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Analisis of A-Kinase Anchoring Protein Interactions With PKA Using Fluorescence Resonance Energy Transfer

Published online by Cambridge University Press:  02 July 2020

M. L. Ruehr ,
D. S. Damron  and
M. Bond
M. L. Ruehr
Affiliation:
Department of Molecular Cardiology, Cleveland, OH44195
D. S. Damron
Affiliation:
Center for Anesthesiology Research, Cleveland Clinic Foundation, Cleveland, OH44195
M. Bond
Affiliation:
Department of Molecular Cardiology, Cleveland, OH44195
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Extract

The clustering of components of a signaling pathway at a specific subcellular location raises the local concentration of the appropriate messengers and serves to amplify the signal. The cAMP dependent-protein kinase (PKA) pathway is regulated by compartmentalization of its components. A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular sites, thus presumably increasing substrate specificity. Phosphorylation of the type II regulatory subunit of PKA (RII) increases its affinity for AKAPs in vitro (1). The purpose of this study was to investigate whether altering the phosphorylation state of RII in live cells changes its affinity for an AKAP. Specifically, we investigated the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid (2), and RII, in response to PKA activators or inhibitors.

Fluorescence resonance energy transfer (FRET) was used to monitor binding events between RII and the catalytic subunit (C) of PKA, Ht31, or Ht31P, a mutated form of Ht31 which does not bind RII.

Type
Recent Advances in Light Microscopy
Information
Microscopy and Microanalysis , Volume 6 , Issue S2: Proceedings: Microscopy & Microanalysis 2000, Microscopy Society of America 58th Annual Meeting, Microbeam Analysis Society 34th Annual Meeting, Microscopical Society of Canada/Societe de Microscopie de Canada 27th Annual Meeting, Philadelphia, Pennsylvania August 13-17, 2000 , August 2000 , pp. 828 - 829
Copyright
Copyright © Microscopy Society of America

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References

References:

1.Zachary, et al., Circulation, in press.Google Scholar
2.Lester, et al., Proc. Natl. Acad. Sci. 94:14942–14947, 1997.CrossRefGoogle Scholar
3.Miyawaki, et al., Nature 388:882–887, 1997.CrossRefGoogle Scholar
4.Ruehr, et al., J. Biol. Chem. 274:33092–33096, 1999.CrossRefGoogle Scholar
5.Yang, et al., Biochem. 34:6267–6271, 1995.CrossRefGoogle Scholar
6.Yang, et al., J. Cell Biol. 142:511–522, 1998CrossRefGoogle Scholar