Protein kinase A-anchoring (AKAP) domains in brefeldin
A-inhibited guanine nucleotide-exchange protein 2 (BIG2).
Li H, Adamik R, Pacheco-Rodriguez G, Moss J, Vaughan M.
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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.