AKAP, a novel cytoskeleton-associated protein that anchorscAMP-dependent protein kinase in dendrites

Abstract

cAMP-dependent protein kinase (PKA) mediates intracellular signaling in response to neurotransmitters such as dopamine and norepinephrine. The type II{dollar}\beta{dollar} isoform is the predominant PKA in mammalian brain. The bulk of this enzyme is tightly associated with the particulate fraction of brain homogenates. The particulate nature of PKA-II{dollar}\beta{dollar} is due to interactions between the regulatory subunit (RII{dollar}\beta{dollar}) and anchor proteins in intracellular structures. A novel brain-enriched RII{dollar}\beta{dollar}-binding protein, AKAP (A Kinase Anchor Protein), has been identified and characterized in two species in the Rubin laboratory (Bregman et al., J. Biol. Chem. 264, 4648-4656, 1989; Bregman et al., J. Biol. Chem. 266, 7207-7213, 1991).;Immunoperoxidase staining disclosed the localization of AKAP 150 and RII{dollar}\beta{dollar} in rat brain. The RII-binding protein is a neuronal dendritic protein that is highly expressed in forebrain regions such as basal ganglia and cerebral cortex. Electron microscopy revealed an association of AKAP 150 with dendritic microtubules. The localization pattern of RII{dollar}\beta{dollar} in the brain is similar to that of AKAP 150. The results suggest that the bifunctional AKAP 150 protein tethers PKA-II{dollar}\beta{dollar} to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.;The gene for bovine AKAP 75 was cloned and expressed in mammalian cells (Hirsch et al., J. Biol. Chem. 267, 2131-2134, 1992). AKAP 75 contains 428 amino acid residues. Site-directed mutagenesis determined that approximately 22 amino acids near the C terminus of AKAP 75 comprise the binding domain for RII{dollar}\beta{dollar}. Several hydrophobic residues were identified as essential for efficient binding activity. Deletion analysis identified two regions near the N terminus that govern the attachment of AKAP 75 to intracellular structures.;The neuron-like cell line RT4-E5 and the glial-like cell line RT4-D6 were used to study the hormonal regulation of AKAP 150 expression. In RT4-E5 cells, glucocorticoids elicited an increase in the abundance of AKAP 150 polypeptide, whereas RII{dollar}\beta{dollar} accumulated following forskolin administration. In RT4-D6 cells, glucocorticoids had no effect, but cAMP or forskolin caused AKAP 150 concentration to decline. Thus, neurotransmitters and steroid hormones might regulate the amounts and intracellular distribution of PKA-II{dollar}\beta{dollar} in a cell-specific manner in the nervous system.