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Upregulation of mTORC2 activation by the selective agonist of EPAC, 8-CPT-2Me-cAMP, in prostate cancer cells: assembly of a multiprotein signaling complex.

Publication ,  Journal Article
Misra, UK; Pizzo, SV
Published in: J Cell Biochem
May 2012

Ligation of cell surface-associated GRP78 by activated α(2) -macroglobulin triggers pro-proliferative cellular responses. In part, this results from activation of adenylyl cyclase leading to an increase in cAMP. We have previously employed the cAMP analog 8-CPT-2Me-cAMP to probe these responses. Here we show in 1-LN prostate cancer cells that 8-CPT-2Me-cAMP causes a dose-dependent increase in Epac1, p-Akt(T308) , p-Akt(S473) , but not p-CREB. By contrast, the PKA activator 6-Benz-cAMP caused a dose-dependent increase in p-CREB, but not Epac1. We measured mTORC2-dependent Akt phosphorylation at S473 in immunoprecipitates of mTOR or Rictor from 1-LN cells. 8-CPT-2Me-cAMP caused a two-threefold increase in p-Akt(S473) and Akt(S473) kinase activity in Rictor immunoprecipitates. By contrast, there was only a negligible effect on p-Akt(T308) in Rictor immunoprecipitates. Silencing Rictor gene expression by RNAi significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of Akt at Ser(473) . These studies represent the first report that Epac1 mediates mTORC2-dependent phosphorylation of Akt(S473) . Pretreatment of these cells with the PI 3-Kinase inhibitor LY294002 significantly suppressed 8-CPT-2Me-cAMP-dependent p-Akt(S473) and p-Akt(S473) kinase activities, and both effects were rapamycin insensitive. This treatment caused a two to threefold increase in S6 Kinase and 4EBP1 phosphorylation, indices of mTORC1 activation. Pretreatment of the cells with LY294002 and rapamycin significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of S6 Kinase and 4EBP1. We further demonstrate that in 8-CPT-2Me-cAMP-treated cells, Epac1 co-immunoprecipitates with AKAP, Raptor, Rictor, PDE3B, and PDE4D suggesting thereby that during Epac1-induced activation of mTORC1 and mTORC2, Epac1 may have an additional function as a "scaffold" protein.

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Published In

J Cell Biochem

DOI

EISSN

1097-4644

Publication Date

May 2012

Volume

113

Issue

5

Start / End Page

1488 / 1500

Location

United States

Related Subject Headings

  • Up-Regulation
  • Thionucleotides
  • TOR Serine-Threonine Kinases
  • Sirolimus
  • Signal Transduction
  • Ribosomal Protein S6 Kinases
  • Regulatory-Associated Protein of mTOR
  • Rapamycin-Insensitive Companion of mTOR Protein
  • RNA-Binding Proteins
  • Proto-Oncogene Proteins c-akt
 

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Journal cover image

Published In

J Cell Biochem

DOI

EISSN

1097-4644

Publication Date

May 2012

Volume

113

Issue

5

Start / End Page

1488 / 1500

Location

United States

Related Subject Headings

  • Up-Regulation
  • Thionucleotides
  • TOR Serine-Threonine Kinases
  • Sirolimus
  • Signal Transduction
  • Ribosomal Protein S6 Kinases
  • Regulatory-Associated Protein of mTOR
  • Rapamycin-Insensitive Companion of mTOR Protein
  • RNA-Binding Proteins
  • Proto-Oncogene Proteins c-akt