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The role of D‐AKAP2 scaffolding in integration of PKA signaling
Author(s) -
Eggers Christopher T.,
Taylor Susan S.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb73-c
Subject(s) - scaffold protein , heterotrimeric g protein , microbiology and biotechnology , immunoprecipitation , signal transducing adaptor protein , gtpase activating protein , protein–protein interaction , pdz domain , protein subunit , bimolecular fluorescence complementation , phosphotyrosine binding domain , protein kinase a , biology , plasma protein binding , chemistry , signal transduction , g protein , phosphorylation , biochemistry , sh2 domain , proto oncogene tyrosine protein kinase src , yeast , gene
Scaffold proteins serve as templates to build complexes of multiple signaling proteins at particular locations in the cell, helping to ensure the proper integration of signaling information. The substrate specificity of the cAMP‐dependent protein kinase (PKA) is in large part determined by its interactions with A‐kinase anchoring proteins (AKAPs), which can act as scaffolds to bring PKA together with substrates and other regulatory proteins. While the majority of AKAPs bind tightly only to type‐II PKA, D‐AKAP2 (AKAP10) has been shown to bind to both type I and II. In addition to a PKA‐binding motif and a C‐terminal PDZ‐binding motif, D‐AKAP2 contains two domains with homology to Regulator of G protein Signaling (RGS) proteins, which typically act as GTPase‐activating proteins for heterotrimeric G proteins. The objectives of this study are to identify binding partners for the RGS domains and to determine the biological function of D‐AKAP2. In vitro binding studies with a panel of G proteins suggested an interaction between the second RGS homology domain and the alpha subunit of G(s). Co‐immunoprecipitation experiments confirmed this result, and fluorescent microscopy in HeLa cells demonstrated co‐localization of the two proteins. A yeast two‐hybrid screen using the first RGS domain identified a subunit of the clathrin adaptor protein complex AP‐1 as a putative target. We have used in vitro pull‐down studies and co‐immunoprecipitation from cell lysates to confirm this interaction. Additionally, we have synthesized peptide spot arrays to define the binding epitope in AP‐1. Together, these results point to a possible role for D‐AKAP2 in integrating G(s) and PKA regulation of protein trafficking. This research was funded by the American Cancer Society and NIH (GM54441).