Identification and characterization of a novel A-kinase-anchoring protein (AKAP120) from rabbit gastric parietal cells

The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (RII) dimer to A-Kinase-anchoring proteins (AKAPs...

Full description

Saved in:
Bibliographic Details
Published in:Biochemical journal 1997-03, Vol.322 ( Pt 3) (3), p.801-808
Main Authors: Dransfield, D T, Yeh, J L, Bradford, A J, Goldenring, J R
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Carrier Proteins
Cloning, Molecular
Cyclic AMP-Dependent Protein Kinase Type II
Cyclic AMP-Dependent Protein Kinases - metabolism
DNA, Complementary - genetics
DNA, Complementary - isolation & purification
Molecular Sequence Data
Parietal Cells, Gastric - metabolism
Proteins - genetics
Proteins - isolation & purification
Proteins - metabolism
Rabbits
Sequence Analysis
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (RII) dimer to A-Kinase-anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have screened a rabbit gastric parietal cell cDNA library and have isolated a single RII-binding protein clone. Sequence analysis revealed an open reading frame coding for 1022 amino acids (AKAP120). Recombinant fragments of the full-length clone were prepared and the RII-binding region mapped to an area between amino acids 489 and 549. This area contained a putative alpha-helical RII-binding region between amino acids 503 and 516. Incubation of [32P]RII with a synthetic peptide of AKAP120-(489-522) completely inhibited the binding of [32P]RII to the recombinant AKAP120 fragments that demonstrated RII binding. In vitro RII-binding affinity studies indicated a high-affinity interaction between AKAP120 and RII with a Kapp between 50 and 120 nM for the three recombinant fragments that bound [32P]RII. RNase-protection analysis revealed that AKAP120 is a widely distributed protein, with the highest levels of mRNA observed in gastric fundus. The presence of this novel high-affinity AKAP in gastric parietal cells suggests that it may regulate RII subcellular sequestration in this cell type.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3220801