cAMP-dependent protein kinase anchor protein 75 (AKAP75): Expression, properties, and intracellular localization in model systems

Abstract

A-Kinase Anchor Proteins (AKAPs) have a C-terminal binding site for the regulatory subunit (RII{dollar}\beta{dollar}) of cAMP-dependent protein kinase II{dollar}\beta{dollar} (PKAII{dollar}\beta{dollar}) and an N-terminal domain that mediates the targeting and attachment of the anchor protein to intracellular structures. In vitro biochemical studies and in situ immunocytochemical analysis (Glantz, S. B., Amat, J. A., and Rubin, C. S., (1992) Mol.Biol. Cell 3 1215-1228) suggest that AKAPs anchor PKAII{dollar}\beta{dollar} at specific sites in the dendritic cytoskeleton of neurons. This arrangement would place PKAII{dollar}\beta{dollar} in proximity with its substrates and create "target sites" for cAMP actions. The foregoing model predicts that (a) RII subunits are freely accessible to AKAPs, (b) PKAII holoenzymes, as well as RII subunits, are anchored and (c) changes in the level of AKAP can alter the intracellular distribution of type II PKAs.;These previously untested propositions were addressed by overexpressing bovine brain AKAP75 in a human cell line (HEK293). Non-transfected cells express a low level of endogenous AKAP79, and {dollar}>{dollar}90% of RII subunits are isolated in the cell cytosol. In contrast, stably transfected cells, which express a 10-fold excess of AKAP75, sequester {dollar}>{dollar}90% of their RII subunits in a particulate pool. Catalytic subunits are also transferred to this pool. AKAP75 accumulates in a cell compartment with biochemical properties characteristic of cytoskeleton. Thus, in intact cells AKAPs have access to and avidly bind cytoplasmic type II PKAs. Moreover, an increase in AKAP content can alter the particulate/cytoplasmic distribution of PKAII{dollar}\beta{dollar} and PKAII{dollar}\alpha{dollar}.;AKAP75 facilitates the targeting and transmission of physiological signals carried by cAMP. The N-terminal anchoring region of AKAP75 consists of two non-contiguous sequences. Elimination of the first targeting module (T1, residues 27-48) produced an RII{dollar}\beta{dollar}-binding protein that was distributed equally between the cytosolic and Triton X-100-insoluble fractions of HEK293 cells. Further excision of a second non-adjacent region of similar size (T2, residues 77-91) generated a high affinity RII{dollar}\beta{dollar}-binding protein that was located predominantly ({dollar}>{dollar}85%) in the cell cytosol. Indirect immunofluorescence staining and confocal microscopy were used to determine the precise intracellular localization of RII{dollar}\beta{dollar}-binding proteins (and RII{dollar}\beta{dollar}) in transfected cells that express wild type AKAP75 and mutated AKAPs with defective T1 or T2 domains. RII{dollar}\beta{dollar} is localized in the cytoplasm of non-transfected cells. Wild type AKAP75 targets RII{dollar}\beta{dollar} to the cortical cytoskeleton. Deletion of the T1 and/or T2 domain results in the re-distribution of AKAP75 throughout the cytoplasm. Wild type AKAP75 and F-actin were colocalized in transfected HEK293 cells. However, AKAP75 remained associated with the plasma membrane when F-actin was disrupted with cytochalasin B.;In effort to identify and characterize specific cytoskeleton proteins interacting with AKAP, a baculovirus expression vector was successfully constructed to express AKAP75 in Sf9 cells. Purified recombinant AKAP75 will be used in blot overlay assays, affinity chromatography, binding assays, and in vitro biochemical assays in order to characterize interacting proteins.;The studies in this thesis demonstrate that a novel anchoring/tethering protein (AKAP75) regulates the localization of RII subunits and the PKAII holoenzyme in intact cells. The results also provide clearcut evidence for the targeting of AKAP75 to the cortical cytoskeleton and the ability of AKAP75 to sequester cytoplasmic PKAII isoforms in situ. The essential roles of the T1 and T2 domains in targeting AKAP75 to the cortical cytoskeleton were also documented.