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)

Actin-fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues. Whereas the N-terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full-length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N-terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P-loop). However, introduction of two different point mutations into this putative P-loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non-muscle cells.
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PMID:A novel type of protein kinase phosphorylates actin in the actin-fragmin complex. 889 48

Cardiolipin- or protease-activated protein kinase, isolated from rat liver cytosol and originally named liver PAK-1, was found to be the natural form of protein kinase N (PKN) by comparing the sequences of 43 tryptic peptides of the purified liver enzyme and determining the corresponding liver cDNA sequence. These analyses also identified (i) Arg-546 as the major site of proteolytic activation, (ii) the protease resistance of the C-terminal extension beyond the catalytic domain, and (iii) in vivo stoichiometric phosphorylation of Thr-778 in the mature enzyme. Homology modeling of the catalytic domain indicated that phosphothreonine 778 functions as an anchoring site similar to Thr-197 in cAMP-dependent protein kinase, which stabilizes an active site compatible with preferred substrate sequences of PAK-1/PKN. Sigmoidal autophosphorylation kinetics and increased S6-(229-239) peptide kinase activity following preincubation with ATP suggested phosphorylation-dependent activation of PAK-1/PKN. The onset of activation corresponded with phosphorylation of the regulatory domain site Ser-377 (located within a spectrin homology region), followed by Thr-504 (within a limited protein kinase C homology region), and, to a lesser extent, Thr-64 (in the RhoA-binding region). Several additional sites in the hinge region adjacent to a PEST protein degradation signal were selectively autophosphorylated following cardiolipin activation. Overall, these observations suggest that the regulation of this class of protein kinase involves complex interactions among phosphorylation-, lipid-, and other ligand-dependent activation events.
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PMID:Phosphorylation events associated with different states of activation of a hepatic cardiolipin/protease-activated protein kinase. Structural identity to the protein kinase N-type protein kinases. 894 81

The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane binding and catalytic function and 2) conformation. In the first part, we show that significantly lower concentrations of Ca2+ are required to effect half-maximal membrane binding than to half-maximally activate the enzyme. The disparity between binding and activation kinetics is most striking for protein kinase C betaII, where the concentration of Ca2+ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent Km for Ca2+ for activation. In addition, the Ca2+ requirement for activation of protein kinase C betaII is an order of magnitude greater than that for the alternatively spliced protein kinase C betaI; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca2+ regulation. In the second part, we use proteases as conformational probes to show that Ca2+dependent membrane binding and Ca2+-dependent activation involve two distinct sets of structural changes in protein kinase C betaII. Three separate domains spanning the entire protein participate in these conformational changes, suggesting significant interdomain interactions. A highly localized hinge motion between the regulatory and catalytic halves of the protein accompanies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ too low to promote catalysis; and exposure of the amino-terminal pseudosubstrate and masking of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to each isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca2+-binding site in the amino-terminal regulatory domain.
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PMID:Ca2+ differentially regulates conventional protein kinase Cs' membrane interaction and activation. 932 30

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.
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PMID:Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: regulation of HSP90-binding activity of FKBP52. 940 42

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca2+- and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca2+- and PS-dependent activity of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca2+- and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-alpha remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-alpha was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-alpha and the tripeptide, but it was associated with an increase in the susceptibility of PKC-alpha to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca2+- and PS- dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.
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PMID:Irreversible inactivation of protein kinase C by glutathione. 957 16

Previously, our laboratories have reported on a new class of highly potent tyrosine kinase inhibitors based on the pyrido[2, 3-d]pyrimidine core template. To understand the structural basis for the potency and specificity, a model for the binding mode of this class of inhibitors to the tyrosine kinase domains of c-Src, PDGFr, FGFr, and EGFr tyrosine kinases was developed from structural information (principally utilizing the catalytic domain of c-AMP-dependent protein kinase as template) and structure-activity relationship (SAR) information. In the resulting docking mode, the pyrido[2,3-d]pyrimidine template shows a hydrogen-bonding pattern identical to that of olomoucine. The 6-aryl substituent of the heterocycle is located deep in the binding cleft in a pocket not used by ATP, which helps to confer high-affinity binding as well as specificity. The 2-anilino and 2-(dialkylamino)alkylamino substituents as well as the 7-urea substituent of inhibitors within this class are located at the entrance of the binding cleft and make contact with residues in the hinge region between the two kinase lobes. This allows considerable variability and bulk tolerance for C-2 and N-7 substituents. The models presented here are consistent with the SAR seen for the inhibition of a number of isolated enzymes and provide a structural basis to explain their specificity. They have been used successfully to design new highly potent protein kinase inhibitors.
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PMID:Development of a binding model to protein tyrosine kinases for substituted pyrido[2,3-d]pyrimidine inhibitors. 959 27

