Abstract
PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr(308) by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308) in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308), which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308) by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein.
MeSH terms
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Adaptor Proteins, Signal Transducing / metabolism*
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Animals
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Cell Extracts
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Cell Line, Tumor
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Cell Membrane / metabolism*
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Endoplasmic Reticulum Chaperone BiP
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Heat-Shock Proteins / metabolism*
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Humans
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Immunoprecipitation
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Male
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Mice, Nude
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Models, Biological
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Phosphorylation
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Phosphothreonine / metabolism
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Protein Binding
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Protein Serine-Threonine Kinases / metabolism*
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Proto-Oncogene Proteins c-akt / chemistry*
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Proto-Oncogene Proteins c-akt / metabolism*
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Pyruvate Dehydrogenase Acetyl-Transferring Kinase
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RNA, Double-Stranded / metabolism
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Regulatory-Associated Protein of mTOR
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Transfection
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alpha-Macroglobulins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Cell Extracts
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Endoplasmic Reticulum Chaperone BiP
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HSPA5 protein, human
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Heat-Shock Proteins
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Hspa5 protein, mouse
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PDK1 protein, human
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Pdk1 protein, mouse
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Pyruvate Dehydrogenase Acetyl-Transferring Kinase
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RNA, Double-Stranded
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RPTOR protein, human
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Regulatory-Associated Protein of mTOR
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alpha-Macroglobulins
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Phosphothreonine
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
Grants and funding
The authors have no support or funding to report.