Cyclic AMP-dependent Protein Kinase Binding to A-kinase Anchoring Proteins in Living Cells by Fluorescence Resonance Energy Transfer of Green Fluorescent Protein Fusion Proteins

Abstract

There is now increasing evidence for clustering and compartmentalization of signaling enzymes with their activators and target proteins. This targeting of enzymes with their substrates may promote specificity of signaling events (1-3). To better understand the physiological role of scaffolding proteins, such as A-kinase anchoring proteins (AKAPs), 1 which target cyclic AMP-dependent protein kinase (PKA) (2, 4), it is essential to monitor in real time the interactions between the components of this signaling complex in living cells. However, to date such measurements have not been reported for the PKA pathway. AKAPs and PKA co-localize in cells. This was first shown by the redistribution of the type II regulatory (RII) and the catalytic subunits of PKA from the cytosolic to the particulate fraction following overexpression of AKAP75 in HEK293 cells (5). In studies in HEK293 cells, S-AKAP84 targeted RII to mitochondria (6). In rat cardiac myocytes, endogenous AKAP100 co-localized with RII in the region of the junctional sarcoplasmic reticulum/transverse tubule (7). A functional role of AKAP targeting of PKA is further indicated by studies in which Ht31 peptide, a competitive inhibitor of AKAP/PKA binding, was introduced into AKAP-overexpressing cells. For example, transfection of RINm5F pancreatic beta cells with Ht31 caused redistribution of RII from a perinuclear to a diffuse cytoplasmic localization and prevented cAMP-dependent insulin secretion (8). Similarly, in cardiac myocytes transfected with AKAP79, microinjection of Ht31 impaired the PKA-dependent increase in L-type Ca 2ϩ current (9). These studies demonstrate a role for AKAPs in the regulation of PKA distribution and in the specificity of PKA function.To date, the binding of RII with full-length AKAPs or with the peptide Ht31 has been demonstrated in vitro by RII overlay assay (10 -12), by band shift assays on nondenaturing gels (12, 13), by equilibrium dialysis measurements (12, 13), and recently by solution NMR measurements of the N-terminal dimerization domain of RII with Ht31 (14). Equilibrium dialysis measurements (12) and surface plasmon resonance 2 showed that Ht31 binds RII with nanomolar affinity. Although these in vitro approaches provide evidence for high affinity RII/AKAP or RII/Ht31 interactions in vitro, the results presented here extend these studies by measuring real-time interactions between these molecules in living cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed: Dept. EXPERIMENTAL PROCEDURES Construction of Vectors for the Expression of GFP-tagged Proteins-The cDNAs for RII␣, Ht31, and Ht31P were gifts from John Scott (Vollum Institute, Howard Hughes Medical Institute, Portland, OR). The construct for Ht31, used in the FRET experiments, encodes residues 418 -718 of the human thyroid RII anchoring protein (...

Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A

“…9A). This compound, which has a hydrophobic sequence homologue to AKAP, has been widely demonstrated to disrupt RII binding to the anchoring protein, both in vitro, by overlay assay (Vijayaraghavan et al, 1997b;Carr et al, 1991;Carr et al, 1992) and NMR analysis (Newlon et al, 1999), and, in vivo, by fluorescence resonance energy transfer techniques (Ruehr et al, 1999). We show here that LY294002 is able to stimulate both RIIβ recruitment to the sperm insoluble compartment (Fig.…”

Selectivity and Regulation of A-kinase Anchoring Proteins in the Heart

“…Vectors for the expression of the GFPtagged proteins RII-EYFP, Ht31-ECFP, and Ht31P-ECFP were constructed as described previously (27). CHO cells were stably transfected with Ht31-ECFP or HT31P-ECFP and then transiently transfected with RII-EYFP as described (27), and expression of Ht31-ECFP, HT31P-ECFP, and RII-EYFP was confirmed by immunoblot analysis. The cDNA for RI␣ was a gift of Dr. Susan Taylor (University of California, San Diego, La Jolla, CA).…”

“…RII recombinant protein was expressed under 2 mM isopropyl-1-thio-␤-D-galactopyranoside induction followed by cAMP-agarose affinity purification (26), and characterized as described (25). Vectors for the expression of the GFPtagged proteins RII-EYFP, Ht31-ECFP, and Ht31P-ECFP were constructed as described previously (27). CHO cells were stably transfected with Ht31-ECFP or HT31P-ECFP and then transiently transfected with RII-EYFP as described (27), and expression of Ht31-ECFP, HT31P-ECFP, and RII-EYFP was confirmed by immunoblot analysis.…”

“…Binding of RII␣ in CHO cell lysates to Ht31, mAKAP, and AKAP15/18 peptide surfaces was then measured by SPR. We previously demonstrated that RII-GFP fusion proteins behave in kinetically similar manner to purified RII in this system (27). RII␣ autophosphorylation resulted in ϳ1-(no change), ϳ7-, and ϳ82-fold increases in binding to Ht31, mAKAP, and AKAP15/18, respectively (Fig.…”

Regulation of CXCR4 Receptor Dimerization by the Chemokine SDF-1α and the HIV-1 Coat Protein gp120: A Fluorescence Resonance Energy Transfer (FRET) Study

“…Since FRET efficiency decreases as the inverse sixth power of distance, the acceptor must be in close proximity to the donor (Ͻ10 nm) (Stryer, 1978). Monitoring energy transfer between the enhanced cyan (ECFP) and yellow (EYFP) versions of the green fluorescent protein has frequently been used to detect molecular interactions between proteins, including both cytoplasmic (Ruehr et al, 1999) and membrane proteins (Zhou et al, 2003) such as GPCRs (Dinger et al, 2003). Using FRET, bioluminescence resonance energy transfer (BRET), and related methods, it has become clear that many GPCRs can dimerize or exist as higher order arrays and that this association can greatly alter their signaling properties .…”