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PubMed1977

Phosphoglycerate mutase from wheat germ: studies with 18O-labeled substrate, investigations of the phosphatase and phosphoryl transfer activities, and evidence for a phosphoryl-enzyme intermediate.

From studies using unlabeled phospho-D-glycerate in solutions enriched in H2(18)O, and from experiments involving [18O]phospho-D-glycerate, it is shown that the intramolecular isomerization of 2- and 3-phospho-D-glycerate that is catalyzed by the phosphoglycerate mutase from wheat germ does not involve an intermediate 2,3-cyclic phosphate. It is also shown that phosphoglycerate mutase catalyzes the hydrolysis of the substrate analogues 2-phosphoglycolate, 2-phospho-D-lactate, 3-phosphohydroxypropionate, phosphoenolpyruvate, and phosphohydroxypyruvate. The substrates 3- and 2-phospho-D-glycerate are not hydrolyzed, nor are 2,3-bisphospho-D-glycerate, 2-phospho-L-lactate, 3-phospho-L-glycerate, or sn-glycerol 3-phosphate. Although no exchange of D-[14C]glycerate into phospho-D-glycerate can be detected, the enzyme catalyzes the transfer of the phosphoryl group from "unnatural" donors such as 2-phosphoglycolate, to the "natural" acceptor, D-glycerate. It is concluded that the intramolecular phosphoryl transfer catalyzed by the wheat germ phosphoglycerate mutase follows a pathway involving a phosphoryl-enzyme intermediate.

Glycerophosphates↗
PubMed1977

Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration.

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

Animals↗
PubMed1991

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26.

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca(2+)-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca(2+) (K(0.5) = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca(2+)-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with (32)P-phosphate. Overall, the results show the existence of a Ca(2+)-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca(2+)-dependent phosphorylation.

Journal Article↗
PubMed2005

A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis.

An Arabidopsis thaliana gene encoding a homologue of the potato alpha-glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.

Arabidopsis↗
PubMed1991

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.

Amino Acid Sequence↗
PubMed1986

Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain.

In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.

Adipose Tissue↗
PubMed1994

Rapid changes in nuclear protein tyrosine phosphorylation after growth hormone treatment in vivo. Identification of phosphorylated mitogen-activated protein kinase and STAT91.

Growth hormone (GH) plays a central role in regulating growth and intermediary metabolism in vertebrates, although the mechanisms by which GH initiates these actions are largely unknown. The GH receptor, a member of the cytokine receptor superfamily, does not demonstrate homology with any known tyrosine kinases. However, addition of GH to cells in vitro has been shown to stimulate tyrosine phosphorylation of various intracellular proteins including mitogen-activated protein kinases (MAP kinases) and the newly described Janus kinase, JAK2. Subsequent steps in GH-mediated signal transduction have not been delineated. In the present study, we have examined early events in GH action in vivo. Hypophysectomized juvenile male rats were treated with GH for 15, 30, or 60 min. Rat liver whole cell and nuclear extracts were prepared and analyzed via SDS-polyacrylamide gel electrophoresis and Western blotting techniques. GH rapidly stimulated the tyrosine phosphorylation of at least 8 nuclear proteins of 205, 91, 83, 80, 65, 53, 44, and 42 kDa, and caused the dephosphorylation of a single approximately 149-kDa protein. Using specific antibodies, we have identified three of these nuclear phosphoproteins as 42- and 44-kDa MAP kinases, and as STAT91, a 91-kDa component of the interferon-stimulated gene factor-3 protein complex. One consequence of the activation of STAT91 in the nucleus is the appearance of GH-stimulated DNA binding activity, as assessed by gel-mobility shift assay using an oligonucleotide containing a c-sis-inducible element from the c-fos promoter. These results show that nuclear protein tyrosine phosphorylation is a prominent early event in GH action in vivo and demonstrate a link between GH-stimulated signal transduction and target gene expression.

Animals↗
PubMed1985

Phosphorylation of proteoglycans. Identification of phosphorylation sites in chondroitin sulfate-rich region of core protein.

