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

Authors
  • Hewang Li
  • Ronald Adamik
  • Gustavo Pacheco-Rodriguez
  • Joel Moss
  • Martha Vaughan
Publisher
The National Academy of Sciences
Publication Date
Feb 05, 2003
Source
PMC
Keywords
Disciplines
  • Biology
License
Unknown

Abstract

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an ≈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, RIα. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RIα, 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 RIα and RIβ, 284–301 (domain B) interacted with RIIα and RIIβ, and 517–538 (domain C) interacted with RIα, RIIα, and RIIβ. 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.

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