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)

Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzyme's affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.
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PMID:In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis. 942 20

The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A Kinase Anchor Protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (M(r) 97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M(r) 109,000 (p109) and M(r) 26,000 (p26, representing the "pro" domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82.
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PMID:Assembly of AKAP82, a protein kinase A anchor protein, into the fibrous sheath of mouse sperm. 944 72

1. Inside-out recordings from rat cortical fused nerve terminals indicate that the most common channel observed was a large conductance K+ (BK) channel with characteristics dissimilar to conventional cell body calcium-activated BK (BKCa) channels. 2. BK channels exhibit mode switching between low (mode 1) and high (mode 2) activity, an effect not influenced by membrane voltage. Increasing internal Ca2+ concentration increased time spent in mode 2 as did application of protein kinase A, an effect not mimicked by protein kinase C or protein kinase G. 3. Mode 1 activity was voltage independent although depolarization increased mode 2 channel activity. Global average channel activity was voltage and Ca2+ dependent. 4. Alkaline phosphatase treatment induced channel activity to reside permanently in mode 2, where activity was voltage and Ca2+ dependent but unaffected by protein kinases A, G or C. 5. Internal application of tetraethylammonium blocked BK channel activity in a manner identical to that reported for BKCa channels. 6. These results indicate that nerve terminal membranes have large conductance K+ channels with significant differences in gating kinetics and regulation of activity compared with BKCa channels of other neuronal preparations. The BK channel subtype may play a unique physiological role specific to the nerve terminal.
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PMID:Mode switching characterizes the activity of large conductance potassium channels recorded from rat cortical fused nerve terminals. 982 14

Lissencephaly, a severe brain malformation, may be caused by mutations in the LIS1 gene. LIS1 encodes a microtubule-associated protein (MAP) that is also part of the enzyme complex, platelet-activating factor acetylhydrolase. LIS1 is also found in a complex with two protein kinases; a T-cell Tat-associated kinase, which contains casein-dependent kinase (CDK) activating kinase (CAK), as well as CAK-inducing activity, and with a spleen protein-tyrosine kinase similar to the catalytic domain of p72syk. As phosphorylation is one of the ways to control cellular localization and protein-protein interactions, we investigated whether LIS1 undergoes this post-translational modification. Our results demonstrate that LIS1 is a developmentally regulated phosphoprotein. Phosphorylated LIS1 is mainly found in the MAP fraction. Phosphoamino acid analysis revealed that LIS1 is phosphorylated on serine residues. Alkaline phosphatase treatment reduced the number of visible LIS1 isoforms. In-gel assays demonstrate a 50-kDa LIS1 kinase that is enriched in microtubule-associated fractions. In vitro, LIS1 was phosphorylated by protein kinase CKII (casein kinase II), but not many other kinases that were tested. We suggest that LIS1 activity may be regulated by phosphorylation.
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PMID:LIS1 is a microtubule-associated phosphoprotein. 1049 Nov 72

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)2D3, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)2D3 treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (Ra 0.54 and 4.92 microm) in the presence of control media or media containing 1,25-(OH)2D3 or 1,25-(OH)2D3 plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A2 inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)2D3 inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)2D3 restored cell number to that seen in the control cultures. Inhibition of phospholipase A2 in the presence of 1,25-(OH)2D3 caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)2D3 on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)2D3 treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)2D3 displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A2 also inhibited the 1,25-(OH)2D3-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)2D3 effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)2 D3 mediate their effects through phospholipase A2, which catalyzes one of the rate-limiting steps in prostaglandin E2 production. Further downstream, prostaglandin E2 activates protein kinase A.
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PMID:Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2. 1061 36