Serine residues, which are the sites of phosphorylation in proteoglycans, were demonstrated to be located on chondroitin sulfate-containing peptides. These peptides appeared to be derived primarily from the chondroitin sulfate-rich region of the proteoglycan core protein. The localization of phosphate moieties in the chondroitin sulfate-containing peptides was observed in all experiments. The phosphate moieties were retained on chondroitin sulfate-containing peptides after the protein core was treated with either papain or trypsin. Two phosphopeptide preparations, derived from chondroitin sulfate-containing peptides of proteoglycan subunits, were extensively purified. These 2 phosphopeptide preparations were shown by carbohydrate analysis to be free of keratan sulfate-containing peptides or peptides from the hyaluronic acid binding region of the core protein. One of the phosphopeptide preparations had a phosphate: serine molar ratio of 0.40. This indicated that nearly one-half of the serine residues were phosphorylated rather than glycosylated. Peptides derived from the core protein that contained keratan sulfate had no phosphate moieties.

Cartilage, Articular↗
PubMed1993

Characterization of a 64-kd protein phosphorylated during chemotactic activation with IL-8 and fMLP of human polymorphonuclear leukocytes. II. Purification and amino acid analysis of phosphorylated 64-kd protein.

Lung giant cell carcinoma-derived chemotactic protein (LUCT)/IL-8 and fMet-Leu-Phe stimulate phosphorylation of a 64-kd protein (p64) in 32P-labeled human polymorphonuclear leukocytes (PMNs). The p64 was purified from cytosol of human PMNs (1.8 x 10(9) cells) by DEAE-Sepharose CL6B column chromatography, hydroxyapatite HPLC, and reverse-phase HPLC. By hydroxyapatite HPLC, p64s were separated and produced two peaks containing both nonphosphorylated and phosphorylated p64. Amino acid composition of each p64 was determined. Each p64 was directly subjected to amino acid sequencing, but the amino acid residue in the amino-terminal of the proteins could not be detected. From the results of amino acid composition and other characters of p64, it is suggested that p64 is identical to l-plastin, which is a leukocyte-specific protein.

Amino Acid Sequence↗
PubMed2004

Immunohistochemical localization of phosphorylated protein kinase R and phosphorylated eukaryotic initiation factor-2 alpha in the central nervous system of SJL mice with experimental allergic encephalomyelitis.

Inflammatory cells enter the CNS and target myelin in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), a model of MS, and inflammation is thought to induce stress responses in the CNS. Protein kinase R (PKR) and eukaryotic initiation factor-2 alpha (eIF2 alpha) undergo phosphorylation in response to stress, and the phosphorylated forms of these proteins play a key role in regulating protein synthesis. The objective of this study was to investigate the expression profile of phospho-PKR and phospho-eIF2 alpha during the course of EAE in order to advance the understanding of the stress response in this disease. In control animals (no encephalitogen with no emulsion; no encephalitogen with emulsion) and in preclinical EAE animals, phospho-PKR immunoreactivity was present in oligodendrocytes and some neurons, whereas, in EAE animals with active disease there was widespread labeling of inflammatory cells, and these cells were present during the recovery period of EAE, albeit to a lesser extent. Double-labeling studies revealed that T cells and a few macrophages were phospho-PKR(+). Phospho-eIF2 alpha immunoreactivity was detected in some oligodendrocytes in hindbrain sections of control animals. In EAE animals with active disease, the number of labeled oligodendrocytes increased, and inflammatory T cells also were labeled. Insofar as phospho-PKR activates nuclear factor-kappa B, it may facilitate cytokines expression by T cells. Alternatively, phospho-PKR and phospho-eIF2 alpha may promote apoptosis as a way to regulate T-cell number in the CNS. The expression of phospho-eIF2 alpha in oligodendrocytes during EAE likely is involved with inhibition of protein translation, which is a protective mechanism used to promote cell survival in response to inflammation.

Animals↗
PubMed1995

Distribution of non-phosphorylated and phosphorylated neurofilament proteins in the spinal cord of an anuran amphibian during development and regeneration.

