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PubMed1995

Stress-induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine-53 adjacent to the DNase I binding loop.

Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyostelium discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger Mono-P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress.

Actins↗

PubMed1989

Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: localization of the site of in vivo and in vitro phosphorylation.

1. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released numerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in vivo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.

Chromatography, High Pressure Liquid↗

PubMed1987

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1.

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.

Animals↗

PubMed1988

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit.

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.

Amino Acid Sequence↗

PubMed1990

Phosphorylation of protein synthesis initiation factor-2. Identification of the site in the alpha-subunit phosphorylated in reticulocyte lysates.

The data presented here show that serine-51 of the alpha-subunit of eukaryotic initiation factor eIF-2 is the only residue phosphorylated by the eIF-2 alpha-specific kinases HCR (haem-controlled repressor) and dsI (double-stranded RNA-activated inhibitor) in vitro. This confirms our earlier finding that serine-48 is not labelled by either kinase. Methodology appropriate for the examination of phosphorylation sites in eIF-2 alpha in whole cells and their extracts has been developed, and used to study the site(s) in eIF-2 alpha labelled in reticulocyte lysates. Only serine-51 became phosphorylated under conditions of haem-deficiency or in the presence of double-stranded RNA. No evidence for a second phosphorylation site on the alpha-subunit was obtained with the lysates and conditions used here.

Amino Acid Sequence↗

PubMed1986

Reaction of Lewy bodies with antibodies to phosphorylated and non-phosphorylated neurofilaments.

The reaction of Lewy bodies in the substantia nigra and the sympathetic ganglia was studied in 10 cases of Parkinson's disease and other Lewy body-related conditions, using monoclonal antibodies to non-phosphorylated (02-40) and phosphorylated (03-44 and 07-5) neurofilaments. Although many Lewy bodies were unstained, a peripheral ring of positive immunoreactivity was frequently observed. This reaction was more common and more intense with antibodies to phosphorylated neurofilaments, suggesting a posttranslational phosphorylation of elements in the Lewy body. Although Lewy bodies appear to have some antigenic sites in common with neurofilaments, certain differences between neurofilaments and Lewy body filaments are emphasized.

Antibodies, Monoclonal↗

PubMed2006

Transcriptional autoregulation by phosphorylated and non-phosphorylated KaiC in cyanobacterial circadian rhythms.

Cyanobacteria are the simplest organisms known to exhibit circadian rhythms, which is the fundamental process of homeostasis adapting to daily environmental changes. The cyanobacterial clock gene products, KaiA, KaiB, and KaiC interact with each other, and regulate KaiC phosphorylation and kaiBC expression in a circadian fashion. Molecular genetic study recently proposed that KaiC protein may enhance and repress transcription of clock genes depending on KaiC's phosphorylation status, however, the precise mechanism is still unknown. We developed a mathematical model for the dynamics of cyanobacterial circadian rhythms focusing on the transcriptional regulation by KaiC. We investigated the model using numerical methods, and predicted the transcriptional regulation mechanism by KaiC. We searched for conditions for generating circadian oscillation and concluded that only two mechanisms of the transcriptional regulation are the possible pictures. One is the Transcriptional Repression Model where KaiC represses transcription of the clock genes after phosphorylation, and the other is the Transcriptional Activation Model where KaiC induces transcription after phosphorylation. The Transcriptional Repression Model includes self-repression similarly to the circadian oscillator models that have been proposed previously, and dynamical oscillation is easy to understand. However, the Transcriptional Activation Model does not include any direct repression in its interactive circuit, and is distinct from the previous ideas for circadian clocks. Subsequent computer simulation showed that the Transcriptional Activation Model explains most of the observed mutant phenotypes, and the Transcriptional Repression Model realizes only a half of them. It was also revealed that oscillations in the Transcriptional Activation Model is much more robust against the disruption by cell division or cell elongation than the Transcriptional Repression Model. It suggests that the Transcriptional Activation Model may reflect the essence of the actual transcriptional mechanism of the kai oscillator in cyanobacteria.

Bacterial Proteins↗

PubMed2001

Preserved phosphorylation of RET receptor protein in spinal motor neurons of patients with amyotrophic lateral sclerosis: an immunohistochemical study by a phosphorylation-specific antibody at tyrosine 1062.

