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The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that
Hsin-Sheng Yang1, Aaron P Jansen, Anton A Komar
1Gene Regulation Section, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA. hyang@ncifcrf.gov
Abstract:
Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5' mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4(D418A), a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4(D418A) bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.
Insights
Programmed cell death 4 (Pdcd4) suppresses tumor growth by inhibiting translation initiation factor 4A (eIF4A) helicase activity. This interaction blocks cap-dependent translation and AP-1 activation, crucial for neoplastic transformation.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Programmed cell death 4 (Pdcd4) is identified as a novel tumor suppressor.
- Pdcd4 inhibits neoplastic transformation and AP-1-dependent transcription.
- The precise molecular mechanisms underlying Pdcd4's tumor-suppressive functions are under investigation.
Purpose of the Study:
- To elucidate the molecular interactions through which Pdcd4 exerts its transformation-suppressing effects.
- To investigate the role of Pdcd4 in regulating translation initiation.
- To determine the link between Pdcd4's interaction with translation factors and its effect on AP-1 activity.
Main Methods:
- Yeast two-hybrid analysis to identify Pdcd4 interacting proteins.
- Immunofluorescent confocal microscopy to determine subcellular localization.
- In vitro helicase activity assays using recombinant proteins.
- In vivo translation assays (cap-dependent and IRES-dependent).
- Analysis of protein-protein interactions using mutant Pdcd4 and eIF4G fragments.
Main Results:
- Pdcd4 was found to associate with eukaryotic translation initiation factors 4A (eIF4A) isoforms (eIF4AI and eIF4AII) and colocalizes with eIF4A in the cytoplasm.
- Recombinant Pdcd4 inhibited the helicase activity of eIF4A and eIF4F, and specifically blocked cap-dependent translation but not IRES-dependent translation.
- A mutant Pdcd4 (D418A) unable to bind eIF4A lost its inhibitory effects on translation and AP-1 transactivation.
- Pdcd4 was shown to interfere with the binding of eIF4A to the C-terminal region of eIF4G, impacting translation initiation complex formation.
Conclusions:
- Pdcd4 suppresses neoplastic transformation by inhibiting eIF4A helicase activity, thereby blocking cap-dependent translation.
- Pdcd4's interaction with eIF4A and its subsequent inhibition of translation are essential for its tumor-suppressive activity and AP-1 transrepression.
- These findings reveal a critical mechanism by which Pdcd4 functions as a tumor suppressor through modulation of protein translation.