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 shortage of organ donors has led to reconsideration for the use of non-heart-beating donors (NHBDs). However, graft injury caused by warm ischemia in livers from NHBDs strongly affects posttransplantation outcome. The aim of the present study is to investigate the role of adenosine A2 receptor with regard to hepatic viability after cold preservation of NHBD livers. Cardiac arrest was induced in Wistar rats by phrenotomy of the anesthetized nonheparinized animal. After 60 minutes, the livers were excised and flushed with 60 mL of histidine-tryptophan-ketoglutarate (HTK) and stored submerged in HTK at 4 degrees C for 24 hours. Reperfusion was performed in vitro after all livers were incubated at 22 degrees C in saline solution to account for the period of slow rewarming during surgical implantation in vivo. Addition of the selective A2-receptor agonist (CGS 21680; 30microg/100 mL) to the preservation solution resulted in a significant reduction to one quarter of the parenchymal enzyme release of alanine aminotransferase or lactate dehydrogenase on reperfusion and promoted a 2-fold increase in hepatic bile production. This salutory effect was accompanied by a significant increase (40%) in the activity ratio of protein kinase A (PKA) in the liver tissue and could be abrogated in large part by the PKA inhibitor, Rp-cAMPs. Stimulation of the adenosine A2 receptor during harvest and storage of the graft improves maintenance of tissue integrity in liver grafts. A major part of this effect, which may represent a promising approach for the use of NHBD grafts, seems to be mediated through activation of PKA.
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PMID:Adenosine A2 receptor stimulation protects the predamaged liver from cold preservation through activation of cyclic adenosine monophosphate-protein kinase A pathway. 1071 20

The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a thermostable multidomain protein. It contains a dimerization domain at the N-terminus followed by an inhibitor site that binds the catalytic C-subunit and two tandem cAMP-binding domains (A and B). Two of the three tryptophans in the RIalpha subunit, Trp188 and Trp222, lie in cAMP-binding domain A while Trp260 lies at the junction between domains A and B. The unfolding of wild-type RIalpha (wt-RI), monitored by intrinsic fluorescence, was described previously [Leon, D. A., Dostmann, W. R. G., and Taylor, S. S. (1991) Biochemistry 30, 3035 (1)]. To determine the environment of each tryptophan and the role of the adjacent domain in folding and stabilization of domain A, three point mutations, W188Y, W222Y, and W260Y, were introduced. The secondary structure of wt-RI and the point mutants has been studied by far-UV circular dichroism spectropolarimetry (CD). The CD spectra of wt-RI and the three point mutants are practically identical, and the thermal unfolding behavior is very similar. Intrinsic fluorescence and iodide quenching in the presence of increasing urea established that: (a) Trp222 is the most buried, whereas Trp188 is the most exposed to solvent; (b) Trp260 accounts for the quenching of fluorescence when cAMP is bound; and (c) Trp222 contributes most to the intrinsic fluorescence of the wt-RI-subunit, while Trp188 contributes least. For wt-RI, rR(W188Y), and rR(W260Y), removal of cAMP causes a destabilization, while excess cAMP stabilizes these three proteins. In contrast, rR(W222Y) was not stabilized by excess cAMP.
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PMID:Probing the multidomain structure of the type I regulatory subunit of cAMP-dependent protein kinase using mutational analysis: role and environment of endogenous tryptophans. 1080 16

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.
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PMID:Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase. 1110 80

Our understanding of the mammalian cell cycle is due in large part to the analysis of cyclin-dependent kinase (CDK) 2 and CDK4/6. These kinases are regulated by E and D type cyclins, respectively, and coordinate the G(1)/S-phase transition. In contrast, little is known about CDK3, a homolog of CDK2 and cell division cycle kinase 2 (CDC2). Previous studies using ectopic expression of human CDK3 suggest a role for this kinase in the G(1)/S-phase transition, but analysis of the endogenous kinase has been stymied by the low levels of protein present in cells and by the absence of an identifiable cyclin partner. Herein we report the presence of a single point mutation in the CDK3 gene from several Mus musculus strains commonly used in the laboratory. This mutation results in the replacement of a conserved tryptophan (Trp-187) within kinase consensus domain IX with a stop codon. The protein predicted to be encoded by this allele is truncated near the T loop, which is involved in activation by CDK-activating kinase. This mutation also deletes motif XI known to be required for kinase function and is, therefore, expected to generate a null allele. In stark contrast, CDK3 from two wild-mice species (Mus spretus and Mus mus castaneus) lack this mutation. These data indicate that CDK3 is not required for M. musculus development and suggest that any functional role played by CDK3 in the G(1)/S-phase transition is likely to be redundant with another CDK.
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PMID:A premature-termination mutation in the Mus musculus cyclin-dependent kinase 3 gene. 1117 11

Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.
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PMID:A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome. 1118 77

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.
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PMID:Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family. 1127 93

Site-directed mutagenesis, gel filtration, and fluorescence spectroscopy approaches were used to study the molecular hinge mechanism involved in the beta-strand-exchanged dimer formation of the cyclin-dependent protein kinase regulatory subunit p13(suc1) from Schizosaccharomyces pombe. Single and double mutants of residues Pro-90 and Pro-92 (P90V, P92V, and P90V/P92V) were prepared and assayed. Substitution of Pro-90 prevented dimer formation by arm exchange. However, single point mutations did not affect the two-state unfolding transition of wild-type p13(suc1) at equilibrium (i.e., wild type, DeltaG degrees (0,un) = 7.38 +/- 0.35 kcal mol(-1), vs P90V, DeltaG degrees (0,un) = 6.71 +/- 0.18 kcal mol(-1)). On the contrary, the double mutant unfolded with a complex transition, and the reaction was best described by a three-state model (N <==> I <==> U). Resolution of the state-dependent (native vs denatured) intrinsic fluorescence decay amplitudes of p13(suc1) showed that with P90V/P92V these parameters were affected at [GuHCl] significantly less than with wild-type and single mutant proteins. Moreover, with the latter products, fluorescence quenching measurements at 1 M GuHCl revealed linear Stern-Volmer plots with quenching constants typical of tryptophan residues located in a native environment (1.6 M(-1) < K(SV) < 2.3 M(-1)). Dissimilarly, with P90V/P92V a significant deviation from linearity of the Stern-Volmer plot was obtained. Nonlinear least-squares analysis of these data resolved the significant contribution of highly solvent-accessible emitting species (K(SV) = 26 M(-1)) consistent with large exposure of the tryptophan residues. These results are compatible with the existence of an intermediate unfolding state of the double mutation product. Thus, while single residue substitution studies give support to the primary role of Pro-90 in the p13(suc1) dimer formation by domain swapping, double residue substitution studies indicate the important role of the conserved repeat, Pro-x-Pro, for the proper beta-strand spatial organization and stability.
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PMID:Structural role of the proline residues of the beta-hinge region of p13suc1 as revealed by site-directed mutagenesis and fluorescence studies. 1143 72

An early development-specific soluble 55 kDa Ca(2+)-dependent protein kinase has been purified to homogeneity from sandalwood somatic embryos and biochemically characterized. The purified enzyme, swCDPK, resolved into a single band on 10% polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK activity was strictly dependent on Ca(2+), K(0.5) (apparent binding constant) for Ca(2+)-activation of substrate phosphorylation activity being 0.7 microM and for autophosphorylation activity approximately 50 nM. Ca(2+)-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC(50) for W7=6 microM, for W5=46 microM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a K(m) value of 1.3 mg/ml for histone III-S and a V(max) value of 0.1 nmol min(-1) mg(-1). The enzyme exhibited high specificity for ATP with a K(m) value of 10 nM. Titration with calcium resulted in the enhancement of intrinsic emission fluorescence of swCDPK and a shift in the lambda(max) emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca(2+)-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca(2+) levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca(2+) activation of this enzyme.
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PMID:Purification and characterization of a Ca(2+)-dependent protein kinase from sandalwood (Santalum album L.): evidence for Ca(2+)-induced conformational changes. 1155 40

Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Here we show that turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homologue of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E.
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PMID:Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. 1156 17

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione-S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic digestion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.
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PMID:Domain-domain associations in cystic fibrosis transmembrane conductance regulator. 1194 May 32


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