Ret oncoprotein is a functional receptor for the glial cell line-derived neurotrophic factor (GDNF) family and it is expressed in motor neurons, playing an important role in the motor neuron function. In this study, we examined the expression of the phosphorylation state of tyrosine residue 1062 (Tyr-1062) of Ret in the spinal cords of amyotrophic lateral sclerosis (ALS), using the phosphorylation state specific antibody at Tyr-1062 of Ret. The immunohistochemical study demonstrated that Tyr-1062 of Ret was phosphorylated to variable extents in the surviving motor neurons of all the ALS as well as controls studied. This is the first report that the phosphorylation of Tyr-1062 occurred in neurons with nononcogenic type of Ret. The Ret-signaling pathway by Tyr-1062 autophosphorylation is constitutively activated via the phosphatidylinositol 3-kinase and/or mitogen-activated protein kinase cascade for motoneuron survival even in the ALS motor neurons, supporting the view that GDNF is a candidate for therapeutic approach to ALS.

Aged↗

PubMed1999

Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27).

The effect of external Ca2+ ([Ca2+]e) on the incorporation of [32P] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+]e increased total 32P-incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total 32P-incorporation between zero [Ca2+]e and 1 mM [Ca2+]e was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1-10 nM; higher concentrations of okadaic acid (50-100 nM) further increased total 32P-incorporation. In zero [Ca2+]e the non-specific channel blocker Co2+ (1 mM) decreased total 32P-incorporation by approximately 30%. The results were compared with a previous study [S.T. Wofchuk, R. Rodnight, Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res. 85 (1995) 181-186] in which it was shown that in immature hippocampal slices zero [Ca2+]e compared with 1 mM [Ca2+]e increased 32P-incorporation into GFAP without changing total incorporation. The difference between the results for total 32P-incorporation obtained in cultured astrocytes and immature brain tissue was found to be related to the concentration of [Ca2+]e in the medium since in slices concentrations of [Ca2+]e higher than 1 mM progressively decreased total incorporation. The difference may reflect a higher Ca2+-permeability of the plasma membrane in cultured astrocytes and/or to the complex structure of the slice tissue. In two-dimensional electrophoresis HSP27, in contrast to GFAP and VIM, was separated into 3 immunodetectable isoforms only two of which were normally phosphorylated. After labelling in the presence of okadaic acid both immunodetectable and phosphorylated HSP27 focussed as a single polypeptide. Phorbol dibutyrate (1 microM) and zero [Ca2+]e stimulated the phosphorylation of both isoforms, but in the case of zero [Ca2+]e the effect on the more acidic isoform was proportionally greater.

Age Factors↗

PubMed1997

Regulatory phosphorylation of plant phosphoenolpyruvate carboxylase: role of a conserved basic residue upstream of the phosphorylation site.

In order to mimic regulatory phosphorylation of the Ser-15 of maize C4-form phosphoenolpyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wild-type PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition.

Asparagine↗

PubMed1998

Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B'' (delta) regulatory subunit in vitro and identification of the phosphorylation sites.

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.

Animals↗

PubMed1997

p21ras farnesyltransferase alpha- and beta-subunits are phosphorylated in PC-12 cells: TGF-beta signaling pathway independent phosphorylation.

Farnesyltransferase (FTase) catalyzes the transfer of a farnesyl isoprenoid to the conserved carboxyl-terminal cysteine residue of proteins terminating with the CAAX sequence. Rat brain FTase is a heterodimer consisting of a 49 kDa alpha-subunit and a 46 kDa beta-subunit. In this report, we show, for the first time, that the beta-subunit of FTase is phosphorylated in vivo and the FTase heterodimer contains phosphorylated alpha/beta-subunits in rat adrenal medulla pheocytochroma PC-12 cells. The presence of the phosphorylated FTase subunits as heterodimer in PC-12 cells which are known to be deficient in TGF-beta signaling pathways argues against the involvement of this pathway in their phosphorylation and heterodimerization.

Alkyl and Aryl Transferases↗

PubMed2000

Differential responses of phosphorylated mitogen-activated protein kinase and phosphorylated cyclic-AMP response element-binding protein immunoreactivity in the rat brain to sub-convulsive pentylenetetrazol.

