Organelle-specific Targeting of Protein Kinase AII (PKAII)

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 (...

Section: Expression Of Fluorescent Fusion Proteins In Transfectedsupporting

confidence: 92%

mentioning

confidence: 99%

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

Ruehr

Zakhary

Damron

et al. 1999

“…2A) that cooperatively governs sequestration of RII␣ and RII␤ subunits (35). Residues 511-530 in DAKAP200 can be aligned with tethering regions of mammalian AKAPs (9,35,42) , and Val 530 are in register with essential hydrophobic residues in previously characterized RII binding sites ( Fig. 2A).…”

Section: Discovery Of Cdnas Encoding a Novel Drosophila Akap-mentioning

confidence: 70%

“…A kinase anchor proteins (AKAPs) guide transmission and routing of cAMP signals to such microenvironments. AKAPs have an avid binding site for regulatory subunits (RII) of PKAII isoforms 2 and distinct domains that target the tethered PKA holoenzyme to docking sites in organelles or cytoskeleton (5,6,9,10). Accumulation of a high concentration of anchored PKAII in proximity with clustered substrate-effector protein promotes efficient reception, amplification, and precisely focused transmission of signals borne by cAMP (5)(6)(7)(8).…”

mentioning

confidence: 99%

Generation of a Novel A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog from a Single Gene

Li¹,

Rossi²,

Hoheisel³

et al. 1999

Self Cite

A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI ϳ3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, ⌬DAKAP200. ⌬DAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. ⌬DAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and ⌬DAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; ⌬DAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.Cyclic AMP-dependent protein kinases (PKAs) 1 mediate actions of hormones that stimulate adenylate cyclase (1-4). Signals carried by cAMP often regulate activities of proteins that accumulate at discrete intracellular locations (e.g. ion channels in plasma membrane) (5-8). A kinase anchor proteins (AKAPs) guide transmission and routing of cAMP signals to such microenvironments. AKAPs have an avid binding site for regulatory subunits (RII) of PKAII isoforms 2 and distinct domains that target the tethered PKA holoenzyme to docking sites in organelles or cytoskeleton (5, 6, 9, 10). Accumulation of a high concentration of anchored PKAII in proximity with clustered substrate-effector protein promotes efficient reception, amplification, and precisely focused transmission of signals borne by cAMP (5-8).Several fundamental questions about AKAP⅐RII complexes remain unresolved. Current models suggest that AKAPs statically associate with docking sites in organelles. However, it is possible that AKAP⅐RII complexes shuttle dynamically between intracellular loci. This proposition has neither been excluded nor systematically investigated. Many physiological processes are coordinately regulated by groups of hormones. Homeostatic control is often achieved through synergistic or antagonistic interactions among signaling pathways that are activated by different second messenger molecules. A key question is how signals transmitted via AKAP⅐PKAII complexes are integrated with inputs from pathways controlled by other second messengers in order to regulate common effector proteins. PKA anchoring may be regulated by pretranslat...

“…

…”

mentioning

confidence: 99%

Characterization of Structural Features That Mediate the Tethering of Caenorhabditis elegans Protein Kinase A to a Novel A Kinase Anchor Protein

Angelo

Rubin

2000

Self Cite

Caenorhabditis elegans protein kinase A (PKAI CE) (5,6,9,10). Clustering of a high concentration of AKAP⅐PKAII complexes in proximity with PKA substrate/effector proteins in cytoskeleton/organelles enables efficient reception, rapid amplification, and precisely focused targeting of cAMP signals. Consequently, PKA-catalyzed phosphorylation of co-localized effector proteins is optimized (11-13). Key tenets of the preceding signaling model have been verified. Disruption of AKAP⅐PKA complexes in situ markedly diminishes the ability of cAMP to regulate such critical physiological processes as ion transport, gene transcription, apoptosis, and hormone secretion (8,(13)(14)(15)(16).Mammals employ Ͼ20 distinct AKAPs to adapt type II PKAs for specialized functions (5)(6)(7)(8)17). Amino acid sequences of the anchor proteins are markedly divergent; thus, AKAPs are functional, not structural, homologs. Different AKAPs accumulate in distinct locations; examples include cytoplasmic surfaces of plasma membrane, mitochondria, Golgi membranes, and centrosomes (5,6,9,10,18). Routing of PKAII to specific microenvironments is governed by unique targeting/anchoring domains in individual AKAPs (9, 19 -22). Primary structures of RII-binding sites in AKAPs are not highly conserved (e.g. see alignments in Refs. 23 and 24). However, the folding pattern and physical properties of amino acids that subserve the ligation of RII subunits are universal properties of AKAPs. The RII-binding region of typical AKAPs comprises 16 -20 contiguous amino acids that have a predicted propensity to fold into an amphipathic ␣-helix (25). The ␣-helix includes six critical, precisely positioned amino acids whose large aliphatic side chains co-operatively create an extended hydrophobic surface (19). The size, hydrophobicity, and configuration of this domain generate a unique receptor site for a complementary, apolar docking surface that is produced from the folding of ϳ30 amino acid residues in RII␣ or RII␤ dimers (26 -29). Robust hydrophobic * This work was supported by National Institutes of Health Grant GM57660 and a stipend from Training Grant GM07620 (to R. A. A). 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.‡ To whom correspondence should be addressed: Dept.