title: Mutational analysis of the DEAD-box RNA helicase eIF4AII characterizes its interaction with transformation suppressor Pdcd4 and eIF4GI.
abstract: Eukaryotic initiation factor (eIF) 4A unwinds secondary and tertiary structures in the 5'-untranslated region of mRNA, permitting translation initiation. Programmed cell death 4 (Pdcd4) is a novel transformation suppressor and eIF4A-binding partner that inhibits eIF4A helicase activity and translation. To elucidate the regions of eIF4A that are functionally significant in binding to Pdcd4, we generated point mutations of eIF4A. Two-hybrid analysis revealed that five eIF4A mutants completely lost binding to Pdcd4 while four eIF4A mutants retained wild-type levels of binding. The residues that, when mutated, inactivated Pdcd4 binding specified ATP binding, ATP hydrolysis, or RNA binding. With the exception of the Q-motif mutant eIF4AP56L, the eIF4A mutants inactivated for Pdcd4 binding were inactivated for binding to eIF4G (GM, GC, or both) and for enhancing translation. Several eIF4A mutants showing wild-type level binding to Pdcd4 were also inactivated for binding to eIF4G and for enhancing translation. Thus, significant dissociation of eIF4A's Pdcd4- and eIF4G-binding regions appears to occur. Because three of the four eIF4A mutants that retained Pdcd4 binding also suppressed translation activity in a dominant-negative manner, the structure that defines the Pdcd4-binding domain of eIF4A may be necessary but is insufficient for translation. A structural homology model of eIF4A shows regions important for binding to Pdcd4 and/or eIF4G lying on the perimeters of the hinge area of eIF4A. A competition experiment revealed that Pdcd4 competes with C-terminal eIF4G for binding to eIF4A. In summary, the Pdcd4-binding domains on eIF4A impact both binding to eIF4G and translation initiation in cells.
authors.affiliation: Laboratory of Cancer Prevention, Bldg. 576, Rm. 101, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Halina
authors.initials: H
authors.lastname: Zakowicz
authors.forename: Hsin-Sheng
authors.initials: HS
authors.lastname: Yang
authors.forename: Cristi
authors.initials: C
authors.lastname: Stark
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Nicole
authors.initials: N
authors.lastname: Laronde-Leblanc
authors.forename: Nancy H
authors.initials: NH
authors.lastname: Colburn
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis
chemical_list: D017931:DNA Primers; C113920:EIF4G1 protein, human; D039603:Eukaryotic Initiation Factor-4G; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039601:Eukaryotic Initiation Factor-4A