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 protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X(n)(R/K)X(n)(S/T)X(n) (S*/T*), were n = 0-3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step, which could lead to a specific sorting and productive translocation in plant cells.
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PMID:Phosphorylation of the transit sequence of chloroplast precursor proteins. 862 59

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.
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PMID:Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance. 888 90

Phosphoenolpyruvate carboxylase (PEPC) kinase was partially purified about 3000-fold from soybean root nodules by a fast-protein liquid chromatography protocol. This protein-serine kinase has an apparent native molecular mass of about 30,000 as estimated by size-exclusion chromatography. Following electrophoresis of this partially purified PEPC-kinase preparation in a denaturing gel containing dephospho maize leaf PEPC as substrate, the in situ renaturation and assay of protein kinase activity revealed two, PEPC-dependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The approximately 32-kDa polypeptide was significantly more active than the approximately 37-kDa catalytic subunit. The activity of this partially purified PEPC kinase, and a less purified sample, was Ca2+-insensitive. This protein kinase preparation was able to phosphorylate purified PEPCs from soybean nodules, maize leaves, and a sorghum recombinant C4 PEPC. In contrast, this PEPC kinase was unable to phosphorylate a phosphorylation-site mutant form of sorghum C4 PEPC (S8Y), two other soybean nodule phosphoproteins [nodulin-26 and nodulin-100 (sucrose synthase)], bovine serum albumin, and histone III-S. Following in vitro phosphorylation of purified dephospho soybean nodule PEPC from stem-girdled plants by the partially purified nodule PEPC kinase, the former's activity and sensitivity to L-malate inhibition increased and decreased, respectively. Notably, the Ca2+-independent PEPC kinase activity in nodules from illuminated plants was markedly greater than that in nodules harvested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in nodules was controlled reversibly by illumination and extended darkness pretreatments of the parent plants, suggesting that photosynthate supply from the shoots is likely responsible for these striking changes in PEPC kinase activity observed in planta in the legume nodule.
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PMID:Phosphoenolpyruvate carboxylase protein kinase from soybean root nodules: partial purification, characterization, and up/down-regulation by photosynthate supply from the shoots. 922 39

Sucrose synthase (SS; EC 2.4.1.13) was radiolabeled in situ by incubating detached soybean nodules with 32Pi. Phosphoamino acid analysis indicated that SS was phosphorylated on a serine residue(s). In-vitro phosphorylation of purified nodule SS by desalted nodule extracts was Ca2+-dependent. This SS-kinase was partially purified (approximately 2200-fold) from nodules harvested from illuminated plants. The molecular mass of the SS-kinase was about 55,000 on a Superdex 75 size-exclusion column or in a denaturing autophosphorylation gel. With either purified nodule SS or Syntide 2 as substrate, exogenous calmodulin and phosphatidylserine showed little or no effect on the in-vitro activity of this partially purified protein kinase. However, its activity was inhibited by W-7. The purified nodule SS-kinase (or CDPK) phosphorylated nodule PEP carboxylase (PEPC; EC 4.1.1.31) in the presence of Ca2+. In contrast, a partially purified nodule PEPC-kinase preparation was incapable of phosphorylating nodule SS. Unlike nodule PEPC [Zhang et al. (1995) Plant Physiol. 108, 1561-1568], the phosphorylation state of SS is not likely modulated in planta by photosynthate supply from the shoots.
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PMID:Seryl-phosphorylation of soybean nodule sucrose synthase (nodulin-100) by a Ca2+-dependent protein kinase. 923 14

Two isoforms of sucrose synthase (SS1 and SS2) from maize (Zea mays, var. Mona) seedlings co-purified with a calcium and phospholipid dependent protein kinase. The enzymatic preparation obtained gave a positive reaction with the antibody against mammalian protein kinase C. Maize sucrose synthase was phosphorylated by the endogenous protein kinase. Also, mammalian protein kinases (protein kinase C and protein kinase A) were able to phosphorylate the 86 kDa subunit of sucrose synthase. When excised seedlings were fed [32P]orthophosphate, sucrose synthase was also phosphorylated. Microsequencing of in vivo labelled enzyme has shown phosphorylation of Ser-15 in SS2. The present work provides evidence that maize sucrose synthase is the physiological substrate of the endogenous calcium and phospholipid dependent protein kinase(s).
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PMID:Phosphorylation of sucrose synthase from maize seedlings. 958 64

A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.
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PMID:An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11. 1005 Mar 18

