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)

A 64-kDa protein was purified from an octyl glucoside/cholate extract of spinach thylakoids. N-Terminal analysis yielded 23 residues of sequence, of which the first 15 were identical to a sequence reported [Gal, A., Herrmann, R. G., Lottspeich, F., & Ohad, I. (1992) FEBS Lett. 298, 33-35] for a protein kinase with specificity toward the photosystem II light-harvesting complex (LHC-II). We report the complete sequence of this 64-kDa protein, deduced from cDNA clones. The transit peptide has a chloroplast import signal at the N-terminus and a C-terminal hydrophobic span bounded by basic amino acids that predicts localization of the protein to the thylakoid lumen. The mature protein sequence is about 50% identical to several polyphenol oxidases (PPOs). Canonical protein kinase motifs are absent, as are sequences characteristic of ATP-binding sites. The mature protein resembles arthropodan hemocyanin (Hc), possessing three major domains. The N-terminal domain is rich in cysteine residues and predicted alpha-helices. The central domain has a conserved motif, N-terminal to a presumptive Cu-A site, that is not found in tyrosinases or Hc and is proposed as the provider of a third imidazole ligand to Cu-A. An unusual 13-residue, glutamine-rich link begins a C-terminal domain containing 7 predicted beta-strands which, by analogy with Hc, may form an antiparallel beta-barrel. We conclude that this 64-kDa polypeptide is a lumenal PPO and the precursor of a 42.5-kDa PPO form described previously [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Spinach thylakoid polyphenol oxidase: cloning, characterization, and relation to a putative protein kinase. 779 29

cAMP response element-binding protein (CREB) and modulator protein (CREM) regulate the transcription of cAMP-responsive genes via phosphorylation by cAMP-dependent protein kinase A. Reverse transcription and polymerase chain amplification of RNA from male germ cells identify an alternatively spliced CREM isoform, CREM delta C-G, lacking four exons including those encoding the protein kinase A-regulated phosphorylation domain and the flanking glutamine-rich transcriptional activation domains. CREM delta C-G retains exons that encode the basic-leucine zipper (bZIP) DNA-binding domain, binds to cAMP response elements (CREs), and competitively inhibits binding of CREB and CREM to CREs. Expression of CREM delta C-G inhibits transcription of a CRE-containing chloramphenicol acetyltransferase reporter plasmid induced by endogenous CREB. Antiserum to CREM detects CREM delta C-G in elongated spermatids from rat testis. These observations indicate that CREM delta C-G is a unique form of a competitive negative regulator of CREB-mediated gene transcription expressed in a maturation-dependent manner in haploid germ cells. The developmental specificity of CREM delta C-G suggests that it may play a role in transcriptional regulation during spermatogenesis.
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PMID:An isoform of transcription factor CREM expressed during spermatogenesis lacks the phosphorylation domain and represses cAMP-induced transcription. 780 53

Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles. Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N., Taylor, S.S., and Sowadski, J. M. (1991) Science 253, 414-420). To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons. Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli. C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP. The Vmax(app) was not affected by the mutation of C alpha E203. The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity. PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203. The PKI alpha R15A mutant was reduced in its inhibition of C alpha. Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results. These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo.
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PMID:Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor. 790 1

cAMP regulates the expression of a number of genes through the protein kinase A-mediated phosphorylation of CREB at Ser-133. The effects of Ser-133 phosphorylation appear to be primarily transmitted through a modulatory kinase-inducible domain in CREB that functions cooperatively with a 120-amino acid glutamine-rich region (Q2) to stimulate transcription. Indeed, the kinase-inducible domain activity alone is not sufficient to sustain a transcriptional response as illustrated by the CREM family of repressors, which contain the kinase-inducible domain but lack the Q2 region. Here we demonstrate that Q2 functions as a potent constitutive activator in vitro. The transcription factor TFIID fraction supports transcriptional activation by Q2, although the "TATA" binding protein alone does not, suggesting that other components of the TFIID complex mediate the response to CREB Q2. In fact, Q2 associates with the TATA binding protein-associated factor dTAFII110. As the transcriptionally inactive CREM alpha and beta proteins lack sequences in Q2 that are necessary for binding dTAFII110, our results suggest that these proteins may repress transcription because they are unable to interact with the basal transcription complex.
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PMID:The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex. 790 13

The mammalian transcriptional activator CREB binds as a dimer to a broad spectrum of inducible promoters. CREB activity is modulated by several signalling agents (protein kinase A [PKA], Ca2+, and transforming growth factor beta) and via functional interactions with cell-specific transcription factors. In addition, CREB can activate transcription constitutively and repress the activity of several other transcriptional activators. The mechanisms that allow CREB to act in such a malleable manner and the role that CREB dimerization might play in this are poorly understood. To probe the latter issue, we have created monomeric forms of CREB by fusing CREB to the DNA-binding domain of a protein (B-cell specific activator protein [BSAP]) that binds to DNA as a monomer. Remarkably, monomeric CREB acts as a potent, constitutive activator under conditions in which native CREB is inducible by PKA. Thus, CREB contains constitutive activation regions that are unable to function in native CREB. Two glutamine-rich domains that are important for native, PKA-inducible CREB activity are required for the constitutive activity of monomeric CREB. In contrast, two elements within the kinase-inducible domain of CREB are dispensable for constitutive activity. We discuss our results in relation to inducible and constitutive CREB activity and the potential modes of action of other activators that directly interact with CREB.
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PMID:A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator. 793 35

