated by phosphorylation. Here, we report how the thermal stability of RRM2 is decreased by the presence of RRM1, indicating that both domains are interacting in solution. In addition, even though no significant structural changes are observed among mutants of HuR RRM12 mimicking phosphorylated species, slight differences in stability are appreciable, which may explain the RNA binding activity of HuR.

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Abbreviations

AREs:

Adenine- and uracile-rich elements

CARM1:

Coactivator-associated arginine methyltransferase 1 protein

CD:

Circular dichroism

Chk2:

Checkpoint 2 kinase

Cdk:

Cyclin-dependent kinase 1

DSF:

Differential scanning fluorimetry

DTT:

Dithiothreitol

ELAV:

Embryonic lethal and abnormal vision

HNS:

Human novel shuttling

HuR:

Human antigen R

HuR FL:

HuR full-length

K D :

Dissociation affinity constant

PKCα:

Protein kinase C α

PKCδ:

Protein kinase C δ

RBP:

RNA binding protein

RMSD:

Root-mean-square deviation

RRM:

RNA recognition motif

RRM12 WT:

RRM12 wild-type

RT-PCR:

Real-time polymerase chain reaction

T m :

Midpoint melting temperature

UTRs:

Untranslated regions

References

  • Abdelmohsen K, Lal A, Kim HH, Gorospe M (2007a) Posttranscriptional orchestration of an anti-apoptotic program by HuR. Cell Cycle 6:1288–1292

    Article  PubMed  CAS  Google Scholar 

  • Abdelmohsen K, Pullmann R Jr, Lal A, Kim HH, Galban S, Yang X, Blethrow JD, Walker M, Shubert J, Gillespie DA, Furneaux H, Gorospe M (2007b) Phosphorylation of HuR by Chk2 regulates SIRT1 expression. Mol Cell 25:543–557

    Article  PubMed  CAS  Google Scholar 

  • Abdelmohsen K, Srikantan S, Yang X, Lal A, Kim HH, Kuwano Y, Galban S, Becker KG, Kamara D, de Cabo R, Gorospe M (2009) Ubiquitin-mediated proteolysis of HuR by heat shock. EMBO J 28:1271–1282

    Article  PubMed  CAS  Google Scholar 

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201

    Article  PubMed  CAS  Google Scholar 

  • Aroca A, Díaz-Quintana A, Díaz-Moreno I (2011) A structural insight into the C-terminal RNA recognition motifs of T-cell intracellular antigen-1 protein. FEBS Lett 585:2958–2964

    Article  PubMed  CAS  Google Scholar 

  • Benoit RM, Meisner NC, Kallen J, Graff P, Hemmig R, Cebe R, Ostermeier C, Widmer H, Auer M (2010) The x-ray crystal structure of the first RNA recognition motif and site-directed mutagenesis suggest a possible HuR redox sensing mechanism. J Mol Biol 397:1231–1244

    Article  PubMed  CAS  Google Scholar 

  • Birney E, Kumar S, Krainer AR (1993) Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res 21:5803–5816

    Article  PubMed  CAS  Google Scholar 

  • Brennan CM, Steitz JA (2001) HuR and mRNA stability. Cell Mol Life Sci 58:266–277

    Article  PubMed  CAS  Google Scholar 

  • Denkert C, Weichert W, Winzer KJ, Muller BM, Noske A, Niesporek S, Kristiansen G, Guski H, Dietel M, Hauptmann S (2004) Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast carcinoma. Clin Cancer Res 10:5580–5586

    Article  PubMed  CAS  Google Scholar 

  • Díaz-Moreno I, Hollingworth D, Frenkiel TA, Kelly G, Martin S, Howell S, García-Mayoral M, Gherzi R, Briata P, Ramos A (2009) Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14–3-3 binding. Nat Struct Mol Biol 16:238–246

    Article  PubMed  Google Scholar 

  • Díaz-Moreno I, Hollingworth D, Kelly G, Martin S, García-Mayoral M, Briata P, Gherzi R, Ramos A (2010) Orientation of the central domains of KSRP and its implications for the interaction with the RNA targets. Nucleic Acids Res 38:5193–5205

    Article  PubMed  Google Scholar 

  • Dixon DA, Tolley ND, King PH, Nabors LB, McIntyre TM, Zimmerman GA, Prescott SM (2001) Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells. J Clin Invest 108:1657–1665

    PubMed  CAS  Google Scholar 

  • Doller A, Huwiler A, Muller R, Radeke HH, Pfeilschifter J, Eberhardt W (2007) Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2. Mol Biol Cell 18:2137–2148

    Article  PubMed  CAS  Google Scholar 

  • Doller A, Pfeilschifter J, Eberhardt W (2008) Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR. Cell Signal 20:2165–2173

    Article  PubMed  CAS  Google Scholar 

  • Doller A, Schlepckow K, Schwalbe H, Pfeilschifter J, Eberhardt W (2009) Tandem phosphorylation of serines 221 and 318 by protein kinase Cdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR. Mol Cell Biol 30:1397–1410

    Article  Google Scholar 

  • Embade N, Fernández-Ramos D, Varela-Rey M, Beraza N, Sini M, de Juan VG, Woodhoo A, Martínez-López N, Rodríguez-Iruretagoyena B, Bustamante FJ, de la Hoz AB, Carracedo A, Xirodimas DP, Rodríguez MS, Lu SC, Mato JM, Martínez-Chantar ML (2011) Mdm2 regulates HuR stability in human liver and colon cancer through neddylation. Hepatology. doi:10.1002/hep.24795

