Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.
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PMID:cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta. 133 41

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.
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PMID:Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta. 201 23

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.
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PMID:Characterization of the protein kinase activities of human platelet supernatant and particulate fractions. 632 54

Specificity of protein kinases and phosphatases may be achieved through compartmentalization with preferred substrates. In neurons, adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase (PKA) is localized at postsynaptic densities by association of its regulatory subunit with an A kinase anchor protein, AKAP79. Interaction cloning experiments demonstrated that AKAP79 also binds protein phosphatase 2B, or calcineurin (CaN). A ternary complex of PKA, AKAP, and CaN was isolated from bovine brain, and colocalization of the kinase and the phosphatase was established in neurites of cultured hippocampal neurons. The putative CaN-binding domain of AKAP79 is similar to that of the immunophilin FKBP-12, and AKAP79 inhibited CaN phosphatase activity. These results suggest that both PKA and CaN are targeted to subcellular sites by association with a common anchor protein and thereby regulate the phosphorylation state of key neuronal substrates.
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PMID:Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. 752 41

In neurons cAMP-dependent protein kinase II beta (PKAII beta) is sequestered in the dendritic cytoskeleton because the regulatory subunit (RII beta) of the enzyme is tightly bound by A Kinase Anchor Proteins (AKAPs). The prototypic neuronal anchor protein AKAP75 has a COOH-terminal 22-residue RII beta binding (tethering) site. A key feature of the tethering site is that several amino acids with large aliphatic side chains mediate the high-affinity binding of RII beta. Mutagenesis, recombinant protein expression, and physicochemical characterization were used to investigate the structural basis for the homodimerization and AKAP75 binding activities of RII beta. Several crucial residues are located in an NH2-terminal region that encompasses amino acids 13-36. Substitution of Ala for Leu13 or Phe36 generates monomeric RII beta subunits that cannot bind AKAP75. The results are not due to general misfolding since mutant RII beta monomers bind cAMP and inhibit the catalytic subunit of PKAII beta with the same affinity and efficacy as wild-type RII beta dimers. Moreover, substitution of Ala for Leu12, Val20, Leu21, Phe31, Leu33, or Leu39 and replacement of Leu13 with Ile or Val did not impair the dimerization reaction. Evidently, large hydrophobic side chains of Leu13 and Phe36 play pivotal roles in stabilizing RII beta-RII beta interactions. A secondary consequence of destabilizing RII beta dimers is the loss of intracellular targeting/anchoring capacity because monomers fail to bind AKAP75. Other NH2-terminal residues directly modulate the affinity of RII beta dimers for the AKAP75 tethering site. Replacement of Val20-Leu21 with Ala-Ala produced a dimeric RII beta protein that binds AKAP75 approximately 4% as avidly as wild-type RII beta. It is possible that the aliphatic side chains of Val20 and Leu21 interact with the essential Leu and Ile residues in the AKAP75 tethering region.
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PMID:Mutagenesis of the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta reveals hydrophobic amino acids that are essential for RII beta dimerization and/or anchoring RII beta to the cytoskeleton. 782 31

The generation of dorso-ventral polarity in Drosophila relies on the formation of a nuclear gradient of the rel/nuclear factor kappa B transcription factor dorsal in the pre-cellular syncitial embryo by a process of differential nuclear localization. It is thought that the gradient is formed by activation at ventral positions of the membrane receptor Toll that in turn causes the local dissociation of dorsal from the cytoplasmic anchor protein cactus. Although Toll is related in its cytoplasmic domain to the interleukin-1 receptor little is known about the signal transduction pathways that lead from Toll to the relocalization of dorsal. In this paper we have used immunofluorescence microscopy as a direct assay of dorsal protein nuclear localization in the Drosophila cell line Schneider 2. We find that increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of dorsal. By contrast, we find that activation of endogenous protein kinase A and expression of wild-type Toll receptors, which activate zen-chloramphenicol acetyltransferase reporter genes in this system, have only a marginal effect on the cellular distribution of the dorsal protein. Treatment of cells with activators of protein kinase C and radical oxygen intermediates, both of which activate nuclear factor kappa B, also has little effect on dorsal protein localization. We propose that different threshold levels of dorsal activation can be established by distinctly regulated signal transduction pathways.
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PMID:Relocalization of Drosophila dorsal protein can be induced by a rise in cytoplasmic calcium concentration and the expression of constitutively active but not wild-type Toll receptors. 790 39

