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)

Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.
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PMID:Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. 151 24

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.
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PMID:Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta. 173 21

In mammalian spermatozoa, most of the type II alpha isoform of cAMP-dependent protein kinase (PKAII alpha) is anchored at the cytoplasmic surface of a specialized array of mitochondria in the flagellar cytoskeleton. This places the catalytic subunits of PKAII alpha in proximity with potential target substrates in the cytoskeleton. The mechanism by which PKAII alpha is anchored at the outer surface of germ cell mitochondria has not been elucidated. We now report the cloning of a cDNA that encodes a novel, germ cell A kinase anchor protein (AKAP) designated S-AKAP84. S-AKAP84 comprises 593 amino acids and contains a centrally located domain that avidly binds regulatory subunits (RII alpha and RII beta) of PKAII alpha and PKAII beta. The 3.2-kilobase S-AKAP84 mRNA and the cognate S-AKAP84 RII binding protein are expressed principally in the male germ cell lineage. Expression of S-AKAP84 is tightly regulated during development. The protein accumulates as spermatids undergo nuclear condensation and tail elongation. The timing of S-AKAP84 expression is correlated with the de novo accumulation of RII alpha and RII beta subunits and the migration of mitochondria from the cytoplasm (round spermatids) to the cytoskeleton (midpiece in elongating spermatids). Residues 1-30 at the NH2 terminus of S-AKAP84 constitute a putative signal/anchor sequence that may target the protein to the outer mitochondrial membrane. Immunofluorescence analysis demonstrated that S-AKAP84 is co-localized with mitochondria in the flagellum.
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PMID:Characterization of S-AKAP84, a novel developmentally regulated A kinase anchor protein of male germ cells. 749 50

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 the human lymphoblastic cell line KE 37, Northern blot analysis with cDNA probes for human regulatory subunits RII alpha RII beta of the cAMP-dependent protein kinase (A-kinase) type II and immunoblotting or immunoprecipitation studies with several antibodies directed against RII alpha and RII beta show that these two isoforms are expressed. The major isoform alpha is mostly cytosolic, whereas the beta isoform appears concentrated in the Golgi-centrosomal area, as judged by immunofluorescence and cell fractionation. Using a 32P-labelled RII overlay on Western blots, a 350-kDa RII-binding protein (AKAP 350) was specifically identified in centrosomes isolated from this cell line, whereas a Golgi fraction has previously been demonstrated to contain an 85-kDa RII-binding protein (AKAP 85). AKAP 350 is highly insoluble and can partially be extracted from centrosomes as a complex of AKAP 350 and RII subunit. AKAP 350 was identified as a specific centrosomal protein previously demonstrated in the pericentriolar material. The potential significance of a specific subcellular distribution for different RII-binding proteins in nonneuronal cells is discussed.
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PMID:A high-affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II in the centrosome of human cells. 844 Mar 20

The cytosolic cAMP activates in eukaryotic cells several isoforms of cAMP-dependent protein kinase (PKAs) involved in signal transduction. The effects of individual PKA isoforms are determined by their cellular localisation, specified through binding to distinct A Kinase Anchor Proteins (AKAPs). A new member of the AKAP family, a membrane-anchored 903 amino acid long protein, designated AKAP149, is characterized in the present work. It is a putative splicing variant of S-AKAP84 with the important new feature of a RNA-binding motif (KH domain). This domain together with the known characteristics of AKAPs suggests the involvement of AKAP149 in the phosphorylation-dependent regulation of RNA-processing.
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PMID:Molecular characterization of AKAP149, a novel A kinase anchor protein with a KH domain. 876 36

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.
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PMID:Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. 900 65

Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with protein kinase A-anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause a loss of PKA modulation of cellular responses. In this report we use S-Ht31, a cell-permeant anchoring inhibitor peptide, to study the role of PKA anchoring in sperm. Our analysis of three species of mammalian sperm detected three isoforms of PKA (RIIalpha, RIIbeta, and RIbeta) and one 110-kDa AKAP. The addition of S-Ht31 to bovine caudal epididymal sperm inhibits motility in a time- and concentration-dependent manner. A control peptide, S-Ht31-P, identical to S-Ht31 except for a proline for isoleucine substitution to prevent amphipathic helix formation, had no effect on motility. The inhibition of motility by S-Ht31 is reversible but only if calcium is present in the suspension buffer, suggesting a role for PKA anchoring in regulating cellular calcium homeostasis. Surprisingly, inhibition of PKA catalytic activity had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation. These data suggest that the interaction of the regulatory subunit of PKA with sperm AKAPs, independent of PKA catalytic activity, is a key regulator of sperm motility and that disruption of this interaction using cell-permeable anchoring inhibitor peptides may form the basis of a sperm-targeted contraceptive.
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PMID:Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility. 903 May 27

Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.
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PMID:Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits. 906 79

Experiments were designed to test the idea that A kinase anchor proteins (AKAPs) tether regulatory subunits (RII) of protein kinase AII (PKAII) isoforms to surfaces of organelles that are bounded by phospholipid bilayers. S-AKAP84, one of three RII-binding proteins encoded by a single-copy murine gene, was studied as a prototypic organelle-associated AKAP. When S-AKAP84 was expressed in HEK293 cells, the anchor protein was targeted to mitochondria and excluded from other cell compartments. The RII tethering site is located in the cytoplasm adjacent to the mitochondrial surface. Endogenous RII subunits are not associated with mitochondria isolated from control cells. Expression of S-AKAP84 in transfected HEK293 cells triggered a redistribution of 15% of total RII to mitochondria. Thus, the tethering region of the organelle-inserted anchor protein is properly oriented and avidly binds RII (PKAII) isoforms in intact cells. Two critical domains in S-AKAP84 were mapped. Residues 1 to 30 govern insertion of the polypeptide into the outer mitochondrial membrane; amino acids 306-325 constitute the RII-binding site. Properties established for S-AKAP84 in vitro and in situ strongly suggest that a physiological function of this protein is to concentrate and immobilize RII (PKAII) isoforms at the cytoplasmic face of a phospholipid bilayer.
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PMID:Organelle-specific targeting of protein kinase AII (PKAII). Molecular and in situ characterization of murine A kinase anchor proteins that recruit regulatory subunits of PKAII to the cytoplasmic surface of mitochondria. 918 49


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