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) migration pattern of wild-type and mutated human androgen receptors (ARs) expressed in COS-1 cells was analyzed. In the absence of hormone, the wild-type AR migrated as a closely spaced 110-112 kDa doublet. Alkaline phosphatase treatment resulted in a single 110 kDa band showing that the 112 kDa upshift reflects receptors phosphorylation. Deletion of the N-terminal amino acids 46-101 or 100-142 resulted in mutant ARs migrating as single protein bands. Three consensus phosphorylation sites in this region were substituted, and the resulting mutated proteins were analyzed. Two Ser-Pro-directed kinase consensus sites at positions Ser-80 and Ser-93 were both necessary for the AR 112 kDa upshift. Substitution of the putative casein kinase II Ser-118 site had no effect on the AR migration pattern. Surprisingly, deletion of the glutamine repeat, located directly N-terminal of the Ser-Pro sites, resulted also in an AR single form. Lengthening of the glutamine repeat caused an increase in the spacing between the two isotypes of the doublet, showing that the number of glutamine residues determines the extent of the upshift. Hormone treatment induced an extra isotype with an apparent molecular mass of 114 kDa, resulting in a 110-112-114 kDa AR triplet. The hormone-induced upshift was dependent on the Ser-80 consensus phosphorylation site. Mutations in the DNA binding domain caused a different distribution of receptor protein over the three AR isotypes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Changes in the abundance of androgen receptor isotypes: effects of ligand treatment, glutamine-stretch variation, and mutation of putative phosphorylation sites. 794 16

Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.
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PMID:Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation. 814 71

Activation of protein kinase A (PKA) by cAMP results in phosphorylation of cAMP response element-binding protein (CREB) and induction of specific gene expression. However, whether CREB participates directly in basal (PKA-independent) transcription is still an open question, and existing studies conflict over the identification of putative basal activation domains. In the present study, the activation domain of CREB, whether fused to the GAL4 DNA binding domain (CRG) or in native CREB, stimulated basal activity of the minimal tk promoter, but not the minimal SV40 early promoter. Cotransfection with PKI, a specific inhibitor of PKA, blocked PKA-induced expression of both promoters, but did not block CRG-mediated basal expression of the tk promoter. In addition, both CRG and a PKA phosphorylation site mutant provided comparable stimulation of basal tk promoter activity. Examination of a series of CREB deletion mutants mapped basal activity to interacting domains, located on either side of the previously identified PKA activation domain (amino acids 98-142). This PKA-independent activity mapped primarily to a bipartite COOH-terminal basal activation domain (amino acids 165-252). Its major component bears no obvious homology to previously identified activation domains, whereas a minor component is glutamine-rich. This COOH-terminal domain acts independently and provides the majority of basal activation but requires an NH2-terminal domain (amino acids 41-86) to provide full basal activity. A repressor domain (amino acids 142-165), deletion of which enhanced both basal and PKA-activated transcription, was also identified. This work establishes that CREB contains distinct basal and PKA-activated domains, that they operate independently for both loss of function and gain of function, and that they work on different promoters in different cell types.
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PMID:Distinct activation domains within cAMP response element-binding protein (CREB) mediate basal and cAMP-stimulated transcription. 839 25

The avian retroviral oncoprotein v-Rel and its cellular homolog c-Rel are members of a family of related site-specific DNA-binding proteins. Towards the carboxy-terminal end of the highly conserved Rel homology (RH) domain in the majority of Rel proteins, there is a consensus recognition sequence for protein kinase A (PK-A). We have investigated the importance of this sequence (Arg-Arg-Pro-Ser) for several functional properties of v-Rel and c-Rel. Disruption of the PK-A sequence by a two amino acid insertion between the arginine and the proline residues completely abolished the ability of v-Rel and c-Rel to bind a kappa B site in vitro. When the phosphorylatable serine in this sequence (Ser-275 in v-Rel, Ser-266 in c-Rel) was replaced by an alanine, DNA binding by v-Rel was not affected, whereas the ability of c-Rel to bind DNA was reduced approximately fourfold by this mutation. Similarly, a serine to tryptophan change greatly reduced the DNA-binding ability of c-Rel, whereas v-Rel was not appreciably affected by this change. When this serine was replaced by an acidic amino acid, DNA binding by v-Rel was reduced approximately twofold and the DNA-binding activity of c-Rel was nearly abolished. Glutaraldehyde cross-linking experiments indicated that mutations at the PK-A recognition site that reduced DNA binding also negatively affected protein oligomerization, which is likely to be responsible for the reduced ability of mutant v-Rel and c-Rel proteins to bind DNA. Domain-swapping experiments showed that structural differences between v-Rel and c-Rel in the central region of the proteins are primarily responsible for the higher sensitivity of c-Rel to a serine to alanine mutation in the PK-A site. One difference between v-Rel and c-Rel, a glutamine to alanine change in v-Rel located three amino acids carboxy-terminal to the PK-A phosphorylatable serine (Ala-278 in v-Rel; Glu-269 in c-Rel), is mainly responsible for the lack of an effect on DNA binding by v-Rel when Ser-275 is replaced by alanine. That is, a v-Rel double mutant (v-275A/278E) showed reduced DNA-binding and transforming abilities as compared with v-Rel and v-275A. Similarly, the mutations in c-Rel that affected DNA binding showed a corresponding effect on the ability of c-Rel proteins to activate transcription in yeast from a reporter gene containing upstream Rel binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:v-Rel and c-Rel are differentially affected by mutations at a consensus protein kinase recognition sequence. 843 55

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.
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PMID:The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. 853 40


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