Abstract
The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted us to examine the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. We used RNA interference (RNAi) to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affected insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3-Phosphoinositide-Dependent Protein Kinases
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Amino Acid Sequence
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Animals
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Conserved Sequence
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Drosophila Proteins / genetics
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Drosophila Proteins / metabolism*
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Drosophila melanogaster / genetics
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Drosophila melanogaster / metabolism*
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Insulin / metabolism
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Intracellular Signaling Peptides and Proteins*
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Models, Biological
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Mutagenesis, Site-Directed
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PTEN Phosphohydrolase
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Peptide Initiation Factors*
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Phosphatidylinositol 3-Kinases / genetics
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Phosphoric Monoester Hydrolases / genetics
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Phosphoric Monoester Hydrolases / metabolism
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Phosphorylation
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-akt
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RNA Interference
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Receptor Protein-Tyrosine Kinases / genetics
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Receptor Protein-Tyrosine Kinases / metabolism*
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Ribosomal Protein S6 Kinases / genetics
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Ribosomal Protein S6 Kinases / metabolism*
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Signal Transduction
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism
Substances
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Carrier Proteins
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Drosophila Proteins
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Insulin
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Intracellular Signaling Peptides and Proteins
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Peptide Initiation Factors
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Phosphoproteins
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Proto-Oncogene Proteins
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Thor protein, Drosophila
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Tumor Suppressor Proteins
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Receptor Protein-Tyrosine Kinases
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tor protein, Drosophila
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3-Phosphoinositide-Dependent Protein Kinases
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Akt1 protein, Drosophila
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Ribosomal Protein S6 Kinases
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Phosphoric Monoester Hydrolases
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PTEN Phosphohydrolase