This study demonstrates that the transformation suppressor Pdcd4 physically associates with the translation initiation factor eIF4A, resulting in inhibition of helicase activity (Fig. ) and translation (Fig. and ). The inhibition of translation requires Pdcd4 binding to eIF4A, as Pdcd4D418A
, a mutant inactivated for binding to eIF4A, has no effect on translation (Fig. ). These findings are in agreement with previous observations that mutational inhibition of the helicase and/or ATPase activity of eIF4A inhibits translation (35
). Therefore, Pdcd4 is not only a novel eIF4A binding protein, it is the first example of a protein that inhibits translation through inactivation of eIF4A activity.
Wild-type Pdcd4 but not the Pdcd4D418A
mutant inhibited AP-1-dependent transcription (Fig. ). Inhibition of AP-1 is sufficient to inhibit tumor promotion, both in the JB6 cell model and in mouse skin carcinogenesis in vivo (9
). The lack of eIF4A binding by the mutant Pdcd4 appears to disable the inhibition of translation and consequently to disable the transrepression of AP-1 that contributes to Pdcd4's suppression of transformation. Pdcd4D418A
is not inactivated for all its activities, as it retains the ability to bind the middle domain of eIF4G (Fig. ). Taken together, these results suggest that the loss of binding to eIF4A results in the loss of a transformation-relevant function.
How does Pdcd4 inhibit translation? A model to explain the mechanism underlying the translational inhibition by Pdcd4 must take into account the findings that (i) Pdcd4 inhibits the helicase activity of eIF4A, (ii) Pdcd4 blocks eIF4A binding to the C-terminal one-third of eIF4G, (iii) Pdcd4 binds to the middle one-third of eIF4G independently of eIF4A binding, and (iv) the Pdcd4 domains for binding to eIF4A and the middle domain of eIF4G are distinct (Fig. ). The C-terminal domain of eIF4G is a regulatory domain for translation, and its binding to eIF4A has been shown to greatly enhance translation (32
). Therefore, prevention of eIF4A binding to the C-terminal domain by Pdcd4 (Fig. ) contributes to inhibition of translation.
FIG. 8. Model of how Pdcd4 inhibits translation. (A) A sandwich model of eIF4A binding to eIF4G is shown (32). The eIF4A molecule binds to the middle and C-terminal one-third of eIF4G. (B) Model for Pdcd4 inhibition of translation. Pdcd4 inhibits the helicase (more ...)
eIF4A exchanges between free eIF4A and eIF4A bound in the eIF4F complex (36
). Biochemical and kinetic studies have shown that eIF4A may proceed through two to three association-dissociation cycles at the beginning of translation initiation to unwind double-stranded RNA (42
). Blocking this process inhibits translation. Pdcd4 binding to the middle one-third of eIF4G independent of eIF4A (Fig. ) suggests that eIF4A may be trapped by Pdcd4 on the middle domain of eIF4G, thus blocking the association-dissociation cycle of eIF4A through eIF4F. The observation (Fig. ) that addition of eIF4A to the rabbit reticulocyte lysate does not relieve inhibition of translation by Pdcd4 further supports this model. eIF4A, when trapped in a Pdcd4-inactivated form bound to eIF4G, would not be displaced by added free eIF4A. Further testing of this model will be important.
Our data do not indicate the stoichiometry for the ratio of eIF4G and eIF4A. One molecule of eIF4A may associate with one molecule of eIF4G to form a “sandwich,” as proposed by Morino et al. (32
) (Fig. ), or two molecules of eIF4A may associate with one molecule of eIF4G. Recently, two groups have proposed a stoichiometry for eIF4G to eIF4A. Korneeva et al. (19
), using surface plasmon resonance techniques and recombinant eIF4G proteins, showed a 1:2 ratio for eIF4G to eIF4A. On the other hand, Li et al. (24
), using immunoprecipitation of endogenous and tagged eIF4A, concluded that 1:1 was the ratio for eIF4G to eIF4A.
Two human Pdcd4 homologs, H731-L and H731, are 96% and 93% identical, respectively, in amino acid sequence to mouse Pdcd4. H731-L and H731 are alternative transcripts of the same gene. H731 lacks 11 amino acids in the N-terminal region that are present in H731-L and Pdcd4. H731 was identified and isolated with the Pr-28 antibody, which recognizes a nuclear antigen in proliferating cells (31
). Recent studies by Yoshinaga et al. (52
) used the human H731 antibody to determine the expression and localization of H731 in several cell lines and tissues. H731 was abundantly expressed and localized in the cytoplasm of cancer cells. In normal cells, however, H731 was localized in the nuclei. These observations are in disagreement with our observations of Pdcd4 localization and differential expression. First, the level of Pdcd4 expression in the (less progressed) JB6 P− cells was about 8- to 10-fold higher than that in JB6 P+ cells (49
), and transformed JB6 cells (unpublished data). Second, the results of immunofluorescent confocal microscopy analysis indicate that Pdcd4 is colocalized with eIF4A in the cytoplasm (Fig. ). In addition, the immunoprecipitation and GST pulldowns showing that Pdcd4 physically interacts with eIF4A (Fig. ) and eIF4G (Fig. ) provide further support for cytoplasmic localization of Pdcd4.