A kinase anchor proteins (AKAPs) have a C-terminal binding site for the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (PKAII beta) and an N-terminal domain that mediates the targeting and attachment of the anchor protein to intracellular structures. In vitro biochemical studies and recent 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 beta at specific sites in the dendritic cytoskeleton of neurons. This arrangement would place PKAII beta 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. We have addressed these previously untested propositions by overexpressing bovine AKAP75 in a human cell line (HEK293). Non-transfected cells express a low level of endogenous AKAP79, and approximately 90% of RII alpha and RII beta subunits are isolated in the cell cytosol. In contrast, stably transfected cells, which express a 10-fold excess of AKAP75, sequester > 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, 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 beta and PKAII alpha.
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PMID:Expression of a kinase anchor protein 75 depletes type II cAMP-dependent protein kinases from the cytoplasm and sequesters the kinases in a particulate pool. 846 92

Cyclic AMP-dependent protein kinase II beta (PKAII beta) is the principal mediator of cAMP action in neurons. A Kinase Anchor Proteins (AKAPs) are enriched in forebrain neurons and have distinct high affinity binding domains for the regulatory subunit (RII beta) of PKAII beta and components of the dendritic cytoskeleton. The selective accumulation of AKAP.RII beta complexes near dendritic microtubules tethers PKAII beta in proximity with adenylate cyclase in the synaptic plasma membrane and cytoskeletal proteins that are substrates for the kinase, thereby creating intraneuronal target sites for signals carried by cAMP. We have characterized the targeting (anchoring) and tethering (RII beta binding) domains of a prototypic anchor protein AKAP75. Deletion of N-terminal residues 27-48 generated a truncated RII beta-binding protein that partitions equally between the cytosol and detergent-insoluble fractions of HEK293 cells. Further removal of a non-adjacent sequence (residues 77-91) produced a cytosolic protein with unimpaired RII beta binding activity. Thus, two noncontiguous domains mediate the intracellular localization of AKAP75. Boundaries for the RII beta tethering domain were mapped to residues 392-413 by scanning mutagenesis. Residues containing long aliphatic side chains are essential for the high affinity binding of RII beta by AKAP75. Contributions of hydrophobic amino acids to tethering activity also depend on the position of the residue in the sequence. Certain conservative mutations that should not alter significantly the overall hydrophobicity or helicity of the tethering region (e.g. replacement of Leu with Ala) diminish the RII beta binding activity of AKAP75.
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PMID:Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the cytoskeleton. 850 14

The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression.
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PMID:The v-Ki-Ras oncogene alters cAMP nuclear signaling by regulating the location and the expression of cAMP-dependent protein kinase IIbeta. 881 Mar

The A126 cell line, in contrast to its PC12 parent, does not differentiate, accumulate nuclear cAMP-dependent protein kinase A (PKA) catalytic subunit, or transcribe cAMP-dependent promoters in response to cAMP. Total PKA is reduced by 50% and is partly resistant to cAMP-induced dissociation in vivo. Unlike PC12, where PKAII is membrane-associated, PKAII is exclusively cytosolic in A126. Cotransfection with the RII anchor protein (AKAP75) and the PKA catalytic subunit (C-PKA) restored cAMP-induced transcription to levels found in PC12. These data indicate that membrane-bound PKAII amplifies cAMP signaling to the nucleus and suggest that cAMP-mediated responses are specified by the type and cellular localization of the PKA isoform.
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PMID:Membrane localization of cAMP-dependent protein kinase amplifies cAMP signaling to the nucleus in PC12 cells. 893 28


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