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Analyzing Phosphorylation-Dependent Regulation of Subcellular Localization and Transcriptional Activity of Transcriptional Coactivator NT-PGC-1α

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Peroxisome Proliferator-Activated Receptors (PPARs)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 952))

Abstract

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a nuclear transcriptional coactivator that regulates the genes involved in energy metabolism. Recent evidence has been provided that alternative splicing of PPARGC1A gene produces a functional but predominantly cytosolic isoform of PGC-1α (NT-PGC-1α). We have demonstrated that transcriptional coactivation capacity of NT-PGC-1α is directly correlated with its nuclear localization in a PKA phosphorylation-dependent manner. In this chapter, we describe quantitative imaging analysis methods that are developed to measure the relative fluorescence intensity of the protein of interest in the nucleus and cytoplasm in a single cell and the frequency distribution of nuclear/cytoplasmic intensity ratios in the population of cells, respectively. This chapter also describes transient cotransfection and dual-luciferase reporter gene assay that examine the ability of coactivators to activate the transcriptional activity of transcription factors.

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Acknowledgments

We thank Anik Boudreau, Jeho Shin, Yagini Joshi, Chelsea Black, and Peter Huypens for their technical contributions, and David Burk and Courtney Cain for their bioimaging support of this project. This work was supported by NIH RO1 DK074772 (TWG), by a P&F award to JSC from the Pennington NORC (NIH 1P30 DK072476), and in part by NIH grant P20-RR021945 (TWG).

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Authors and Affiliations

  1. Laboratory of Nutrient Sensing and Adipocyte Signaling, Pennington Biomedical Research Center, Baton Rouge, LA, USA

    Ji Suk Chang & Thomas W. Gettys

Authors
  1. Ji Suk Chang
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  2. Thomas W. Gettys
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Corresponding author

Correspondence to Thomas W. Gettys .

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Editors and Affiliations

  1. School of Pharmacy, University of Missouri-Kansas City, Charlotte Street 2464, Kansas City, 64108, USA

    Mostafa Z. Badr

  2. School of Pharmacy, University of Missouri-Kansas City, Charlotte Street 2464, Kansas City, 64108, Missouri, USA

    Jihan A. Youssef

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Chang, J.S., Gettys, T.W. (2013). Analyzing Phosphorylation-Dependent Regulation of Subcellular Localization and Transcriptional Activity of Transcriptional Coactivator NT-PGC-1α. In: Badr, M., Youssef, J. (eds) Peroxisome Proliferator-Activated Receptors (PPARs). Methods in Molecular Biology, vol 952. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-155-4_11

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  • DOI: https://doi.org/10.1007/978-1-62703-155-4_11

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-154-7

  • Online ISBN: 978-1-62703-155-4

  • eBook Packages: Springer Protocols

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