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)

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.
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PMID:Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family. 186 33

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.
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PMID:Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. 192 62

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.
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PMID:Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart. 154 21

Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.
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PMID:Molecular cloning and expression of glycogen synthase kinase-3/factor A. 216 70

Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.
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PMID:A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin. 226 35

Five rabbit cDNAs, encoding four conventional protein kinase Cs (PKCs), alpha, beta I, beta II, and gamma, and a novel PKC-related protein (nPKC epsilon) were transfected into COS cells. Antisera raised against a bacterially synthesized fragment of PKC alpha or nPKC epsilon and against a chemically synthesized peptide of PKC beta I or beta II, specifically identified the corresponding species in the transfected cells. All four PKCs and nPKC epsilon expressed by transfection served as phorbol ester receptors. Phorbol 12,13-dibutyrate (PDBu)-binding activities of all PKCs and nPKC epsilon required phospholipid but not magnesium. The phosphatidylserine requirement for the activity of nPKC epsilon is independent of Ca2+ and similar to that for PKC alpha observed at 0.03 mM Ca2+. Calcium dependence of the binding activity was observed only for the four conventional PKCs. Scatchard plot analysis clearly showed that the dissociation constants of PDBu for all four PKCs were nearly the same (approximately 25 nM) in the presence of Ca2+, and that the value for nPKC epsilon was slightly higher (84 nM) and independent of Ca2+. The latter value is comparable to those observed in several cell types under conditions of Ca2+ chelation. Translocation of conventional PKC alpha to the membranes was induced with phorbol ester in a Ca2+-dependent manner, whereas the PDBu-stimulated translocation of nPKC epsilon did not require Ca2+. These results, together with previous studies on the enzymological characteristics of nPKC epsilon (Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53, 731-741), suggest that nPKC epsilon plays an important role in a transmembrane signaling pathway distinct from that involving conventional PKCs.
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PMID:Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C. 229 8

The effect of 2-aminopurine (2AP), an inhibitor of the RNA-dependent P1/eIF-2 protein kinase, on the expression of the reovirus serotype 1 Lang strain S1 and S4 genes in transfected simian COS cells was examined. In the absence of 2AP, the s4-encoded sigma 3 gene product was expressed about five times more efficiently than the s1-encoded sigma 1 gene product. When COS cells were treated with 2AP, the synthesis of the sigma 1 polypeptide was increased about fivefold compared to that in untreated cells even though s1 mRNA levels were not detectably altered. In contrast to the increased translational efficiency of the s1 mRNA observed in 2AP-treated cells, the translational efficiency of the s4 mRNA was not affected by 2AP treatment. However, the cytoplasmic accumulation of s4 mRNA was transiently decreased by 2AP treatment. These results demonstrate that the expression of the reovirus S1 and S4 genes in transient transfection assays is differentially affected by 2AP. Furthermore, when considered together with the prior observation that the reovirus s1 mRNA is a potent activator of the RNA-dependent protein kinase relative to the s4 mRNA which is a very poor activator, the results are consistent with the suggestion that the differential translational efficiency of the reovirus s1 and s4 mRNAs in vivo may be attributed in part to their differential ability to activate the P1/eIF-2 protein kinase.
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PMID:Biosynthesis of reovirus-specified polypeptides. 2-aminopurine increases the efficiency of translation of reovirus s1 mRNA but not s4 mRNA in transfected cells. 233 Jun 70

A chimeric G alpha subunit cDNA, referred to as G alpha s/i(38), was constructed containing the complete 5'-untranslated region of G alpha s, the first 356 codons of the rat G alpha s and the last 36 codons and 428 base pairs of the 3'-untranslated region of the rat G alpha i cDNA. Transient expression of the G alpha s/i(38) protein in COS cells allowed detection of a chimeric protein which was recognized by antibodies generated against an internal G alpha s sequence as well as antibodies recognizing the carboxyl terminus of G alpha i2. Chinese hamster ovary cell clones stably expressing the chimeric G-protein alpha subunit transcript (G alpha s/i(38] demonstrated 1.5-2.5-fold constitutively elevated cyclic AMP levels and a 3-4-fold increase in the activity ratio of cyclic AMP-dependent protein kinase, although expression of the chimeric polypeptide could not be demonstrated presumably because of low expression of the mutant alpha s. Expression of the rat G alpha s transcript yielded clones that were similar to wild-type Chinese hamster ovary cells in regard to cyclic AMP levels and protein kinase activity. In the presence of methyl isobutylxanthine, a cyclic AMP phosphodiesterase inhibitor, cyclic AMP levels in clones expressing the G alpha s/i(38) transcript were 10-15-fold higher than G alpha s expressing clones. Adenylyl cyclase activation by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) in membranes from clones expressing the G alpha s/i(38) transcript demonstrated a diminished lag time for maximal activation, indicating an increased relative GDP dissociation rate for the chimeric G alpha subunit and an increase in total adenylyl cyclase activity relative to wild-type G alpha s expressing clones. Cholate extracts from membranes of G alpha s/i(38) expressing clones, when mixed with cyc- S49 membranes, reconstituted an increased GTP gamma S-stimulated adenylyl cyclase activity and a diminished lag time for maximal activation compared to cholate extracts prepared from G alpha s-expressing clones. The G alpha s/i(38) construct confers a dominant constitutive activation of adenylyl cyclase when expressed in cells in the presence of a background of wild-type G alpha s.
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PMID:Expression of a G alpha s/G alpha i chimera that constitutively activates cyclic AMP synthesis. 246 29

The primary structure of the zeta subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The zeta subspecies of protein kinase C consists of 592 amino acid residues with the calculated molecular mass of 67,740 Da and has regulatory and protein kinase domains in its amino- and carboxyl-terminal halves, respectively. Although all members of the protein kinase C family so far identified have a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence in the regulatory domain, the zeta subspecies contains only one set of this sequence. Northern (RNA)-blot hybridization analysis indicated that two major RNA transcripts of the zeta subspecies with different lengths may be generated by the use of different polyadenylylational signals. The enzyme was expressed in COS-7 cells by transfection with the cDNA construct encoding its whole sequence. It showed an approximate molecular mass of 64,000 Da upon SDS/PAGE. The enzyme activity was significantly dependent on phospholipid but was independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein. The zeta subspecies expressed in COS-7 cells did not appear to show binding activity of phorbol ester. The structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other subspecies of protein kinase C. Perhaps, this subspecies belongs to another entity of the enzyme family.
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PMID:Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties. 247 89

The G-protein GS couples hormone-activated receptors with adenylyl cyclase and stimulates increased cyclic AMP synthesis. Transient expression in COS-1 cells of cDNAs coding for the GS alpha-subunit (alpha S) or alpha S cDNAs having single amino acid mutations Gly49----Val or Gly225----Thr elevated cyclic AMP levels, resulting in the activation of cyclic AMP dependent protein kinase. Stable expression in Chinese hamster ovary cells of alpha S Val49 cDNA resulted in a small constitutive elevation of cyclic AMP that was sufficient to persistently activate cyclic AMP dependent protein kinase activity 1.5-2-fold over basal activity. Stable expression of wild-type alpha S or alpha S Thr225 in Chinese hamster ovary cells was less effective in sustaining elevated cyclic AMP synthesis and kinase activation compared to alpha SVal49.
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PMID:Mutation of glycine 49 to valine in the alpha subunit of GS results in the constitutive elevation of cyclic AMP synthesis. 254 81


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