The changes in the neurofilament medium and high molecular weight subunits (NF150 and NF200) in the developing and transected spinal cords of bullfrog tadpoles were studied. A monoclonal antibody recognizing the nonphosphorylated epitope of NF150, NF150D, stained the neuronal cell bodies and axons, whereas other antibodies against the phosphorylated NFs, NF150P or NF200P, labeled chiefly the axons. During development, the intensity of axonal staining by the anti-NF150D in the ventral fasciculi in younger tadpoles appeared stronger than older animals, but the reverse was seen for NF150P and NF200P. Complete signal transection of stage IV tadpoles resulted in degeneration and then regeneration of the cord tissue of both cut ends. Each stump lengthened by about 350 microns in the 4 weeks after the lesion. In the proximal stumps, the levels of NF150P or NF200P in the ventral axons at 550-350 microns proximal to the transection site increased notably by about 24-73% of the control value 7-28 days post-transection; however, the content of NF150D was decreased. The densities of NF150D and NF150P protein spots on the Coomassie blue-stained two-dimensional gels of the normal and injured cords also displayed alterations similar to the immunocytochemical data. Intense labeling by the anti-NF150P or NF200P was present in the cell bodies of axotomized motor neurons in the ventral horn. The results suggest that central axonal regeneration may be accompanied by upregulated phosphorylated neurofilament proteins.

Animals↗
PubMed1982

Immunoblot identification of phosphorylated basic proteins of rat and rabbit CNS and PNS myelin: evidence for four phosphorylated basic proteins and P2 in rat PNS myelin.

The immunoblot technique permits the visualization of proteins following their separation on acrylamide gels, transfer to cellulose nitrate sheets and subsequent staining with antiserum. We have utilized this technique to demonstrate the presence of four basic proteins in rat PNS myelin with molecular weights of 21K, 18K, 17K, and 14K. Similarly, we demonstrated the presence of two basic proteins in rabbit PNS myelin (molecular weights of 21K and 18K). Exposure of the immunostained cellulose nitrate strips to X-ray film revealed the phosphorylation of four and two basic proteins in rat and rabbit PNS myelin, respectively. These basic proteins were present in the CNS myelin of the two species and were also phosphorylated. Because of the sensitivity of the immunoblot technique, it was also possible for us to visualize the P2 protein in both rat and rabbit PNS myelin.

Animals↗
PubMed1986

Impaired insulin receptor phosphorylation in skeletal muscle membranes of db/db mice: the use of a novel skeletal muscle plasma membrane preparation to compare insulin binding and stimulation of receptor phosphorylation.

A method has been developed to isolate skeletal muscle plasma membranes from mice in good yield without harsh extraction procedures. The method involves perfusion of mouse hindquarters with a calcium-deficient buffer containing collagenase and hyaluronidase. This is followed by gentle disruption, filtration, and differential centrifugations. The entire procedure takes about six hours and the yield is approximately 4 mg. protein from 10 g. equivalent of hindquarter muscle. The preparation contained predominantly plasma membranes based on specific activities of marker enzymes, electron microscopic data, and specific binding sites for insulin and a -adrenergic ligand. Studies using such preparations from lean, 4-5 week old and 12-20 week old db/db mice showed marked reduction in the phosphorylation of the 95 kDa subunit of the insulin receptor of the obese mice with no change in insulin binding. In addition, there was a progressive reduction in insulin sensitivity in stimulating receptor phosphorylation in the db/db mice.

Animals↗
PubMed1987

Phosphorylation of protamines by protein kinase C: involvement of sites which are phosphorylated in vivo and are not affected by cAMP-dependent protein kinase.

Most fish protamines contain two phosphorylatable sites both of which incorporate phosphate in vivo. Here we show that in two protamines (salmine A1 and clupeine Y1) the site more distant from the N-terminus (residues 20-21) is unaffected by cAMP-dependent protein kinase while it represents the main target for protein kinase C. Such a phosphorylation is typically independent of Ca2+ and phospholipids: responsiveness to these effectors however is conferred by previous fragmentation of protamine with thermolysin. These results suggest that Ca2+, phospholipid-independent phosphorylation of protamine by protein kinase C might have physiological relevance and shed light on the structural basis for the specificity of such an unique process.

Amino Acid Sequence↗
PubMed1983

Phosphorylated metabolites and effects of amphotericin B and ouabain on phosphorylation state in amphibian cornea.

