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)

In this study we have used mature, primary cultured mouse cerebellar granule cells (CGCs) to initiate our studies on the mechanisms governing neuronal trafficking of GABAA receptors (GABARs). Initially the steady-state distribution of GABAR alpha1, alpha6, beta2 and beta3 subunits between the cell surface and cell interior was quantified. Cell surface proteins were modified with a membrane-impermeable cross-linking agent, bis(sulfosuccinimidyl)suberate (BS3) or the proteolytic enzyme, chymotrypsin. The proportion of unmodified (intracellular) and modified (cell surface) subunits was quantified by immunoblotting. We found that 51% of alpha6, 74% of alpha1, and 83% of beta2/3 were expressed at the cell surface, thus identifying a sizeable intracellular pool of alpha6 in contrast to the low levels of intracellular alpha1 and beta2/3. Chronic activation of protein kinase A (PKA) in CGCs in vitro, post-transcriptionally up-regulated expression of alpha1, beta2 and beta3 but not beta6. This was paralleled by an increase in the BZ-S subtype of [3H]Ro154513 binding sites. GABAR alpha1 was increased at the cell surface and in the cell interior, beta2 was increased almost exclusively at the cell surface whilst beta3 was increased almost exclusively in the cell interior. The intracellular pool of alpha6 was not affected. Thus, GABAR subunits are subject to differentially regulated trafficking, affording yet greater scope for GABAR diversity and plasticity.
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PMID:Differential cell surface expression of GABAA receptor alpha1, alpha6, beta2 and beta3 subunits in cultured mouse cerebellar granule cells influence of cAMP-activated signalling. 1190 22

Isoprenoid inhibitors are being evaluated as agents for the treatment of cancer. Their antitumor activity is attributed to inhibition of post-translational modification of Ras, which is crucial for its translocation and attachment to the plasma membrane, and ultimate involvement in signal transduction. However, whether blocking of Ras is solely responsible for the observed antitumor activity is unresolved. In this report, we propose an alternate mechanism. Using breast tumor models, we show that agents possessing a lactone moiety, including statins (such as lovastatin) and the isoprenoid inhibitors (such as FTI-277 and GGTI-298), mediate their cell cycle inhibitory activities by blocking the chymotrypsin activity of the proteasome in vitro. This results in the accumulation of cyclin-dependent kinase inhibitors p21 and p27 with subsequent G(1) arrest. Cells devoid of p21 were refractory to the growth-inhibitory activity of lovastatin, FTI-277, and GGTI-298. However, in these p21 null cells, isoprenylation of key substrates of farnesyl transferase (such as Ras) and of geranylgeranyl transferase (such as RAP-1) were inhibited by FTI-277 and GGTI-298, respectively, suggesting that although both these isoprenoid inhibitors reached and inhibited their intended targets, inhibition of the isoprenylation of Ras and RAP-1A are not sufficient to mediate G(1) arrest. We also show that the cell cycle effects can be attributed to the functional lactone moiety of the aforementioned agents. Collectively, our data suggest that FTI and GGTI and other agents containing an active lactone moiety mediate G(1) arrest via inhibition of the proteasome and up-regulation of p21, independent of the inhibition of isoprenylation of Ras or RAP-1.
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PMID:Farnesyl and geranylgeranyl transferase inhibitors induce G1 arrest by targeting the proteasome. 1642 40

The malarial parasite Plasmodium falciparum has two nucleosome assembly proteins, PfNapS and PfNapL (Chandra, B. R., Olivieri, A., Silvestrini, F., Alano, P., and Sharma, A. (2005) Mol. Biochem. Parasitol. 142, 237-247). We show that both PfNapS and PfNapL interact with histone oligomers but only PfNapS is able to deposit histones onto DNA. This property of PfNapS is divalent cation-dependent and ATP-independent. Deletion of the terminal subdomains of PfNapS abolishes its nucleosome assembly capabilities, but the truncated protein retains its ability to bind histones. Both PfNapS and PfNapL show binding to the linker histone H1 suggesting their probable role in extraction of H1 from chromatin fibers. Our data suggests distinct sites of interaction for H1 versus H3/H4 on PfNapS. We show that PfNapS and PfNapL are phosphorylated both in vivo and in vitro by casein kinase-II, and this modification is specifically inhibited by heparin. Circular dichroism, fluorescence spectroscopy, and chymotrypsin fingerprinting data together suggest that PfNapL may undergo very small and subtle structural changes upon phosphorylation. Specifically, phosphorylation of PfNapL increases its affinity 3-fold for core histones H3, H4, and for the linker histone H1. Finally, we demonstrate that PfNapS is able to extract histones from both phosphorylated and unphosphorylated PfNapL, potentially for histone deposition onto DNA. Based on these results, we suggest that the P. falciparum NapL is involved in the nucleocytoplasmic relay of histones, whereas PfNapS is likely to be an integral part of the chromatin assembly motors in the parasite nucleus.
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PMID:The two Plasmodium falciparum nucleosome assembly proteins play distinct roles in histone transport and chromatin assembly. 1663 44

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.
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PMID:Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins. 1697 Sep 5

The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. In 1999, FDA allowed a health claim on food labels stating that a daily diet containing 25 grams of soy protein, also low in saturated fat and cholesterol, may reduce the risk of heart disease [corrected] This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor.
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PMID:The biochemical and functional food properties of the bowman-birk inhibitor. 1827 67

