PDCD4 inhibits translation initiation by binding to eIF4A using both its MA3 domains

Abstract

Programmed Cell Death 4 (PDCD4) is a protein known to bind eukaryotic initiation factor 4A (eIF4A), inhibit translation initiation, and act as a tumor suppressor. PDCD4 contains two C-terminal MA3 domains, which are thought to be responsible for its inhibitory function. Here, we analyze the structures and inhibitory functions of these two PDCD4 MA3 domains by x-ray crystallography, NMR, and surface plasmon resonance. We show that both MA3 domains are structurally and functionally very similar and bind specifically to the eIF4A N-terminal domain (eIF4A-NTD) using similar binding interfaces. We found that the PDCD4 MA3 domains compete with the eIF4G MA3 domain and RNA for eIF4A binding. Our data provide evidence that PDCD4 inhibits translation initiation by displacing eIF4G and RNA from eIF4A. The PDCD4 MA3 domains act synergistically to form a tighter and more stable complex with eIF4A, which explains the need for two tandem MA3 domains.apoptosis ͉ eIF4G ͉ protein ͉ NMR ͉ x-ray crystallography P DCD4 is a tumor-suppressor protein that is up-regulated on induction of apoptosis (1) and down-regulated in certain aggressive tumors (2). PDCD4 is controlled by protein kinase S6K1 and the ubiquitin ligase SCF ␤TRCP , and its degradation is necessary for efficient protein translation in vivo, which is a prerequisite for cell growth and, consequently, for cell proliferation (3).PDCD4 is known to bind two eukaryotic translation initiation factors eIF4A and eIF4G (4-6). eIF4A is an RNA helicase that works as a subunit of eIF4F, a complex composed of eIF4G and eIF4E. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G (7,8). eIF4G has two independent binding sites for eIF4A (9), one in the conserved middle domain (eIF4G-m, HEAT1/MIF4G) ( Fig. 1), and the other in the adjacent second HEAT domain (eIF4G-MA3, HEAT2/MA3) (reviewed in ref. 10). NMR binding studies have shown that eIF4G-m interacts mainly with the C-terminal domain of eIF4A (eIF4A-CTD) (11), whereas eIF4G-MA3 binds to the N-terminal domain of eIF4A (eIF4A-NTD) and only weakly to eIF4A-CTD (A.M., C.S., K.A.E., and G.W., unpublished work). Mutation and deletion analysis indicates that the interaction of eIF4A with eIF4G-m is necessary for translation, whereas the interaction of eIF4A with eIF4G-c (eIF4G-MA3ϩHEAT3 domain) plays a modulatory role (12).PDCD4 contains two MA3 domains after an N-terminal segment of little known function (1). MA3 is a well conserved ␣-helical motif with typically 3-5 helical hairpins and is a subtype of HEAT domains. A single MA3 domain is found in eIF4G-c (eIF4G-MA3, Fig. 1), which has been reported to also bind eIF4A (10, 13). Recently, crystal and solution structures of mouse PDCD4's Cterminal MA3 domain (MA3-c) were reported (14, 15). MA3-c was shown to bind eIF4A, compete with eIF4G-c, and was sufficient to inhibit translation initiation. However, some questions remained. First, the function of the N-terminal MA3 domain (MA3-m) was unclear. Mutations of conserved amino acid residues in either MA3-c or ...

From unwinding to clamping — the DEAD box RNA helicase family

“…b | Schematic structural representation of the EJC core factors, MLN51, MAGOH and Y14, and ATP bound to the two helicase core domains of eIF4AIII 15 tumour suppressor protein, inhibits translation by binding to eIF4A 102 . One PDCD4 mole cule binds two eIF4A protomers, inhibiting enzymatic activity by trapping the DEAD box protomers in an inactive conformatio n and preventing their binding to eIF4G 103,104 (FIG. 4).…”

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation

“…As a translation regulator, Pdcd4 interacts with the eukaryotic translation initiation factor eIF4A, a RNA helicase that catalyzes the unwinding of mRNA secondary structures in 5 0 -untranslated regions (UTRs) (Yang et al, 2003a(Yang et al, , 2004. Binding of Pdcd4 to eIF4A is mediated by the MA-3 domains, whose structure and complex formation with eIF4A have been analyzed in detail (LaRondeLeBlanc et al, 2007;Waters et al, 2007Waters et al, , 2011Suzuki et al, 2008;Chang et al, 2009;Loh et al, 2009). Because binding of Pdcd4 to eIF4A inhibits the helicase activity of eIF4A (Yang et al, 2003a;Yang et al, 2004), it is believed that Pdcd4 functions as a suppressor of cap-dependent translation of mRNAs with structured 5 0 -UTRs.…”

Expression patterns of the tumor suppressor PDCD4 and correlation with β-catenin expression in gastric cancers

“…PDCD4/ Pdcd4 protein associates with eIF4A, which binds to eIF4G in the initiation complex eIF4F and inhibits the RNA helicase activity of eIF4A, thereby inhibiting cap-dependent translation (24)(25)(26)(27).…”