[en] Background
Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown.
Methods/Findings
In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values.
Conclusions/Significance
Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.
Research center :
Laboratoire de Chimie Biologique Structurale, ULiège, Belgium Centre de Biophysique Moléculaire, Orléans, France CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Tavel, Laurette ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique structurale
Jaquillard, Lucie; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Karsisiotis, Andreas; Centre National de la Recherche Scientifique - CNRS > CBM-Orléans
Saab, Fabienne; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Jouvensal, Laurence; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Brans, Alain ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Delmas, Agnès; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Schoentgen, Françoise; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Cadene, Martine; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Damblon, Christian ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique structurale
Language :
English
Title :
Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry
Publication date :
27 April 2012
Journal title :
PLoS ONE
eISSN :
1932-6203
Publisher :
Public Library of Science, San Franscisco, United States - California
ANR - Agence Nationale de la Recherche [FR] LNCC - Ligue Nationale Contre le Cancer [FR] Cancéropôle du Grand Ouest [FR] CNRS - Centre National de la Recherche Scientifique [FR] ULiège. Patrimoine - Université de Liège. Patrimoine [BE] EU - European Union [BE]
Funding number :
ANR-08-BLAN-0033; EU-NMR program (RII3-026145)
Funding text :
Régions Bretagne, Centre, Pays de la Loire et Poitou-Charentes (AO2007-2010)
Yeung K, Seitz T, Li S, Janosch P, McFerran B, et al. (1999) Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 401: 173-177.
Yeung KC, Rose DW, Dhillon AS, Yaros D, Gustafsson M, et al. (2001) Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation. Mol Cell Biol 21: 7207-7217.
Klysik J, Theroux SJ, Sedivy JM, Moffit JS, Boekelheide K, (2008) Signaling crossroads: the function of Raf kinase inhibitory protein in cancer, the central nervous system and reproduction. Cell Signal 20: 1-9.
Lee HC, Tian B, Sedivy JM, Wands JR, Kim M, (2006) Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells. Gastroenterology 131: 1208-1217.
Sagisaka T, Matsukawa N, Toyoda T, Uematsu N, Kanamori T, et al. (2010) Directed neural lineage differentiation of adult hippocampal progenitor cells via modulation of hippocampal cholinergic neurostimulating peptide precursor expression. Brain Res 1327: 107-117.
Zhu S, Mc Henry KT, Lane WS, Fenteany G, (2005) A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration. Chem Biol 12: 981-991.
Dahl A, Eriksson PS, Persson AI, Karlsson G, Davidsson P, et al. (2003) Proteome analysis of conditioned medium from cultured adult hippocampal progenitors. Rapid Commun Mass Spectrom 17: 2195-2202.
Odabaei G, Chatterjee D, Jazirehi AR, Goodglick L, Yeung K, et al. (2004) Raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis. Adv Cancer Res 91: 169-200.
Kolch W, (2000) Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 351: 289-305.
Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, et al. (2000) Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. Mol Cell Biol 20: 3079-3085.
Saab F, Routier S, Mérour J-Y, Bénéteau V, Schoentgen F, (2010) Comparison of the efficiencies of two TR-FRET methods to detect in vitro natural and synthesized inhibitors of the Raf/MEK/ERK signaling pathway. Int J High Throughput Screen 1: 81-98.
Trakul N, Menard RE, Schade GR, Qian Z, Rosner MR, (2005) Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation. J Biol Chem 280: 24931-24940.
Corbit KC, Trakul N, Eves EM, Diaz B, Marshall M, et al. (2003) Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J Biol Chem 278: 13061-13068.
Lorenz K, Lohse MJ, Quitterer U, (2003) Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 426: 574-579.
Keller ET, (2004) Metastasis suppressor genes: a role for raf kinase inhibitor protein (RKIP). Anticancer Drugs 15: 663-669.
Fu Z, Smith PC, Zhang L, Rubin MA, Dunn RL, et al. (2003) Effects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis. J Natl Cancer Inst 95: 878-889.
Keller ET, Fu Z, Yeung K, Brennan M, (2004) Raf kinase inhibitor protein: a prostate cancer metastasis suppressor gene. Cancer Lett 207: 131-137.
Hagan S, Al-Mulla F, Mallon E, Oien K, Ferrier R, et al. (2005) Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis. Clin Cancer Res 11: 7392-7397.
Dangi-Garimella S, Yun J, Eves EM, Newman M, Erkeland SJ, et al. (2009) Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J 28: 347-358.
Chatterjee D, Bai Y, Wang Z, Beach S, Mott S, et al. (2004) RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis. J Biol Chem 279: 17515-17523.
