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PMID: 12571360 Published · ppublish English Journal Article

Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2).

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 100 ·No. 4 ·2003-02-18 ·Pages 1627-32

Li H, Adamik R, Pacheco-Rodriguez G, Moss J, Vaughan M

Abstract

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.

MeSH Terms
Amino Acid Sequence Base Sequence Binding Sites Blotting, Western Brefeldin A/pharmacology Cell Line Cell Membrane/metabolism Cloning, Molecular Cyclic AMP-Dependent Protein Kinases/metabolism DNA Primers DNA, Complementary Guanine Nucleotide Exchange Factors/antagonists & inhibitors,chemistry,metabolism Humans Precipitin Tests Protein Transport Two-Hybrid System Techniques
Chemicals
ARFGEF2 protein, human DNA Primers DNA, Complementary Guanine Nucleotide Exchange Factors Brefeldin A Cyclic AMP-Dependent Protein Kinases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Li Hewang
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Adamik Ronald
Pacheco-Rodriguez Gustavo
Moss Joel
Vaughan Martha
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0337678100
Published
2003-02-18
Epub
2003-00-05
Pages
1627-32
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC149883
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.0337678100 PMC Full Text View journal details
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