The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO(2)), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential beta5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (beta1 caspase-like, beta2 trypsin-like, and beta5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.
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PMID:Revealing the dynamics of the 20 S proteasome phosphoproteome: a combined CID and electron transfer dissociation approach. 1857 62

The present study was designed to characterize the urinary bladder-derived relaxant factor that was demonstrated by acetylcholine-induced relaxation response in a coaxial bioassay system consisting of rat bladder as the donor organ and rat anococcygeus muscle as the assay tissue. The concentration-dependent relaxation to acetylcholine (10 nM-1 mM) was inhibited by atropine but was not altered by the antagonists of calcitonin gene-related peptide (CGRP 8-37), vasoactive intestinal peptide (VIP 6-28), tachykinin NK1 (L-732138), tachykinin NK2 (MEN-10376), tachykinin NK3 (SB-218795), purinergic P2 (PPADS) and adenosine (CGS 15943) receptors as well as alpha-chymotrypsin. Adenylate cyclase inhibitor SQ-22536 and protein kinase A inhibitor KT-5720 significantly inhibited the acetylcholine response while guanylate cyclase inhibitor ODQ, and protein kinase C inhibitor H-7 did not have any effect. The P2X agonist alpha,beta-methylene ATP (10 nM-0.1 mM) also produced concentration-dependent relaxation response that was inhibited by PPADS, SQ-22536 and KT-5720 in the coaxial bioassay system. In bladder strips, acetylcholine and alpha,beta-methylene ATP elicited concentration-dependent contractions that were not altered in the presence of SQ-22536 and KT-5720. In conclusion, the urinary bladder-derived relaxant factor that was recognized by the coaxial bioassay system is neither a peptide of the bladder neurons nor a purinergic mediator but adenylate cyclase and protein kinase A are involved in its release and/or relaxant effect. Furthermore, activation of purinergic P2X receptors besides the muscarinic receptors leads to the release of this factor.
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PMID:Rat urinary bladder-derived relaxant factor: studies on its nature and release by coaxial bioassay system. 1862 Oct 43

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.
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PMID:Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry. 1954

The catalytic subunit of recombinant wild-type cyclic adenosine monophosphate-dependent protein kinase A (PKA) has been analyzed by a combination of 1D gel electrophoresis, in-gel digestion by trypsin, chymotrypsin, or endoproteinase AspN, and nano-ultraperformance liquid chromatography--MS/MS. The MS/MS spectra were annotated by MASCOT and the annotations were manually controlled. Using Ga(III)-immobilized metal ion affinity chromatography (IMAC), in addition to the four established autophosphorylation sites of the catalytic subunit of recombinant PKA, pSer10, pSer139, pThr197, and pSer338, six new phosphorylated residues have been characterized--pSer14, pThr48, pSer53, pSer212, pSer259, and pSer325. The established phosphorylation sites all are part of a PKA consensus motif and were found to be almost completely modified. In contrast, the newly detected sites were only partially phosphorylated. For estimation of their degree of phosphorylation, a method based on signal intensity measurements was used. For this purpose, signal intensities of all phospho- and non-phosphopeptides containing a particular site were added for estimation of site-specific phosphorylation degrees. This addition was performed over all peptides observed in the different digestion experiments, including their different charge states. pThr48 and pSer259 are located within PKA consensus motifs and were observed to be phosphorylated at 20% and 24%, respectively. pSer14 and pSer53 are located within inverted PKA consensus motifs and were found to be phosphorylated around 10% and 15%, respectively. The sequence environments of pSer212 and pSer325 have no similarity to the PKA consensus motif at all and were observed to be phosphorylated at about 5% or lower. All newly observed phosphorylation sites are located at the surface of the native protein structure of the PKA catalytic subunit. The results add new information on the theme of site-specific (auto)phosphorylation by PKA.
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PMID:Analysis of autophosphorylation sites in the recombinant catalytic subunit alpha of cAMP-dependent kinase by nano-UPLC-ESI-MS/MS. 1959 Aug 56

The largest component of the human heart, the left ventricle (LV), plays a major role in delivering blood throughout the body. Therefore, an in-depth detailed quantitative proteome analysis of the human LV is a valuable resource. For this purpose, a multifaceted proteomics approach combining differential sample fractionations (gel, strong cation exchange (SCX)), enzymatic digestions (trypsin, chymotrypsin, LysN), and peptide fragmentation techniques (CID and ETcaD) was used to enhance protein sequence coverage, identification confidence and quantitative abundance determination. Using stringent criteria, 3584 distinct proteins could be identified from the latest well-annotated Swissprot database (23,000 entries). Commutatively, the over 130,000 identified MS/MS spectra were used to assess concentrations of each identified LV protein through a combination of spectral counting methods. Among the most concentrated proteins, many currently used biomarkers for detection of myocardial infarction reside. These cardiac leakage markers have a good diagnostic power, but their prognostic potential seems limited. Discovery of markers that represent etiological determinants of cardiac disease require a shift of focus towards the signaling proteome. Therefore, a protein-class centered quantitative analysis of kinases, phosphatases and GTPases was adopted. These comparative analyses revealed many cardiac involved kinases (PKA, CaMKII, ERK) to reside among the most abundant signaling proteins, and also to mediate many observed in vivo phosphorylation sites. The abundance chart of signaling proteins may assist in identifying novel functional pathways, for instance through the abundant, but relatively little known, kinases STK38L and OXSR1. The obtained quantitative protein library of the human left ventricle is a valuable resource to isolate signaling based, putative biomarkers with concentrations likely to be detectable in plasma.
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PMID:Proteome-wide protein concentrations in the human heart. 2059 66


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