  • Fan XC, Steitz JA (1998) HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc Natl Acad Sci U S A 95:15293–15298

    Article  PubMed  CAS  Google Scholar 

  • Gorospe M (2003) HuR in the mammalian genotoxic response: post-transcriptional multitasking. Cell Cycle 2:412–414

    Article  PubMed  CAS  Google Scholar 

  • Heinonen M, Bono P, Narko K, Chang SH, Lundin J, Joensuu H, Furneaux H, Hla T, Haglund C, Ristimaki A (2005) Cytoplasmic HuR expression is a prognostic factor in invasive ductal breast carcinoma. Cancer Res 65:2157–2161

    Article  PubMed  CAS  Google Scholar 

  • Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL repository and associated resources. Nucleic Acids Res 37:D387–D392

    Article  PubMed  CAS  Google Scholar 

  • Kim HH, Gorospe M (2008) Phosphorylated HuR shuttles in cycles. Cell Cycle 7:3124–3126

    Article  PubMed  CAS  Google Scholar 

  • Kim HH, Abdelmohsen K, Lal A, Pullmann R Jr, Yang X, Galban S, Srikantan S, Martindale JL, Blethrow J, Shokat KM, Gorospe M (2008a) Nuclear HuR accumulation through phosphorylation by Cdk1. Genes Dev 22:1804–1815

    Article  PubMed  CAS  Google Scholar 

  • Kim HH, Yang X, Kuwano Y, Gorospe M (2008b) Modification at HuR(S242) alters HuR localization and proliferative influence. Cell Cycle 21:3371–3377

    Article  Google Scholar 

  • Li H, Park S, Kilburn B, Jelinek MA, Henschen-Edman A, Aswad DW, Stallcup MR, Laird-Offringa IA (2002) Lipopolysaccharide-induced methylation of HuR, an mRNA-stabilizing protein, by CARM1. Coactivator-associated arginine methyltransferase. J Biol Chem 277:44623–44630

    Article  PubMed  CAS  Google Scholar 

  • Li H, Shi H, Wang H, Zhu Z, Li X, Gao Y, Cui Y, Niu L, Teng M (2010) Crystal structure of the two N-terminal RRM domains of Pub1 and the poly(U)-binding properties of Pub1. J Struct Biol 171:291–297

    Article  PubMed  CAS  Google Scholar 

  • Ma WJ, Cheng S, Campbell C, Wright A, Furneaux H (1996) Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J Biol Chem 271:8144–8151

    Article  PubMed  CAS  Google Scholar 

  • Mazroui R, Di Marco S, Clair E, von Roretz C, Tenenbaum SA, Keene JD, Saleh M, Gallouzi IE (2008) Caspase-mediated cleavage of HuR in the cytoplasm contributes to pp 32/PHAP-I regulation of apoptosis. J Cell Biol 180:113–127

    Article  PubMed  CAS  Google Scholar 

  • Meisner NC, Hintersteiner M, Mueller K, Bauer R, Seifert JM, Naegeli HU, Ottl J, Oberer L, Guenat C, Moss S, Harrer N, Woisetschlaeger M, Buehler C, Uhl V, Auer M (2007) Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR. Nat Chem Biol 3:508–515

    Article  PubMed  CAS  Google Scholar 

  • Niesen FH, Berglund H, Vedadi M (2007) The use of Differential Scanning Fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221

    Article  PubMed  CAS  Google Scholar 

  • Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  PubMed  CAS  Google Scholar 

  • Privalov PL (1979) Stability of proteins: small globular proteins. Adv Protein Chem 33:167–241

    Article  PubMed  CAS  Google Scholar 

  • Santoro MM, Bolen DW (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethane sulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27:8063–8068

    Article  PubMed  CAS  Google Scholar 

  • Sengupta S, Jang BC, Wu MT, Paik JH, Furneaux H, Hla T (2003) The RNA-binding protein HuR regulates the expression of cyclooxygenase-2. J Biol Chem 278:25227–25233

    Article  PubMed  CAS  Google Scholar 

  • Sreerama N, Woody RW (2000) Estimation of protein secondary structure from Circular Dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260

    Article  PubMed  CAS  Google Scholar 

  • Vitali F, Henning A, Oberstrass FC, Hargous Y, Auweter SD, Erat M, Allain FH (2006) Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling. EMBO J 25:150–162

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Tanaka Hall TM (2001) Structural basis for recognition of AU-rich element RNA by the HuD protein. Nat Struct Biol 8:141–145

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Dr. M. Gorospe (NIH, Baltimore, USA) and Dr. J. A. Steitz (Yale University, New Haven, USA) for providing the HuR vectors. We are grateful to Prof. Miguel A. De la Rosa and Dr. Antonio Díaz-Quintana for critical reading of the manuscript. For financial support we thank the Andalusian Government (P07-CVI-02896) and the Spanish Scientific Council Fellowship (JAEpre_08_00375).

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Authors and Affiliations

  1. Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja, Universidad de Sevilla-CSIC, Americo Vespucio 49, 41092, Sevilla, Spain

    Rafael Manfred Scheiba, Ángeles Aroca & Irene Díaz-Moreno

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  1. Rafael Manfred Scheiba
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  2. Ángeles Aroca
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  3. Irene Díaz-Moreno
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Correspondence to Irene Díaz-Moreno.

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Scheiba, R.M., Aroca, Á. & Díaz-Moreno, I. HuR thermal stability is dependent on domain binding and upon phosphorylation. Eur Biophys J 41, 597–605 (2012). https://doi.org/10.1007/s00249-012-0827-3

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  • DOI: https://doi.org/10.1007/s00249-012-0827-3

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