Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with Raptor

PLoS One. 2014 Feb 6;9(2):e88373. doi: 10.1371/journal.pone.0088373. eCollection 2014.

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

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Animals
  • Cell Extracts
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Endoplasmic Reticulum Chaperone BiP
  • Heat-Shock Proteins / metabolism*
  • Humans
  • Immunoprecipitation
  • Male
  • Mice, Nude
  • Models, Biological
  • Phosphorylation
  • Phosphothreonine / metabolism
  • Protein Binding
  • Protein Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins c-akt / chemistry*
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Pyruvate Dehydrogenase Acetyl-Transferring Kinase
  • RNA, Double-Stranded / metabolism
  • Regulatory-Associated Protein of mTOR
  • Transfection
  • alpha-Macroglobulins / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Extracts
  • Endoplasmic Reticulum Chaperone BiP
  • HSPA5 protein, human
  • Heat-Shock Proteins
  • Hspa5 protein, mouse
  • PDK1 protein, human
  • Pdk1 protein, mouse
  • Pyruvate Dehydrogenase Acetyl-Transferring Kinase
  • RNA, Double-Stranded
  • RPTOR protein, human
  • Regulatory-Associated Protein of mTOR
  • alpha-Macroglobulins
  • Phosphothreonine
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt

Grants and funding

The authors have no support or funding to report.