An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchorin...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2011-11, Vol.108 (48), p.19105-19106
Main Authors: Means, Christopher K., Lygren, Birgitte, Langeberg, Lorene K., Jain, Ankur, Dixon, Rose E., Vega, Amanda L., Gold, Matthew G., Petrosyan, Susanna, Taylor, Susan S., Murphy, Anne N., Ha, Taekjip, Santana, Luis F., Tasken, Kjetil, Scott, John D.
Format: Article
Language:English
Subjects:
A Kinase Anchor Proteins - genetics
A Kinase Anchor Proteins - metabolism
Adaptor Proteins, Signal Transducing - genetics
Adaptor Proteins, Signal Transducing - metabolism
Analysis of Variance
Animals
Binding sites
Biological Sciences
Blotting, Western
Cell Line
Cells
Cloning, Molecular
Cyclic AMP-Dependent Protein Kinases - metabolism
Humans
Immunoprecipitation
Mass Spectrometry
Mice
Mitochondria
Mitochondria - metabolism
Mitochondrial Proteins - metabolism
Models, Molecular
Molecules
Mutagenesis, Site-Directed
PNAS Plus
PNAS PLUS (AUTHOR SUMMARIES)
Protein Binding - genetics
Protein Conformation
Proteins
Surface Plasmon Resonance
Transfection
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Summary:A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10–100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925–949 and 1,140–1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pull-down photobleaching experiments show that 41 ± 10% of SKIP sequesters two YFP-RI dimers, whereas 59 ± 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1107182108