A Novel Function of the MA-3 Domains in Transformation and Translation Suppressor Pdcd4 Is Essential for Its Binding to Eukaryotic Translation Initiation Factor 4A

ABSTRACT Αn α-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4E249K, Pdcd4D253A, Pdcd4D414K, Pdcd4D418A, Pdcd4E249K,D414K, and Pdcd4D253A,D418A) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4D253A and Pdcd4D253A,D418A do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4D253A, Pdcd4D418A, or Pdcd4D235A,D418A. Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

[1]  M. Kozak Effects of long 5' leader sequences on initiation by eukaryotic ribosomes in vitro. , 1991, Gene expression.

[2]  N. Gray,et al.  Regulation of protein synthesis by mRNA structure , 1994, Molecular Biology Reports.

[3]  S. Schreiber,et al.  A Signaling Pathway to Translational Control , 1996, Cell.

[4]  P. Slonimski,et al.  Sequence of the genes TIF1 and TIF2 from Saccharomyces cerevisiae coding for a translation initiation factor. , 1988, Nucleic acids research.

[5]  K. Anderson,et al.  Identification of genes regulated by Dexamethasone in multiple myeloma cells using oligonucleotide arrays , 2002, Oncogene.

[6]  C. Norbury,et al.  Translation initiation and its deregulation during tumorigenesis , 2002, British Journal of Cancer.

[7]  N. Colburn,et al.  A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-κB or ODC transactivation , 2001, Oncogene.

[8]  N. Sonenberg,et al.  Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F , 1990, Molecular and cellular biology.

[9]  L. Azzoni,et al.  Differential transcriptional regulation of CD161 and a novel gene, 197/15a, by IL-2, IL-15, and IL-12 in NK and T cells. , 1998, Journal of immunology.

[10]  R. Abramson,et al.  The ATP-dependent interaction of eukaryotic initiation factors with mRNA. , 1987, The Journal of biological chemistry.

[11]  N. Sonenberg,et al.  Dominant negative mutants of mammalian translation initiation factor eIF‐4A define a critical role for eIF‐4F in cap‐dependent and cap‐independent initiation of translation. , 1994, The EMBO journal.

[12]  Jennifer L. Knies,et al.  Pdcd4 suppresses tumor phenotype in JB6 cells by inhibiting AP-1 transactivation , 2003, Oncogene.

[13]  M. Niepel,et al.  Secondary structure in the 5′‐leader or 3′‐untranslated region reduces protein yield but does not affect the functional interaction between the 5′‐cap and the poly(A) tail , 1999, FEBS letters.

[14]  Z. Dong,et al.  Progressive elevation of ap-1 activity during preneoplastic-to-neoplastic progression as modeled in mouse jb6 cell variants. , 1995, International journal of oncology.

[15]  N. Sonenberg,et al.  The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure. , 2001, RNA.

[16]  N. Sonenberg,et al.  Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF‐4A. , 1992, The EMBO journal.

[17]  K. Klempnauer,et al.  The transformation suppressor protein Pdcd4 shuttles between nucleus and cytoplasm and binds RNA , 2003, Oncogene.

[18]  W. Merrick,et al.  Modulation of the Helicase Activity of eIF4A by eIF4B, eIF4H, and eIF4F* , 2001, The Journal of Biological Chemistry.

[19]  A. De Benedetti,et al.  eIF4E expression in tumors: its possible role in progression of malignancies. , 1999, The international journal of biochemistry & cell biology.

[20]  E. Koonin,et al.  Eukaryote-specific domains in translation initiation factors: implications for translation regulation and evolution of the translation system. , 2000, Genome research.

[21]  Z. Dong,et al.  Transformation reversion induced in JB6 RT101 cells by AP-1 inhibitors. , 1995, Carcinogenesis.

[22]  N. Sonenberg,et al.  The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis , 1993, Molecular and cellular biology.

[23]  C. Proud Translation. Turned on by insulin. , 1994, Nature.

[24]  T. Pestova,et al.  The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection. , 2002, Genes & development.

[25]  N. Sonenberg,et al.  The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation , 2003, Molecular and Cellular Biology.

[26]  A. Komar,et al.  eIF4A: the godfather of the DEAD box helicases. , 2002, Progress in nucleic acid research and molecular biology.

[27]  C. Hellen,et al.  Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry , 1996, Molecular and cellular biology.

[28]  N. Sonenberg,et al.  Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A , 1997, Molecular and cellular biology.

[29]  C. Proud Turned on by insulin , 1994, Nature.

[30]  M. Kozak,et al.  How do eucaryotic ribosomes select initiation regions in messenger RNA? , 1978, Cell.

[31]  T. Honjo,et al.  Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. , 1995, Gene.

[32]  M. Hentze,et al.  Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent. , 1998, RNA.

[33]  M. Kozak,et al.  Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs , 1989, Molecular and cellular biology.

[34]  H. Kizaki,et al.  Molecular cloning of the genes suppressed in RVC lymphoma cells by topoisomerase inhibitors. , 1996, Biochemical and biophysical research communications.

[35]  C. Hellen,et al.  Physical Association of Eukaryotic Initiation Factor 4G (eIF4G) with eIF4A Strongly Enhances Binding of eIF4G to the Internal Ribosomal Entry Site of Encephalomyocarditis Virus and Is Required for Internal Initiation of Translation , 2000, Molecular and Cellular Biology.

[36]  N. Sonenberg,et al.  A new translational regulator with homology to eukaryotic translation initiation factor 4G , 1997, The EMBO journal.

[37]  H. Trachsel,et al.  The mouse protein synthesis initiation factor 4A gene family includes two related functional genes which are differentially expressed. , 1988, The EMBO journal.

[38]  N. Colburn,et al.  Activator protein 1 (AP-1)– and nuclear factor κB (NF-κB)–dependent transcriptional events in carcinogenesis , 2000 .

[39]  M. Patel,et al.  Human dihydrolipoamide dehydrogenase gene transcription is mediated by cAMP-response element-like site and TACGAC direct repeat. , 2001, The international journal of biochemistry & cell biology.

[40]  C. Ponting Novel eIF4G domain homologues linking mRNA translation with nonsense-mediated mRNA decay. , 2000, Trends in biochemical sciences.

[41]  Iver Petersen,et al.  Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis , 2003, The Journal of pathology.

[42]  D. Mckay,et al.  Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[43]  N. Colburn,et al.  Differentially expressed protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[44]  N. Sonenberg,et al.  Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region , 2000, Molecular and Cellular Biology.

[45]  R. Carmody,et al.  DUG is a novel homologue of translation initiation factor 4G that binds eIF4A. , 2002, Biochemical and biophysical research communications.