Growth hormone (GH) regulates transcription factors associated with c-fos, including C/EBPbeta. Two forms of C/EBPbeta, liver-activating protein (LAP) and liver inhibitory protein (LIP), are dephosphorylated in GH-treated 3T3-F442A fibroblasts. GH-induced dephosphorylation of LAP and LIP is reduced when cells are preincubated with phosphatidylinositol 3'-kinase (PI3K) inhibitors. GH activates Akt and inhibits glycogen synthase kinase-3 (GSK-3). Lithium, a GSK-3 inhibitor, increases GH-dependent dephosphorylation of LAP and LIP. Both are in vitro substrates of GSK-3, suggesting that GSK-3 inactivation contributes to GH-promoted dephosphorylation of C/EBPbeta. Alkaline phosphatase increases binding of LAP homodimers and decreases binding of LIP homodimers to c-fos, suggesting that dephosphorylation of C/EBPbeta modifies their ability to bind DNA. Both alkaline phosphatase- and GH-mediated dephosphorylation comparably increase binding of endogenous LAP in 3T3-F442A cells. In cells overexpressing LAP and GSK-3, LAP binding decreases, suggesting that GSK-3-mediated phosphorylation interferes with LAP binding. Expression of constitutively active GSK-3 reduced GH-stimulated c-fos promoter activity. These studies indicate that PI3K/Akt/GSK-3 mediates signaling between GH receptor and the nucleus, promoting dephosphorylation of C/EBPbeta. Dephosphorylation increases binding of LAP complexes to the c-fos promoter and may contribute to the participation of C/EBPbeta in GH-stimulated c-fos expression.
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PMID:Growth hormone regulates phosphorylation and function of CCAAT/enhancer-binding protein beta by modulating Akt and glycogen synthase kinase-3. 1127 38

When osteoblasts are cultured on surfaces of increasing microroughness, they exhibit decreases in proliferation, increases in differentiation and local factor production, and enhanced response to 1alpha,25(OH)(2)D(3). The cells interact with surfaces through integrins, which signal by the same pathways used by 1alpha,25(OH)(2)D(3), including protein kinase C via phospholipase C and protein kinase A via phospholipase A(2). This provides opportunities for crosstalk that may contribute to the synergistic effects of surface roughness and the vitamin D metabolite. Because these pathways converge at mitogen-activated protein kinase (MAPK), we tested the hypothesis that the extracellular signal-regulated kinase (ERK1/2) subclass of MAPKs mediates the effects of surface roughness and 1alpha,25(OH)(2)D(3). MG63 osteoblast-like osteosarcoma cells were cultured on commercially pure Ti disks with various surface roughnesses: pretreatment (PT; 0.6 microm average roughness [Ra]), coarse grit-blasted and acid-etched (SLA; 4 microm RA), and titanium plasma-sprayed (TPS; 5.2-microm R(a)). At confluence, cells were treated for 24 h with control media or media containing 10(-7) M 1alpha,25(OH)(2)D(3). One-half of the cultures received 1 microm or 10 microm PD98059, a specific inhibitor of the ERK family of MAPKs. PD98059 alone did not affect proliferation, osteocalcin production, or production of transforming growth factor-beta1 or nitric oxide, regardless of the surface roughness. Alkaline phosphatase was reduced by the inhibition of the ERK family kinases on all surfaces to a comparable extent. However, when PD98059 was added to the cultures with 1alpha,25(OH)(2)D(3), the effects of the seco-steroid were blocked, including the synergistic increases seen in MG63 cells cultured on SLA or TPS. These results indicate that ERK1/2 MAPK is required for the maintenance of alkaline phosphatase at control levels and that the effects of 1alpha,25(OH)(2)D(3) are mediated by ERK1/2. However, the effects of surface roughness are not due to the ERK family of MAPKs. This suggests that alternative pathways may be used, including those mediated by other MAPK subclasses.
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PMID:Osteoblast response to titanium surface roughness and 1alpha,25-(OH)(2)D(3) is mediated through the mitogen-activated protein kinase (MAPK) pathway. 1137 60

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogen-regulated genes.
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PMID:The transcriptional repressor Sp3 is associated with CK2-phosphorylated histone deacetylase 2. 1217 73

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.
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PMID:Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues. 1237 1

Characterization of the phosphate effect on ethylene production by Penicillium digitatum is reported. A low level of phosphate (0.001 millimolar) was about 200 to 500 times as effective as a high phosphate level (100 millimolar) in stimulating ethylene production and the stimulation was readily reversed by addition of phosphate. This phosphate effect did not operate in static cultures. The precursor of ethylene in the stimulated low phosphate system was glutamate but not alpha-ketoglutarate, which is a precursor in static systems. Actinomycin D and cycloheximide effectively inhibited the low phosphate/high ethylene-producing system. Alkaline phosphatase and protein kinase activities were higher in low than in high phosphate systems. We suggest that phosphate level regulates ethylene production by P. digitatum and that the regulation involves a phosphorylation or dephosphorylation reaction of some enzyme system associated with ethylene production. Phosphate-mediated control of ethylene production may also involve the transcriptional and translational machinery of the fungal cell. P. digitatum apparently can produce widely different levels of ethylene by different pathways, depending on culture conditions under which it is grown.
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PMID:Characterization of the Phosphate-mediated Control of Ethylene Production by Penicillium digitatum. 1666 Sep 14


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