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)

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) and other signaling enzymes to distinct subcellular organelles. Using the yeast two-hybrid approach, we demonstrate that Rab32, a member of the Ras superfamily of small molecular weight G-proteins, interacts directly with the type II regulatory subunit of PKA. Cellular and biochemical studies confirm that Rab32 functions as an AKAP inside cells. Anchoring determinants for PKA have been mapped to sites within the conserved alpha5 helix that is common to all Rab family members. Subcellular fractionation and immunofluorescent approaches indicate that Rab32 and a proportion of the cellular PKA pool are associated with mitochondria. Transient transfection of a GTP binding-deficient mutant of Rab32 promotes aberrant accumulation of mitochondria at the microtubule organizing center. Further analysis of this mutant indicates that disruption of the microtubule cytoskeleton results in aberrantly elongated mitochondria. This implicates Rab32 as a participant in synchronization of mitochondrial fission. Thus, Rab32 is a dual function protein that participates in both mitochondrial anchoring of PKA and mitochondrial dynamics.
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PMID:Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics. 1218 51

Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto alpha-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.
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PMID:Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS). 1220 84

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). We examine the functional importance of PKA anchoring during Drosophila oogenesis by analyzing membrane integrity and actin structures in mutants with disruptions in Akap200, an AKAP. In wild-type ovaries, Pka-RII and Akap200 localized to membranes and to the outer rim of ring canals, actin-rich structures that connect germline cells. In Akap200 mutant ovaries, Pka-RII membrane localization decreased, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also had enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline resulted in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, we examined genetic interactions with other genes that are known to regulate ring canal morphology. Akap200 mutations suppressed the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism.
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PMID:An A-kinase anchoring protein is required for protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila. 1222 1

Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinase anchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes with N-methyl-d-aspartic acid or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315-360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315-360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330-357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity.
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PMID:Mapping the protein phosphatase-2B anchoring site on AKAP79. Binding and inhibition of phosphatase activity are mediated by residues 315-360. 1235 62

Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger-mediated signaling events. Localization of the cAMP-dependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). This study focused on recent advances that further our understanding of AKAPs, with particular emphasis on the bidirectional regulation of signaling events by AKAP signaling complexes and their contribution to the control of actin reorganization events.
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PMID:Intracellular targeting of protein kinases and phosphatases. 1247 80

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.
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PMID:Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy. 1250 94

We have used differential display to profile and compare the mRNAs expressed in the hippocampus of freely moving animals after the induction of long-term potentiation (LTP) at the perforant path-dentate gyrus synapse with control rats receiving low-frequency stimulation. We have combined this with in situ hybridization and have identified A-kinase anchoring protein of 150 kDa (AKAP-150) as a gene selectively up-regulated during the maintenance phase of LTP. AKAP-150 mRNA has a biphasic modulation in the dentate gyrus following the induction of LTP. The expression of AKAP-150 was 29% lower than stimulated controls 1 h after the induction of LTP. Its expression was enhanced 3 (50%), 6 (239%) and 12 h (210%) after induction, returning to control levels by 24 h postinduction. The NMDA receptor antagonist CPP blocked the tetanus-induced modulation of AKAP-150 expression. Interestingly, strong generalized stimulation produced by electroconvulsive shock did not increase the expression of AKAP-150. This implies that the AKAP-150 harbours a novel property of selective responsiveness to the stimulation patterns that trigger NMDA-dependent LTP in vivo. Its selective up-regulation during LTP and its identified functions as a scaffold for protein kinase A, protein kinase C, calmodulin, calcineurin and ionotropic glutamate receptors suggest that AKAP-150 encodes is an important effector protein in the expression of late LTP.
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PMID:LTP but not seizure is associated with up-regulation of AKAP-150. 1254 70

GABA(A) receptors, the key mediators of fast synaptic inhibition in the brain, are predominantly constructed from alpha(1-6), beta(1-3), gamma(1-3), and delta subunit classes. Phosphorylation by cAMP-dependent protein kinase (PKA) differentially regulates receptor function dependent upon beta subunit identity, but how this kinase is selectively targeted to GABA(A) receptor subtypes remains unresolved. Here we establish that the A-kinase anchoring protein 150 (AKAP150), directly binds to the receptor beta1 and beta3, but not to alpha1, alpha2, alpha3, alpha6, beta2, gamma2, or delta subunits. Furthermore, AKAP79/150 is critical for PKA-mediated phosphorylation of the receptor beta3 subunit. Together, our observations suggest a mechanism for the selective targeting of PKA to GABA(A) receptor subtypes containing the beta1 or beta3 subunits dependent upon AKAP150. Therefore, the selective interaction of beta subunits with AKAP150 may facilitate GABA(A) receptor subtype-specific functional modulation by PKA activity which may have profound local effects on neuronal excitation.
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PMID:A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA(A) receptors by cAMP-dependent protein kinase via selective interaction with receptor beta subunits. 1259 41

Compartmentalization of the cAMP-dependent protein kinase (PKA) is coordinated through association with A-kinase anchoring proteins (AKAPs). A defining characteristic of most AKAPs is a 14- to 18-aa sequence that binds to the regulatory subunits (RI or RII) of the kinase. Cellular delivery of peptides to these regions disrupts PKA anchoring and has been used to delineate a physiological role for AKAPs in the facilitation of certain cAMP-responsive events. Here, we describe a bioinformatic approach that yields an RII-selective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a K(d) of 0.4 nM and binds RI with a K(d) of 277 nM. AKAP-IS associates with the type II PKA holoenzyme inside cells and displaces the kinase from natural anchoring sites. Electrophysiological recordings indicate that perfusion of AKAP-IS evokes a more rapid and complete attenuation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computer-based and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.
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PMID:Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring. 1267 69

Microtubule-associated protein-2 (MAP2) is a brain specific A-kinase anchoring protein that targets the cyclic AMP-dependent protein kinase holoenzyme (PKA) to microtubules. Phosphorylation of MAP2 by different protein kinases is crucial for neuronal growth. The N-terminus of MAP2 contains the binding site for regulatory subunit II of cAMP-dependent protein kinase (PKA-RIIbeta). Using homologous recombination, we created a mutant line of mice (delta1-158) that express truncated MAP2 lacking the N-terminal peptide and the PKA binding site. Deletion of the PKA binding site from the MAP2 gene resulted in decreased efficiency of MAP2 phosphorylation. Biochemical and immunohistochemical studies demonstrate major changes in the morphology of hippocampal neurons in delta1-158 mice. Behavioral tests indicate that delta1-158 mice were impaired (exhibited less conditioned freezing) relative to Wild-Type (WT) controls during a test of contextual, but not during auditory cue, fear conditioning when tested at 8 weeks or 8 months of age. The delta1-158 mice displayed a heightened sensitivity to shock at 8 weeks, but not at 8 months of age. We conclude that PKA binding to MAP2 and MAP2 phosphorylation is essential for the selective development of contextual memory.
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PMID:Deletion of the N-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory. 1276 72


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