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)

CaVPT, a target protein of Ca2(+)-vector from amphioxus muscle, was purified from its complex with CaVP after dissociation by 6 M urea and chromatographies on DEAE-cellulose and calmodulin-Sepharose. The amino acid sequence of CaVPT has been determined. The protein is composed of 243 residues and possesses an unblocked N terminus. Its molecular weight is 26,621, distinctly lower than the apparent molecular weight deduced from electrophoresis on sodium dodecyl sulfate-containing gels. CaVPT contains a potential Asn-linked glycosylation site, four potential protein kinase C phosphorylation sites, and two casein kinase II phosphorylation sites. From the sequence the following three particular domains can be inferred: a collagen-like N-terminal segment, rich in Pro and Ala, that resembles the N-terminal segment of skeletal muscle myosin light chain kinase; next to it (from residues 33 to 50) is located a strongly amphiphilic and basic alpha-helical segment which likely binds the calcium vector protein since a proteolytic cut after Arg50, occurring occasionally during the purification of CaVPT, impairs the binding to immobilized calmodulin. This segment is followed by two immunoglobulin folds. The two immunoglobulin folds typically belong to the C2 subclass and particularly resemble those present in the neural cell surface adhesion molecules NCAM, L1, F11, MAG, TAG-1, fasciclin II, and amalgam. Recently, the presence of immunoglobulin folds of this type has been reported in some intracellular muscular proteins, namely in smooth muscle myosin light chain kinase, striated muscle C protein and titin, as well as in the nematode 600-kDa protein twitchin. From this structural study we can formulate the working hypothesis that CaVPT acts on the structure of the thick filament in muscle or regulates, perhaps via other immunoglobulin fold-containing proteins.
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PMID:Primary structure of the target of calcium vector protein of amphioxus. 224 56

In our previous report we showed cytochrome b5 to be a competitive inhibitor of cAMP-dependent protein kinase (PKA) for interaction with cytochrome P450 (P450). While P450 was phosphorylated, cytochrome b5 was not. The phosphorylation of P450 resulted in an inhibition of its catalytic activity. In this report we attempt to determine the relationship between phosphorylation of P450 from phenobarbital-induced rat and its destruction. The results indicate there is a considerable alteration of P450 IIB1 when it is put into the phosphorylation medium. This includes destruction, i.e., loss of the hemoprotein nature (Soret peak), as well as denaturation, conversion of a proportion of the P450 to P420. The extent of phosphorylation correlated best with the amount of destroyed hemoprotein, and not with the formation of P420. There did not appear to be phosphorylation-dependent formation of apo-P450. Further, prior conversion of the P450 to P420 using sodium deoxycholate showed the same extent of phosphorylation as before the conversion. Thus, intact P450 is not required for phosphorylation nor is phosphorylation a prerequisite for hemoprotein destruction. P450 CAM (CIA1), which has the PKA substrate recognition sequence internalized, likewise undergoes conversion to P420 but this denaturation does not result in phosphorylation. Destruction of CIA1 with 6 M urea, however, did permit phosphorylation by PKA. P450 IIB1 destruction was greatly diminished by cytochrome b5. This stabilization resulted in a decreased degree of phosphorylation as well as an increase in negative ellipticity in circular dichroism, indicative of an increase in the proportion of alpha-helical content in the P450. Suggestions are made that this structural modification caused by cytochrome b5 stabilizes the P450 against denaturation as well as against destruction and phosphorylation. Further, when the P450 IIB1 was kept stable as P450 in the absence of cytochrome b5 and without loss of hemoprotein during the incubation period, using phosphate-glycerol buffer containing 0.4% Emulgen 911, the phosphorylation of the P450 was greatly diminished, with only minor effects on the protein kinase reaction itself. These results suggest that the protein kinase reaction itself. These results suggest that the protein kinase substrate recognition sequence is not readily accessible to PKA in mammalian P450 IIB1 but requires a destabilization of the protein for phosphorylation to take place.
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PMID:Relationship between phosphorylation and cytochrome P450 destruction. 227 44

The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
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PMID:Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. 230 51

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. "High dose" cycloheximide (200 micrograms/ml) inhibited flow immediately. "Low dose" cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder. 244 2

In 32Pi-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. It was purified from bovine neutrophil cytosol by a series of chromatographic steps, including ion exchange on DE-52 cellulose and Mono Q, and filtration on Bio-Gel P60 in the presence of mercaptoethanol and urea. The apparent molecular mass of the purified protein, assessed by SDS-PAGE and mercaptoethanol by reference to protein markers, ranged between 20 and 23 kDa, depending on the percentage of polyacrylamide and conditions of migration. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Some properties of the 23-kDa protein, including its amino acid composition, were determined. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of four discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [gamma-32P]ATP in the presence of bovine neutrophil PKC supplemented with Ca2+, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. The apparent KM of ATP was 9 microM. The 23-kDa protein was also phosphorylated by PKM, the catalytic fragment of PKC obtained after removal of the regulatory domain, but not by cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization. 251 5