The major phosphorylation site of maize sucrose synthase (SuSy) is well conserved among plant species but absent in the deduced peptide sequence of the tomato SuSy cDNA (TOMSSF). In this study, we report the in vitro phosphorylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogenous Ca2+-dependent protein kinase activity. Two distinct 32P-labeled peptides detected in the tryptic peptide map of in vitro 32P-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid analysis of the major 32P-labeled peptide revealed the presence of a SuSy isozyme in young tomato fruit having the N-terminus phosphorylation site present in other plant species. By using Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two 32P-labeled tomato SuSy isoforms were separated from a nonradiolabeled SuSy fraction by using a pH gradient. The major 32P-SuSy isoform was phosphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabeled SuSy fraction was indicated by a higher retention during Fe(III)-IMAC and by tryptic peptide mapping analysis. Kinetic analyses of SuSy isoforms purified by Fe(III)-IMAC have revealed that phosphorylation of the major phosphorylation site of tomato fruit SuSy was not sufficient by itself to modulate tomato SuSy activity, whereas the affinity for UDP increased about threefold for the multiphosphorylated SuSy isoform.
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PMID:Purification of tomato sucrose synthase phosphorylated isoforms by Fe(III)-immobilized metal affinity chromatography. 1022 51

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.
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PMID:Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings. 1063 3

We searched for genes encoding members of the group-3 SNF1-related protein kinase (SnRK3) family in the Arabidopsis thaliana database, and seven independent sequences were identified. Transcripts of two of them were found to accumulate differentially upon treatment with light, cytokinins and sugars. Full-length cDNAs were isolated and designated as AtSR1 and AtSR2; they encode polypeptides of 442 and 429 amino acids with relative molecular masses of 50.3 kDa and 48.2 kDa, respectively. In etiolated seedlings, no transcripts of either gene were observed. However, upon exposure to light or cytokinins, transcripts of AtSR1 but not AtSR2 began to accumulate. The induction with light was greatly reduced in the presence of a cytokinin antagonist, suggesting that cytokinins are involved in light-signaling pathways. In contrast, transcription of AtSR2, but not of AtSR1, was greatly increased by sucrose, as well as glucose and fructose. AtSR2 expressed in E. coli efficiently phosphorylated sucrose synthase in the presence of manganese ions. These results suggest that, although SnRK3 proteins may generally be involved in sugar metabolism, expression of AtSR1 and AtSR2 is differentially and distinctly regulated by various external signals, and AtSR2 may function in the regulation of sucrose synthase by specific phosphorylation.
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PMID:Two novel genes encoding SNF-1 related protein kinases from Arabidopsis thaliana: differential accumulation of AtSR1 and AtSR2 transcripts in response to cytokinins and sugars, and phosphorylation of sucrose synthase by AtSR2. 1121 22

Plant SNF1-related protein kinase (SnRK1) phosphorylates 3-hydroxy-3-methylglutaryl-Coenzyme A, nitrate reductase and sucrose phosphate synthase in vitro, and is required for expression of sucrose synthase in potato tubers and excised leaves. In this study, a barley (Hordeum vulgare) endosperm cDNA, SnIP1, was isolated by two-hybrid screening with barley SnRK1b, a seed-specific form of SnRK1. The protein encoded by the SnIP1 cDNA was found to interact with barley SnRK1b protein in vitro. Southern analysis suggested that barley contains a single SnIP1 gene or small gene family. SnIP1 transcripts were detected in RNA isolated from leaf, root and mid-maturation seed. Sequence similarity searches against protein, nucleotide and expressed sequence tag databases identified hitherto uncharacterized sequences related to SnIP1 from maize (Zea mays, accession number AI691404), arabidopsis (Arabidopsis thaliana. AC079673 and AB016886) and poplar (Populus balsamifera, AI166543). No homologous sequences were identified from outside the plant kingdom, but weak sequence similarity was found between the SnIP1 peptide and yeast (Saccharomyces cerevisiae) SNF4 and its mammalian homologue AMPKy. Nevertheless, SnIP1 failed to complement a yeast snf4 mutant. SnIP1 was found to have little overall sequence similarity with the PV42 family of SNF4-like plant proteins, but proteins of both the SnIP1 and PV42 families contain a conserved hydrophobic sequence we named the SnIP motif.
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PMID:Identification of SnIP1, a novel protein that interacts with SNF1-related protein kinase (SnRK1). 1200 97


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