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 unique nature of the proline side-chain imposes severe constraints on the polypeptide backbone, and thus it seems likely that it plays a special structural or functional role in the architecture of proteins. We have investigated the role of proline residues in suc1, a member of the cyclin-dependent kinase (cks) family of proteins, whose known function is to bind to and regulate the activity of the major mitotic cdk. The effect on stability of mutation to alanine of all but two of the eight proline residues is correlated with their conservation within the family. The remaining two proline residues are located in the hinge loop between two beta-strands that mediates a domain-swapping process involving exchange of a beta-strand between two monomers to form a dimer pair. Mutation of these proline residues to alanine stabilises the protein. cdk binding is unaffected by these mutations, but dimerisation is altered. We propose, therefore, that the double-proline motif is conserved for the purpose of domain swapping, which suggests that this phenomenon plays a role in the function of cks proteins. Thus, the conservation of the proline residues is a good indicator of their roles in suc1, either in the stabilisation of the native state or in performing functions that are as yet unknown. In addition, the strain resulting from two of the proline residues was relieved successfully by mutation of the preceeding residue to glycine, suggesting a general method for designing more stable proteins.
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PMID:Sequence conservation provides the best prediction of the role of proline residues in p13suc1. 1092 2

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.
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PMID:The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase Ialpha-like isoform. 1093 Apr 56

Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.
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PMID:The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7. 1093 49

The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.
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PMID:A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis. 1094 47

A combination of site-directed labeling and time-resolved fluorescence anisotropy was used to further elucidate the structure and underlying dynamic features of the type I regulatory (R(I)(alpha)) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic (C)-subunit binding on the backbone flexibility around seven sites of cysteine substitution and fluorescein maleimide labeling (Thr(6)Cys, Leu(66)Cys, Ser(75)Cys, Ser(81)Cys, Ser(99)Cys, Ser(145)Cys, and Ser(373)Cys) in the R(I)(alpha) subunit were assessed. Focusing on the fast rotational correlation time, the results indicate that most of the interdomain segment connecting the dimerization/docking (D/D) and tandem cAMP-binding domains is probably weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu(66)Cys) that could allow the structured interdomain/cAMP-binding and D/D domains to pivot about each other. Finally, cAMP binding dramatically reduces the backbone flexibility around only the two sites of cysteine substitution in the cAMP-binding domains, suggesting a selective structural stabilization caused by cAMP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.
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PMID:Consequences of cAMP and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit. 1111 51

A-kinase anchoring proteins (AKAPs) are a heterogeneous family of scaffolding proteins that regulate the compartmentalization of signaling components, in particular that of the broad specificity kinase PKA. Here we describe the identification of a new member of this gene family, termed Xenopus gravin-like (Xgl), which encodes a highly acidic protein of 268 kDa that shares extensive homology with human Gravin and murine SSeCKS. Xgl is zygotically expressed in a highly dynamic fashion. During gastrulation Xgl is expressed in posterior mesoderm of the dorsal blastopore lip. During neurulation expression is transiently detected in the forebrain, two bilateral neuroectodermal stripes and the notochord. At tailbud stages expression commences in the mandibular neural crest and the roof of the spinal cord from where neural crest cells migrate into the intersomitic region. In addition expression is detected in the heart and the anterior aspect of the chordoneural hinge.
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PMID:Xgravin-like (Xgl), a novel putative a-kinase anchoring protein (AKAP) expressed during embryonic development in Xenopus. 1116 90

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

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE (gamma-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation/dephosphorylation.
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PMID:Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif. 1206 Jul 53

Here we present a novel technique for the alignment of flexible proteins. The method does not require an a priori knowledge of the flexible hinge regions. The FlexProt algorithm simultaneously detects the hinge regions and aligns the rigid subparts of the molecules. Our technique is not sensitive to insertions and deletions. Numerous methods have been developed to solve rigid structural comparisons. Unlike FlexProt, all previously developed methods designed to solve the protein flexible alignment require an a priori knowledge of the hinge regions. The FlexProt method is based on 3-D pattern-matching algorithms combined with graph theoretic techniques. The algorithm is highly efficient. For example, it performs a structural comparison of a pair of proteins with 300 amino acids in about 7 s on a 400-MHz desktop PC. We provide experimental results obtained with this algorithm. First, we flexibly align pairs of proteins taken from the database of motions. These are extended by taking additional proteins from the same SCOP family. Next, we present some of the results obtained from exhaustive all-against-all flexible structural comparisons of 1329 SCOP family representatives. Our results include relatively high-scoring flexible structural alignments between the C-terminal merozoite surface protein vs. tissue factor; class II aminoacyl-tRNA synthase, histocompatibility antigen vs. neonatal FC receptor; tyrosine-protein kinase C-SRC vs. haematopoetic cell kinase (HCK); tyrosine-protein kinase C-SRC vs. titine protein (autoinhibited serine kinase domain); and tissue factor vs. hormone-binding protein. These are illustrated and discussed, showing the capabilities of this structural alignment algorithm, which allows un-predefined hinge-based motions.
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PMID:Flexible protein alignment and hinge detection. 1211 93

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.
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PMID:A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves. 1212 55


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