Molecular and Cellular Biochemistry

, Volume 141, Issue 2, pp 129–134 | Cite as

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

  • Prakash Sista
  • Sharon Edmiston
  • James W. Darges
  • Simon Robinson
  • David J. Burns
Article

Abstract

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)

Key words

protein kinase C reporter assays human growth hormone modulators signal transduction TPA response element (TRE) AP1 

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Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Prakash Sista
    • 1
  • Sharon Edmiston
    • 1
  • James W. Darges
    • 1
  • Simon Robinson
    • 1
  • David J. Burns
    • 1
  1. 1.Sphinx Pharmaceuticals CorporationDurhamUSA

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