Maki M, Matsukawa N, Yuasa H, Otsuka Y, Yamamoto T, et al. (2002) Decreased expression of hippocampal cholinergic neurostimulating peptide precursor protein mRNA in the hippocampus in Alzheimer disease. J Neuropathol Exp Neurol 61: 176-185.
Hickox DM, Gibbs G, Morrison JR, Sebire K, Edgar K, et al. (2002) Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2. Biol Reprod 67: 917-927.
Moffit JS, Boekelheide K, Sedivy JM, Klysik J, (2007) Mice lacking Raf kinase inhibitor protein-1 (RKIP-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury. J Androl 28: 883-890.
Hahm JR, Ahn JS, Noh HS, Baek SM, Ha JH, et al. (2010) Comparative analysis of fat and muscle proteins in fenofibrate-fed type II diabetic OLETF rats: the fenofibrate-dependent expression of PEBP or C11orf59 protein. BMB Rep 43: 337-343.
Serre L, Vallee B, Bureaud N, Schoentgen F, Zelwer C, (1998) Crystal structure of the phosphatidylethanolamine-binding protein from bovine brain: a novel structural class of phospholipid-binding proteins. Structure 6: 1255-1265.
Simister PC, Burton NM, Brady RL, (2011) Phosphotyrosine recognition by the Raf kinase inhibition protein. Forum Immunopath Dis Ther 2: 59-70.
Banfield MJ, Barker JJ, Perry AC, Brady RL, (1998) Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction. Structure 6: 1245-1254.
Bucquoy S, Jolles P, Schoentgen F, (1994) Relationships between molecular interactions (nucleotides, lipids and proteins) and structural features of the bovine brain 21-kDa protein. Eur J Biochem 225: 1203-1210.
Atmanene C, Laux A, Glattard E, Muller A, Schoentgen F, et al. (2009) Characterization of human and bovine phosphatidylethanolamine-binding protein (PEBP/RKIP) interactions with morphine and morphine-glucuronides determined by noncovalent mass spectrometry. Med Sci Monit 15: 178-187.
Granovsky AE, Clark MC, McElheny D, Heil G, Hong J, et al. (2009) Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism. Mol Cell Biol 29: 1306-1320.
Shemon AN, Heil GL, Granovsky AE, Clark MC, McElheny D, et al. (2010) Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands. PLoS One 5: e10479.
Shemon AN, Eves EM, Clark MC, Heil G, Granovsky A, et al. (2009) Raf kinase inhibitory protein protects cells against locostatin-mediated inhibition of migration. PLoS One 4: e6028.
Dadvar P, Kovanich D, Folkers GE, Rumpel K, Raijmakers R, et al. (2009) Phosphatidylethanolamine-binding proteins, including RKIP, exhibit affinity for phosphodiesterase-5 inhibitors. ChemBioChem 10: 2654-2662.
Park S, Rath O, Beach S, Xiang X, Kelly SM, et al. (2006) Regulation of RKIP binding to the N-region of the Raf-1 kinase. FEBS Lett 580: 6405-6412.
Rath O, Park S, Tang HH, Banfield MJ, Brady RL, et al. (2008) The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK. Cell Signal 20: 935-941.
Zuiderweg ERP, (2002) Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41: 1-7.
Farmer BT 2nd, Venters RA, (1996) Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins. J Biomol NMR 7: 59-71.
Traut TW, (1994) Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140: 1-22.
Kroslak T, Koch T, Kahl E, Höllt V, (2001) Human Phosphatidylethanolamine-binding Protein Facilitates Heterotrimeric G Protein-dependent Signaling. J Biol Chem 276: 39772-39778.
Ribas C, Penela P, Murga C, Salcedo A, García-Hoz C, et al. (2007) The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling. Biochim Biophys Acta 1768: 913-922.
Marley J, Lu M, Bracken C, (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20: 71-75.
Limbach PA, Crain PF, McCloskey JA, (1995) Molecular mass measurement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry. J Am Soc Mass Spectrom 6: 27-39.
Jaquillard L, Saab F, Schoentgen F, Cadene M, Improved accuracy of low-affinity protein-ligand equilibrium dissociation constants determined by ESI-MS. J Am Soc Mass Spec (in press).
Yi C, Peng Y, Guo C, Lin D, (2011) 1H, 13C, 15N backbone and side-chain resonance assignments of the human Raf-1 kinase inhibitor protein. Biomol NMR Assign 5: 63-66.
Salzmann M, Wider G, Pervushin K, Senn H, Wuthrich K, (1999) TROSY-type triple-resonance experiments for sequential NMR assignments of large proteins. J Am Chem Soc 121: 844-848.
Meyer B, Peters T, (2003) NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew Chem Int Ed 42: 864-890.
