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)

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

Protein phosphorylation is a primary means of mediating signal transduction events that control cellular processes. Accordingly, the activities of protein kinases and phosphatases are highly regulated. One level of regulation is that the subcellular distribution of several kinases and phosphatases is restricted by association with targeting proteins or subunits. This mechanism promotes rapid and preferential modulation of specific targets within a defined microenvironment in response to diffusible second messengers. The type II cAMP-dependent protein kinase (PKA) is targeted by association of its regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). To date, 36 unique AKAPs have been identified. Each of these proteins contains a conserved amphipathic helix responsible for AKAP association with cellular structures. Disruption of PKA/AKAP interaction with peptides patterned after the amphipathic helix region blocks certain cAMP responses, including the modulation of glutamate receptor ion-channel activity in neurons and transcription of cAMP-responsive genes. Yeast two-hybrid screening methods have identified neuronal specific AKAP79-binding proteins including the beta isoform of the phosphatase 2B, calcineurin. Biochemical and immunological studies have confirmed the two-hybrid results and identified additional members of this multienzyme signaling complex, including certain protein kinase C isoforms. These findings are consistent with colocalization of CaN, PKC, and type II PKA by AKAP79 and suggest a novel model for reversible phosphorylation in which the opposing kinase and phosphatase actions are colocalized in a signal transduction complex by association with a common anchor protein.
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PMID:Dissection of protein kinase and phosphatase targeting interactions. 921 Feb 33

Activation of protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development in Drosophila. Thus, PKA-anchor protein complexes may be involved in controlling these crucial biological processes. Evaluation of this proposition requires knowledge of PKA binding/targeting proteins in the fly. We now report the discovery and characterization of cDNAs encoding a novel, Drosophila A kinase anchor protein, DAKAP550. DAKAP550 is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. It binds regulatory subunits (RII) of both mammalian and Drosophila PKAII isoforms. The tethering region of DAKAP550 includes two proximal, but non-contiguous RII-binding sites (B1 and B2). The B1 domain (residues 1406-1425) binds RII approximately 20-fold more avidly than B2 (amino acids 1350-1369). Affinity-purified anti-DAKAP550 IgGs were exploited to demonstrate that the anchor protein is expressed in many cells in nearly all tissues throughout the lifespan of the fly. However, DAKAP550 is highly enriched and asymmetrically positioned in subpopulations of neurons and in apical portions of cells in gut and trachea. The combination of RII (PKAII) binding activity with differential expression and polarized localization is consistent with a role for DAKAP550 in creating target loci for the reception of signals carried by cAMP. The DAKAP550 gene was mapped to the 4F1.2 region of the X chromosome; flies that carry a deletion for this portion of the X chromosome lack DAKAP550 protein.
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PMID:Molecular characterization of a novel A kinase anchor protein from Drosophila melanogaster. 933 42

Distinct A Kinase Anchor Proteins (AKAPs) immobilize and concentrate protein kinase A II (PKAII) isoforms at specific intracellular locations. AKAP121 binds and targets PKAIIalpha to the cytoplasmic surface of mitochondria. Mechanisms that control expression of this mitochondrial AKAP are unknown. We have cloned cDNA for rat AKAP121 and show that AKAP121 protein expression is regulated by thyroid stimulating hormone (TSH) and cAMP. Differentiated thyroid cells (TL5) accumulate AKAP121 upon incubation with TSH or a cAMP analog. Levels of total and newly synthesized AKAP121 mRNA also increased after treatment. AKAP121 mRNA accumulated in the presence of cycloheximide, suggesting that transcription of the anchor protein gene is directly controlled by cAMP and PKA. AKAP121 is induced with similar kinetics when an unrelated, spermatocyte-derived cell line (GC-2) is incubated with 8-chlorophenylthio-cAMP. Thus, AKAP121 concentration may be controlled by hormones that activate adenylate cyclase. This mode of regulation could provide a general mechanism for (a) enhancing the sensitivity of distal organelles to cAMP and (b) shifting the focus of cAMP-mediated signaling from cytoplasm to organelles.
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PMID:Expression of a kinase anchor protein 121 is regulated by hormones in thyroid and testicular germ cells. 972 70

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.
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PMID:The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells. 1003 48

The A126 cell line, a derivative of PC12, is defective in cAMP-induced transcription and does not differentiate in the presence of cAMP. In these cells overexpression of a cAMP-dependent protein kinase (PKA) anchor protein, AKAP75, and of the PKA catalytic subunit substantially increased the fraction of PKAII bound to the membrane, stimulated the transcription of cAMP-induced genes, and induced terminal differentiation. Conversely, wild type PC12 cells expressing a derivative of the AKAP75 protein, AKAP45, which binds the PKA regulatory subunits RII, but fails to locate them to the membranes, induced translocation of PKAII to the cytosol. These cells did not efficiently accumulate PKA catalytic subunit in the nuclei when stimulated with cAMP, did not transcribe cAMP-induced genes, and failed to differentiate when exposed to cAMP. These data indicate that membrane-bound PKA positively controls the transcription of cAMP-induced genes and differentiation in PC12 cells.
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PMID:Membrane-bound cAMP-dependent protein kinase controls cAMP-induced differentiation in PC12 cells. 1055 10

Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIalpha and RIIbeta subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE).R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu(236), Ile(248), and Leu(252)) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His(254)-Arg(255)-Lys(256)) at the C terminus of the tethering site set an upper limit on affinity for R(CE.) A central dipeptide (Phe(243)-Ser(244)) contributes critical and distinctive properties of the tethering site. Ser(244) is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe(243) ensures maximal R(CE) binding activity, thereby supporting a "gatekeeper" function of Ser(244). Substitution of Phe(243)-Ser(244) with Leu-Val generated an RII-specific AKAP. R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys(23), Val(27), Ile(32), and Cys(44)) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu(20), Leu(35), Val(36), Ile(40), and Ile(41) have little impact on complex formation. Tyr(26) is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIalpha. RIalpha (PKAIalpha) is tightly sequestered by AKAP(CE) in vitro (K(D) = approximately 10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.
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PMID:Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms. 1066 Jun 5

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.
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PMID:Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase. 1098 83

Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath. In many cases, DFS is familial, suggesting a genetic component. Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase. We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS. No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3. Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A. Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm. Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm. Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans.
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PMID:Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath. 1122 5

We have reported that a novel c-Myc-binding protein, AMY-1 (associate of Myc-1), stimulated the transcription activity of c-Myc. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human HeLa cDNA library, and a clone encoding cAMP-dependent protein kinase anchor protein 149 (AKAP149), was obtained. AMY-1 was found to bind in vitro and in vivo to the regulatory subunit II binding region of AKAP149 and S-AKAP84, a splicing variant of AKAP149 expressed in the testis. AMY-1 was expressed postmeiotically in the testis, as S-AKAP84 was expressed. Furthermore, S-AKAP84 and regulatory subunit II, a regulatory subunit of cAMP-dependent protein kinase, made a ternary complex in cells, and AMY-1 was localized in the mitochondria of HeLa and sperm in association with AKAP149 and S-AKAP84, respectively. These results suggest that AMY-1 plays a role in spermatogenesis.
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PMID:AMY-1, a c-Myc-binding protein, is localized in the mitochondria of sperm by association with S-AKAP84, an anchor protein of cAMP-dependent protein kinase. 1148 2


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