The phosphorylated metabolite content of endothelial denuded frog corneas and toad corneal epithelial cells was measured. The extract of frog corneas contained adenine, uridine and guanine nucleotides, in addition to various glycolytic intermediates, orthophosphate (Pi) and creatine phosphate. The relationship between the phosphorylation state: PS = [ATP]/[ADP] X [Pi] and the rate of active ion transport was studied in the toad cornea. This was considered by measuring the effects of 10(-5) M-amphotericin B and 10(-2) M-ouabain on the adenine nucleotide and Pi content of toad epithelial cells since these drugs selectively stimulate and inhibit active ion transport and metabolic rates, respectively. In order to calculate PS values, we derived and validated a relationship for converting content in terms of nmol/mg protein into concentration (mM). Incubation with amphotericin B in six different cell suspensions resulted in decreases of the ATP and ADP concentrations from 2.3 to 1.2 mM and 0.7 and 0.4 mM, respectively. The Pi concentration increased more than the ATP decrease from 6.2 to 12.2 mM. The average of these individual changes in each of the suspensions resulted in a significant decrease of the calculated PS from 599 +/- 226 to 342 +/- 127 M-1. However, ouabain increased the ATP concentration from 2.3 to 2.6 mM and decreased the ADP and Pi concentrations from 0.7 to 0.3 mM and 6.2 to 5.4 mM, respectively. The average of these individual changes in each of the suspensions resulted in a significant increase of the PS from 599 +/- 226 to 2975 +/- 1639 M-1. These results support the current notion that the PS is the feedback signal synchronizing metabolic rates with the energetic requirements of active Na and K transport.

Adenine Nucleotides↗
PubMed1995

Activation of the MAP kinase homologue RK requires the phosphorylation of Thr-180 and Tyr-182 and both residues are phosphorylated in chemically stressed KB cells.

A MAP kinase homologue, termed the reactivating kinase (RK), lies in a signalling pathway which mediates cellular responses to stress. Here we demonstrate that the stress-induced activation of the RK in human KB cells is accompanied by the phosphorylation of Thr-180 and Tyr-182, and that the phosphorylation of both residues is required for the activation of this enzyme.

Amino Acid Sequence↗
PubMed1994

Manipulation of ion-pairing reagents for reversed-phase high-performance liquid chromatographic separation of phosphorylated opioid peptides from their non-phosphorylated analogues.

The use of reversed-phase high-performance liquid chromatography for the separation of a mixture of 14 phosphorylated and non-phosphorylated enkephalins is described. The influence of two homologous series of hydrophobic ion-pairing reagents, consisting of perfluorinated carboxylic (trifluoroacetic, pentafluoropropionic and hexafluorobutyric) acids and sodium salts of sulfonic (butane-, hexane- and heptane-) acids, on the retention of enkephalin peptides was investigated. The incorporation of the phosphate group reduces retention time in proportion with the resulting change in hydrophobicity of the peptide. All peptides exhibit increase in retention time with increase in the counter ion hydrophobicity. The increase is proportional to the number of positively charged groups present in a peptide. Phosphopeptides show small increases in retention times than their corresponding non-phospho derivatives. The near-neighbor effect of the Tyr-O-phosphate group is responsible for suppression of the ion-pairing interaction of the mobile phase counter ions with the positively charged terminal amino group of enkephalins.

Amino Acid Sequence↗
PubMed1987

Chick oviduct progesterone receptor phosphorylation: characterization of a copurified kinase and phosphorylation in primary cultures.

A protein kinase activity was copurified with the chick oviduct progesterone receptor. The enzyme is magnesium dependent and can use the B subunit of progesterone receptor or histones as substrates. The physiochemical parameters of the kinase were determined [pI approximately 5.3; Stokes radius approximately 7.2 nm; sedimentation coefficient (S 20,w) approximately 5.6] and compared to those of the purified B subunit. The results were consistent with the presence of an unique enzyme distinct from the receptor itself. The physiological significance of receptor phosphorylation was investigated in oviduct cells grown in primary culture. Cells were labeled with [32P]orthophosphate in presence or absence of progesterone and the receptor components were immunoprecipitated with a specific polyclonal antibody. Although progesterone treatment lead to the attachment of most of the receptor (approximately 80%) to nuclear structures, the 32P-labeled B subunit was only recovered in the cytosol fraction. Different procedures to extract the nuclear receptor did not allow detection of any 32P-labeled form in the nuclear-soluble fractions, suggesting that the B subunit was not further phosphorylated upon the exposure of cells to progesterone.

Animals↗