It is unknown whether the two highly identical proteins, Pdcd4 and H731L, localize differently. The nuclear localization of H731 might be attributed to differential tissue specificity or to the use of different antibodies. It is noteworthy that the Pdcd4 antibody used in the present studies shows high specificity (49
), whereas the H731 antibody recognizes several proteins ranging in molecular mass from 51 to 64 kDa (5
Comparison of the Pdcd4 protein sequence with proteins in the GenBank database reveals two α-helical MA-3 domains that are located from amino acids 163 to 284 and amino acids 326 to 449 (Fig. ). The MA-3 domain extends over approximately 120 amino acids with 80 to 85% consensus secondary structure; although there are many conserved amino acids in the MA-3 domain, no specific consensus sequence has been reported (2
). In human and mouse eIF4G, the MA-3 domain is located within the second eIF4A binding domain (amino acids 1201 to 1441) (32
) (Fig. ), implying that the MA-3 domain may play an essential role in binding eIF4A. The finding that Pdcd4 prevents eIF4A from binding to the C-terminal one-third of eIF4G (Fig. ) supports this hypothesis. Indeed, deletion or mutation of either MA-3 domain in the Pdcd4 protein dramatically inactivated the binding to eIF4A in a mammalian two-hybrid assay (Fig. and unpublished data), indicating that the MA-3 domain is required for binding eIF4A.
Several tumors and tumor cell lines show elevated levels of translation initiation factors such as eIF4E (7
), eIF4A (10
), and eIF4G (3
). Overexpression of eIF4E (22
) or eIF4G (11
) resulted in transformation of NIH 3T3 cells, suggesting that translation factors may function as oncogenes. Therefore, downregulation or inactivation of translation factors may suppress transformation. How does Pdcd4 suppress tetradecanoyl phorbol acetate-induced neoplastic transformation in JB6 cells? A small number of molecular events are known to be required for tumor promoter-induced transformation of JB6 P+ cells and tumorigenesis in vivo. Among these required molecular events are activation of transcription factors AP-1, NF-κB, and serum response element as well as ornithine decarboxylase activation (15
). Of these events, wild-type Pdcd4 inhibits only AP-1 activation (49
The mRNAs that are translational targets of Pdcd4 are unknown. One possibility is that Pdcd4 inhibits the translation of AP-1 proteins or of enzymes or coactivators required for their activation. Mitogen treatment of cells greatly stimulates the translation of a group of so-called “translationally repressed” mRNAs (4
). This group of mRNAs are often involved in cell proliferation. Included are mRNAs for growth factors, growth promotion genes, and proto-oncogenes (7
) that contain long GC-rich 5′ untranslated regions having the potential to form stable secondary structure(s) at the 5′ end. Translation of this group of mRNAs may be inefficient and highly dependent on the eIF4A helicase activity (20
). Inhibiting or decreasing eIF4A helicase activity would be expected to limit translation of the translationally repressed mRNAs resulting in the suppression of cell growth or transformation. For instance, mutation of eIF4A in Schizosaccharomyces pombe
inhibited translation of cdc25
but not of cdc2
and arrested cells in the G2
phase. Deletion of the 5′ untranslated region of cdc25
In a related study, we found an inverse relationship between Pdcd4 expression and proliferation within a number of tissues, but especially the cervical epithelia of mice during estrus, which includes a cyclical period of actively proliferating cervical epithelium (A. Jansen, unpublished data). Recent studies by Svitkin et al. (47
), with mRNAs varying in stability of secondary structure and eIF4A mutants, showed that the more stable the secondary structure within the 5′ untranslated region of mRNA, the lower the efficiency of translation. These results further support the idea that the requirement for eIF4A in translation is proportional to the stability of the secondary structure within the 5′ untranslated region.
In summary, suppression of eIF4A helicase activity and/or interference with eIF4A binding to eIF4G by Pdcd4 may suppress the translation of a set of mRNAs that limits the activation of AP-1 or other molecular events required for transformation in JB6 cells. Identification of the genes that are most sensitive to translational inhibition by Pdcd4 will be important.