Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.
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PMID:In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium. 812 14

1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography. 2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin-dependent protein kinase and casein kinases. 3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis. 4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca2+/calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).
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PMID:Rat liver endoplasmic reticulum protein kinases. 818 36

The fungal metabolite BE-23372M is a structurally novel protein kinase inhibitor. Its IC50 for epidermal growth factor (EGF) receptor kinase was 0.03 microM. IC50 values of BE-23372M for other protein tyrosine kinases, erbB-2, p43v-abl, insulin receptor kinase, and p60c-src were 0.42, 1.0, 3.3, and 4.5 microM, respectively, and the IC50 for protein kinase C, a serine/threonine kinase, was 4.1 microM. Cdc2 kinase, casein kinases I and II and cAMP-dependent protein kinase were not inhibited by 20 microM BE-23372M. A kinetic study showed that BE-23372M was competitive with respect to the substrate peptide and to ATP. Autophosphorylation of solubilized EGF receptor kinase was clearly inhibited by 0.1 microM BE-23372M. Autophosphorylation of EGF receptor in A431 cells was also inhibited. These results show that BE-23372M is a potent and specific EGF receptor kinase inhibitor. It should be a valuable tool for EGF receptor kinase research.
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PMID:BE-23372M, a novel and specific inhibitor for epidermal growth factor receptor kinase. 818 23

Following in situ renaturation and assay of protein kinase activity after denaturing electrophoresis of relatively impure samples of maize phosphoenolpyruvate carboxylase (PEPC) kinase, a approximately 30-kDa polypeptide was implicated as the best candidate for the PEPC kinase catalytic subunit. This kinase's apparent native molecular weight was estimated at 28,000 by gel filtration on a calibrated Superose 12 column (HR 10/30), suggesting that the isolated PEPC kinase is monomeric. This protein-serine kinase was partially purified about 4000-fold from illuminated maize leaves by ammonium sulfate precipitation and sequential chromatography on Ultrogel AcA 54, hydroxylapatite, blue dextran-agarose, and an analytical AcA 54 column. Analysis by denaturing electrophoresis revealed that a 30-kDa polypeptide copurified with PEPC kinase activity during the final step. This highly purified kinase had an apparent Km (PEPC subunit) of 2.5 microM and a Km (total ATP) of 40 microM at pH 8.0, its pH optimum. Upon in vitro phosphorylation of darkform (dephospho) C4 PEPC at Ser-15 (maize PEPC) or Ser-8 (sorghum), the malate sensitivity of the target enzyme decreased significantly. The maize PEPC kinase activity was markedly inhibited by L-malate, a negative allosteric effector of its protein substrate, in a concentration- and pH-dependent manner. Comparative phosphorylation studies with the catalytic subunit of mammalian cAMP-dependent protein kinase and casein revealed that a significant part of the malate inhibition of PEPC kinase activity in vitro was due to this effector's interaction with PEPC. The activity of both the highly purified PEPC kinase and a less pure sample prepared rapidly in the presence of various protease inhibitors was insensitive to Ca2+ chelation or addition. It is concluded that the approximately 30-kDa maize PEPC kinase is a low abundance, Ca(2+)-independent protein-serine kinase that activates its target enzyme by the exclusive phosphorylation of the regulatory serine residue near the N terminus and the resulting decrease in feedback inhibition by L-malate.
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PMID:Partial purification and characterization of phosphoenolpyruvate carboxylase protein-serine kinase from illuminated maize leaves. 834 24

We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.
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PMID:Identification of a serine/threonine-specific protein phosphatase from the archaebacterium Sulfolobus solfataricus. 838 14

Stimulation of fibroblasts with serum growth factors results in the rapid activation of a set of immediate-early genes, among them 3CH134. We have purified a bacterially expressed form of the 3CH134-encoded polypeptide and demonstrated that it has intrinsic protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in vitro. This activity is optimal at pH 7.5, is sensitive to vanadate and cysteinyl modifying agents, and is insensitive to a panel of serine/threonine phosphatase inhibitors. Purified 3CH134 protein displays a high degree of selectivity among the tyrosine-phosphorylated polypeptide substrates tested. Under our assay conditions, the rates of dephosphorylation are in the order EDNDYINASL peptide < myelin basic protein < reduced, carboxyamidomethylated, and maleylated lysozyme (RCML) < p42mapk. There is a 200-fold range in rates for these substrates, with p42mapk dephosphorylated 15-fold more rapidly than RCML. Although 3CH134 is most closely related to the tyrosine/serine dual-specificity phosphatase VH1, we failed to detect any 3CH134-directed activity on casein or RCML phosphorylated on serine/threonine residues by cAMP-dependent protein kinase. Since 3CH134 expression is controlled transcriptionally and posttranscriptionally, it may represent a class of PTPases whose activity is regulated at the level of protein synthesis and degradation.
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PMID:The growth factor-inducible immediate-early gene 3CH134 encodes a protein-tyrosine-phosphatase. 838 79

Casein kinase II (CKII) is composed of a catalytic subunit (alpha) and a regulatory subunit (beta) that combine to form an alpha 2 beta 2 holoenzyme. The alpha-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme, and the addition of purified beta subunit stimulates its activity against casein (C. Cochet and E. M. Chambaz, 1983, J. Biol. Chem. 258, 1403-1406). Here we report a kinetic analysis of the phosphorylation of various protein and peptide substrates by the alpha subunit and the holoenzyme of Drosophila melanogaster CKII. We demonstrate that the alpha subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (RII) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as alpha-casein and the synthetic peptide RRREEETEEE. The Km of the alpha subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the alpha subunit alone. While the Km values of the alpha subunit and the holoenzyme for calmodulin are similar, the Vmax of the alpha subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the alpha subunit contains the necessary determinants for CKII substrate specificity, the beta subunit can either inhibit or activate it, in a substrate-dependent manner. Finally, we also demonstrate that polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the alpha subunit as well.
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PMID:Phosphorylation of calmodulin by the catalytic subunit of casein kinase II is inhibited by the regulatory subunit. 842 62

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40-43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent Km values have been determined to be 15 microM for ATP, 1.2 microM for S6 and 10 microM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n - 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.
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PMID:A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase. 859 70

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
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PMID:A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II. 944 23

Ser/Thr protein kinases play important roles in signal transduction pathways that control the proliferation and differentiation of eukaryotic cells. In this paper, we present evidence that emodin, an anthraquinone derivative, selectively inhibits casein kinase II (CKII), a Ser/Thr kinase, as a competitive inhibitor. The results with ethyl acetate extracts of the rhizomes of Rheum palmatum showed that emodin significantly inhibited the activity of cyclin B/cdc2 protein kinase (cdc2). We measured IC50 values for emodin on the activities of several Ser/Thr protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC), cdc2, casein kinases I (CKI) and CKII. Interestingly, emodin inhibited CKII activity with an IC50 value of 2 microM, which was two to three orders of magnitude lower than those against the other kinases. Enzyme kinetic assays showed that emodin inhibited CKII activity as a competitive inhibitor against ATP with a Ki value of 7.2 microM. Collectively, we suggest that emodin is a selective CKII inhibitor, whose action mechanism is mediated through competitively binding to the ATP binding site.
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PMID:Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, selectively inhibits the activity of casein kinase II as a competitive inhibitor. 1008 37


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