Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.
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PMID:Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase. 812 92

Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with A-Kinase Anchoring Proteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RIIalpha or RIIbeta, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RIIalpha decreased AKAP-binding up to 24 +/- 3 (n = 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RIIalpha reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n = 4) whereas introduction of an additional proline at position 6 in RIIbeta increased or conferred binding toward these anchoring proteins. Therefore, we conclude that beta-branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and prolines at positions 6 and 7 increase or stabilize RIIalpha interaction with selected anchoring proteins.
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PMID:Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. Classification Of side chain determinants for anchoring and isoform selective association with AKAPs. 891 May 53

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.
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PMID:Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding. 1076 1

The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.
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PMID:A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase. 1127 82

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.
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PMID:Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes. 1182 86

We have reported that a novel c-Myc-binding protein, AMY-1, binds to cAMP-dependent protein kinase-anchoring protein 149 (AKAP149) and its splicing variant, AKAP84 and is localized in the mitochondria in a complex with RII, a regulatory subunit of cAMP-dependent protein kinase (PKA) (Furusawa, M., Ohnishi, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2001) J. Biol. Chem. 276, 36647-36651). In this study, we further found that AMY-1 competitively bound to either AKAP95 or AKAP84 in the nucleus and the cytoplasm, respectively, in a concentration-dependent manner of either AKAP. Like AKAP84, AMY-1 was found to bind to the RII-binding region of AKAP95 in vivo and in vitro and to make a ternary complex with RII. It was also found that the formation of the complex of AMY-1 with AKAP84/95 and RII prevented a catalytic subunit from binding to this AKAP complex, leading to suppression of PKA activity. These findings suggest that AMY-1 is an important modulator of PKA.
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PMID:AMY-1 interacts with S-AKAP84 and AKAP95 in the cytoplasm and the nucleus, respectively, and inhibits cAMP-dependent protein kinase activity by preventing binding of its catalytic subunit to A-kinase-anchoring protein (AKAP) complex. 1241 7

Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Calpha and Cbeta are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.
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PMID:Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing. 1759 3