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

UK Misra, SV Pizzo - Journal of cellular biochemistry, 2012 - Wiley Online Library
UK Misra, SV Pizzo
Journal of cellular biochemistry, 2012Wiley Online Library
Ligation of cell surface‐associated GRP78 by activated α2‐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‐AktT308, p‐AktS473, but
not p‐CREB. By contrast, the PKA activator 6‐Benz‐cAMP caused a dose‐dependent …
Abstract
Ligation of cell surface‐associated GRP78 by activated α2‐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‐AktT308, p‐AktS473, 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‐AktS473 and AktS473 kinase activity in Rictor immunoprecipitates. By contrast, there was only a negligible effect on p‐AktT308 in Rictor immunoprecipitates. Silencing Rictor gene expression by RNAi significantly suppressed 8‐CPT‐2Me‐cAMP‐induced phosphorylation of Akt at Ser473. These studies represent the first report that Epac1 mediates mTORC2‐dependent phosphorylation of AktS473. Pretreatment of these cells with the PI 3‐Kinase inhibitor LY294002 significantly suppressed 8‐CPT‐2Me‐cAMP‐dependent p‐AktS473 and p‐AktS473 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. J. Cell. Biochem. 113: 1488–1500, 2012. © 2011 Wiley Periodicals, Inc.
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