The possible advantage of using multiple phospho-specific antibodies to study changes in brain activity was assessed. For this purpose, rats were injected intraperitoneally with either a control treatment or 15 mg/kg pentylenetetrazol. The sub-convulsive dose of pentylenetetrazol did not induce marked behavioural effects. Ten minutes after treatment, the rats were perfused and the brains were dissected. Adjacent brain sections were immunohistochemically stained for phosphorylated cyclic-AMP response element-binding protein and phosphorylated mitogen-activated protein kinase. Opposite effects of pentylenetetrazol treatment were observed on the immunoreactivity of these two antibodies within the hypothalamic paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. In these regions, pentylenetetrazol treatment increased phosphorylated mitogen-activated protein kinase immunoreactivity, but decreased phosphorylated cyclic-AMP response element-binding protein immunoreactivity. These findings show that changes in the activity of a brain nucleus can be accompanied by differential changes in the activity of two signal transduction pathways, which can be detected immunohistochemically. Therefore, the use of multiple phospho-specific antibodies may enhance our potential to monitor changes in brain activity.

Animals↗

PubMed1996

Spatiotemporal dynamics of CREB phosphorylation: transient versus sustained phosphorylation in the developing striatum.

The cAMP response element-binding protein (CREB) is a plasticity-associated transcription factor that can potentially integrate cAMP and calcium signals at the gene activation level. We tested for convergent Ser-133 phosphorylation of CREB via dopamine D1/D5 receptors and L-type calcium channels in organotypic cultures of neonatal striatum. We found such convergence only transiently. Sustained CREB phosphorylation by D1/D5 receptor and L-type channel agonists was targeted to opposite (striosome and matrix) cellular phenotypes. Subsequent expression of the CRE-containing gene, c-fos, matched the divergent patterns of sustained CREB phosphorylation, and both divergent patterns could be switched by inhibition of phosphatases, including calcineurin. Control of the duration of CREB phosphorylation may be a critical regulator of CRE-mediated gene expression by dopamine and calcium.

Animals↗

PubMed1990

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change.

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).

Adenosine Diphosphate↗

PubMed1996

Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer.

The sodium-potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E.Nae), reacts with 0.01-2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 +/- 0.9) x 10(2) s-1 and K0.5 = 75 +/- 25 microM (ph 7.4, 25 degrees C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd=Kf+Kr+9.2 x 10(2) s-1. However, the formation of phosphoenzyme from E.Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd=(4.2 +/- 0.8) x 10(2) s-1. This result show that phosphoryl transfer from bound ATP to the enzyme is not the rate limiting step for phosphoenzyme formation from E.Na3. The result is consistent with a rate-limiting conformational change of the E.Na3.ATP intermediate that is followed by rapid phosphoryl transfer, with kcat > or = 3000 s-1.

Adenosine Diphosphate↗

PubMed1988

Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis.

A cascade of protein phosphorylation, initiated by autophosphorylation of the CheA protein, may be important in the signal transduction pathway of bacterial chemotaxis. A proteolytic fragment of CheA cannot autophosphorylate, but can still transfer phosphate to proteins that generate excitation and adaptation signals. The site of CheA phosphorylation is His 48; mutants altered at this position are non-chemotactic. Similar mechanisms of transient protein phosphorylation and phosphoryl group transfer seem to be involved in processing sensory data and in activating specific gene expression.

Amino Acid Sequence↗

PubMed1984

Phosphorylation of ovine rhodopsin. Identification of the phosphorylated sites.

Light-dependent phosphorylation of sheep opsin was obtained in purified discs to which was added a partially purified preparation of rhodopsin kinase. A maximum ratio of 1.8 mol of phosphate/mol of rhodopsin bleached was obtained. Perturbing the lipid bilayer did not alter the phosphorylation ratio. Dephosphorylation in both segments and discs was only achieved when the supernatant fraction from a retina homogenate was added. Complete dephosphorylation required the presence of the detergent dodecyltrimethylammonium bromide in the incubation medium. Treatment of phosphorylated disc membranes with Staphylococcal aureus V8 proteinase generated two membrane-bound fragments, only one of which (V8-S, Mr 12 000) was labelled, together with a soluble seven-residue peptide that contained [32P]phosphoserine. Peptide sequencing, together with subdigestion procedures, localized the phosphorylation sites to serine residues at positions 334, 338 and 343 in the whole sequence and threonine residues at positions 335 and 336.

Amino Acid Sequence↗