title: Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP.
abstract: Eukaryotic initiation factor 4E (eIF4E) binds the 5'-cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [gamma-32P]8-azidoguanosine 5'-triphosphate (8-N3GTP) demonstrated cross-linking at Lys-119 in the S4-H2 loop which is distant from the m7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951-961; Friedland et al. (1997) Protein Sci. 6, 125-131]. Modeling studies indicate that 8-N3GTP cross-linked with Lys-119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4-H2 loop produced proteins with a 5-10-fold higher affinity for m7GTP than wild-type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5'-ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.
authors.affiliation: Department of Medicine, Genetics Program of the Winship Cancer Center, Emory University School of Medicine, 165 Michael Street, Room 201, Atlanta, GA 30322, USA.
authors.forename: Taly
authors.initials: T
authors.lastname: Spivak-Kroizman
authors.forename: Diana E
authors.initials: DE
authors.lastname: Friedland
authors.forename: Christine
authors.initials: C
authors.lastname: De Staercke
authors.forename: Kim M
authors.initials: KM
authors.lastname: Gernert
authors.forename: Dixie J
authors.initials: DJ
authors.lastname: Goss
authors.forename: Curt H
authors.initials: CH
authors.lastname: Hagedorn
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000071156:Promyelocytic Leukemia Protein; D011487:Protein Conformation; D012314:RNA Cap Analogs / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010448:Peptide Initiation Factors; D000071156:Promyelocytic Leukemia Protein; D012314:RNA Cap Analogs; D012315:RNA Caps; D011994:Recombinant Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; C017516:7-methylguanosine triphosphate