Among the four classes of chitinase, a class II chitinase had not yet been reported for rice. We have isolated and characterized a class II acidic chitinase, Rcht2, from rice (Oryza sativa L. cv. Cheongcheongbyeo). The protein consists of a single polypeptide chain of 261 amino acid residues and includes a putative signal sequence of 29 amino acids at its N-terminus. It has a calculated molecular mass of 27,642 Da and an isoelectric point of 5.56. The Rcht2 chitinase lacks the cysteine-rich and hinge domains in the N-terminal region of the protein, which is the criterion for its classification as a class II chitinase. Comparison of the genomic and the cDNA sequence revealed that the coding region of Rcht2 consist of three exons of 301, 112, and 370 bp separated by two introns of 89 and 984 bp. In suspension-cultured rice cells, the transcript level of Rcht2 was dramatically increased by treatment with both glycol chitin and fungal elicitor. The application of protein phosphatase 1 and 2A inhibitors, calyculin A and okadaic acid, effectively abolished the induction of Rcht2 in response to fungal elicitor. In contrast, the activation of Rcht2 transcript was not inhibited by both cycloheximide and protein kinase inhibitors. These results demonstrate that protein dephosphorylation events play a crucial role in the elicitor-mediated induction of Rcht2 in rice cells, while de novo protein synthesis is not required for induction.
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PMID:A new class II rice chitinase, Rcht2, whose induction by fungal elicitor is abolished by protein phosphatase 1 and 2A inhibitor. 961 19

Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R2C2) and of a Delta1-91RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the RII PKA isoforms.
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PMID:Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the cAMP-dependent protein kinase by neutron contrast variation. 980 12

p13(suc1) (suc1) is a member of the CDC28 kinase specific family of cell cycle regulatory proteins that bind to the cyclin-dependent kinase CDK2 and regulate its activity. suc1 has two distinct conformational and assembly states, a compact globular monomer and a beta strand-exchanged dimer. The dimerisation is an example of domain-swapping, and is mediated by a molecular hinge mechanism that is conserved across the entire CKS family. It has been proposed that the function of suc1 may be modulated by the dimerisation process with monomer-dimer switching occurring in response to a change in the cell environment. We have investigated the stability and folding of suc1 as a first step in determining the mechanism and functional role of the strand exchange. Suc1 unfolds reversibly at equilibrium in a two-state manner with a free energy of unfolding of 7.2 kcal mol-1. The kinetics of folding and unfolding are complex, and double-jump stopped-flow methods revealed that there are at least three parallel folding pathways arising from distinct unfolded and partly folded, intermediate states. The major population of unfolded species fold rapidly according to a three-state mechanism, D1->I1->N, with a rate constant for the formation of native species, N, from the intermediate, I1, of 65 s-1 in water. Two minor populations of unfolded molecules fold more slowly. Folding of one population is limited by proline isomerisation in a partly folded state, and some expansion of the protein is required for isomerisation to occur. The other population could be assigned to rate-limiting isomerisation of the peptidyl-proline bond of residue 90, which is located in the molecular hinge. A minor, fast phase was detected in the unfolding kinetics that corresponds to unfolding of a small population of a distinct native-like form. Heterogeneity was removed upon mutation of Pro90 to Ala. The unfolding kinetics of the strand-exchanged dimer were also investigated and showed that the dimer unfolds at the same rate as the monomer.
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PMID:Stability and folding of the cell cycle regulatory protein, p13(suc1). 981 33

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.
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PMID:Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase. 1091 58


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