Evidence is presented that distinct cellular signal transduction pathways involving cAMP-dependent protein kinase-A and phorbol ester-stimulated protein kinase-C coordinately modulate gene transcription through common as well as distinct cis-acting elements and DNA-binding proteins. When transfected and expressed in HeLa and placental JEG-3 cells, fusion reporter plasmids that differ only by a single base deletion or addition to interconvert the octameric cAMP-responsive element TGACGTCA (CRE) to form the heptameric phorbol ester-responsive element TGACTCA (TRE) are differentially regulated by cAMP and phorbol esters [12-O-tetradecanoyl phorbol-14-acetate (TPA)]. Transcription directed by the CRE is stimulated by cAMP and not TPA, although the basal expression mediated by this element in JEG-3 and HeLa cells is augmented by endogenous protein kinase-C activity. In contrast, TRE mediates transcriptional responses to both cAMP and TPA, and the two agents together give synergistic responses. Inhibition of cAMP-dependent protein kinase-A by expression of a minigene encoding a peptide inhibitor of A-kinase abolishes the response of TRE to cAMP alone as well as the cAMP-induced component of the synergistic response to treatment with both TPA and cAMP. Desensitization of the protein kinase-C dependent pathway by prolonged exposure of cells to phorbol esters eliminates the TPA-induced transcription by TRE and inhibits the TPA-induced component of the synergistic response to both cAMP and TPA. Therefore, both protein kinases, A and C, are involved in transcriptional activation by the TRE; the function of either kinase alone results in a moderate level of activity, but the combined results of both functionally stimulated kinases are synergistically positive. Electrophoretic mobility shift assays using whole extracts of JEG-3 cells indicate that a common factor(s) binds both TRE and CRE; however, another factor(s) that binds to the CRE will not bind to the TRE. Further, a latent regulatory enhancer element (URE) located upstream of the CRE's in the human alpha gonadotropin gene, although inactive when paired alone with the alpha 100 promoter, induces basal and stimulated transcriptional activity of both CRE and TRE on the average of 10- to 20-fold. The data support the existence of a gene regulatory network consisting of related cis-acting elements and DNA-binding proteins whose transcriptional activities are regulated by the convergent actions of protein kinases-C and -A.
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PMID:Distinct adenosine 3',5'-monophosphate and phorbol ester-responsive signal transduction pathways converge at the level of transcriptional activation by the interactions of DNA-binding proteins. 254 58

A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.
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PMID:Purification of an active EGF receptor kinase with monoclonal antireceptor antibodies. 257 26

The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E. coli. The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea. The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide. The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay. This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function.
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PMID:Expression of the kinase domain of mouse protein kinase C in E. coli. 267 45

Rat liver fructose-1,6-bisphosphatase was phosphorylated with [32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. After digestion with trypsin, two peptides were isolated containing 68 and 32% of the total radioactivity, respectively. The former was found to contain the sequence Ala-Lys-Ser(P)-Arg-Pro-Ser(P)-Leu-Pro. In this fragment, Ser-341, but not Ser-338, had earlier been reported to be a phosphorylation site. The other peptide contained phosphorylated Ser-356. It was demonstrated that all the protein-bound [32P]phosphate was distributed evenly between these three serines in the native enzyme regardless of the degree of phosphorylation. Preservation of the three-dimensional structure, however, was needed to obtain phosphorylation of Ser-356. Peptides containing each phosphorylatable serine residue were sequentially removed by digesting the enzyme with chymotrypsin which cleaved off Ser-356, denaturing it with urea, digesting it further with chymotrypsin, thus removing Ser-341, and finally treating it with trypsin which eliminated the rest of the radioactivity which was bound to Ser-338. Kinetic studies of fructose-1,6-bisphosphatase digested in this manner revealed that phosphorylation of Ser-338 decreased the apparent Km for fructose 1,6-bisphosphatase, whereas phosphorylation of Ser-341 decreased the inhibitory effect of AMP and fructose 2,6-bisphosphatase, Phosphorylation of Ser-356 did not affect these parameters.
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PMID:Rat liver fructose-1,6-bisphosphatase. Identification of serine 338 as a third major phosphorylation site for cyclic AMP-dependent protein kinase and activity changes associated with multisite phosphorylation in vitro. 282 3

A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorylation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).
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PMID:Protein kinase activity associated with the nuclear lamina. 283 26


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