scholarly journals The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus

1986 ◽  
Vol 239 (1) ◽  
pp. 205-211 ◽  
Author(s):  
M Katan ◽  
M J McGarvey ◽  
W S Stevely ◽  
D P Leader
Keyword(s):  
Protein Kinase ◽  
Ribosomal Protein ◽  
Time Course ◽  
Pseudorabies Virus ◽  
Deae Cellulose ◽  
Protein Kinase Activity ◽  
Ribosomal Protein S6 ◽  
Infected Cells

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.

Protein kinase activity and ribosome phosphorylation in ethionine-treated rats

1979 ◽  
Vol 57 (3) ◽  
pp. 209-215 ◽  
Author(s):  
Margaret A. Treloar ◽  
Robert Kisilevsky
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Ribosomal Protein ◽  
Kinase Activity ◽  
Synthetic Activity ◽  
Clear Correlation ◽  
Protein Kinase Activity ◽  
Phosphoprotein Phosphatase ◽  
Ribosomal Protein S6

The regulation of protein synthesis at the level of the ribosome was investigated using the model system of ethionine-induced inhibition of protein synthesis. The phosphorylation of ribosomal protein S6 was examined in vivo during ethionine intoxication and during the adenine-induced reversal of ethionine intoxication. The extent of phosphorylation of S6 correlated well with protein synthetic activity observed after ethionine, and ethionine followed by adenine treatments. No clear correlation was observed in the ethionine system between cyclic adenosine 3′:5′-monophosphate concentration or the activity of ribosomal protein kinase and the phosphorylation of ribosomal protein S6. A role for a cyclic adenosine 3′:5′-monophosphate-dependent ribosomal phosphoprotein phosphatase is postulated.

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

1983 ◽  
Vol 3 (7) ◽  
pp. 621-629 ◽  
Author(s):  
Michael J. McGarvey ◽  
David P. Leader
Keyword(s):  
Protein Kinase ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Casein Kinase ◽  
Protein Kinase Activity ◽  
Similar Reaction ◽  
Histone Kinase

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
Author(s):  
Markus Frederich ◽  
Li Zhang ◽  
James A. Balschi
Keyword(s):  
Protein Kinase ◽  
Metabolic Inhibitors ◽  
Protein Kinase Activity ◽  
Α Subunit ◽  
Rat Hearts ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

scholarly journals Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.

1981 ◽  
Vol 91 (1) ◽  
pp. 167-174 ◽  
Author(s):  
R M Lewis ◽  
D L Nelson
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Excitable Membrane ◽  
Protein Kinase Activity ◽  
Paramecium Tetraurelia ◽  
Phosphoprotein Phosphatase ◽  
Endogenous Protein ◽  
Endogenous Substrates

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.

5′-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

2011 ◽  
Vol 32 (2) ◽  
pp. 197-209 ◽  
Author(s):  
Yugo Tsuchiya ◽  
Fiona C. Denison ◽  
Richard B. Heath ◽  
David Carling ◽  
David Saggerson
Keyword(s):  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Calcium Signalling ◽  
Metabolic Stress ◽  
Protein Kinase Activity ◽  
Adult Rat ◽  
Protein Kinase C Inhibitor ◽  
Amp Activated Protein Kinase

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4 min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic ‘metabolic stress’. Inactivation of AMPK by adrenaline was abolished by 1 μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.

scholarly journals Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis

2005 ◽  
Vol 73 (12) ◽  
pp. 8334-8344 ◽  
Author(s):  
Ranadhir Dey ◽  
Arup Sarkar ◽  
Nivedita Majumder ◽  
Suchandra Bhattacharyya (Majumdar) ◽  
Kaushik Roychoudhury ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Leishmania Donovani ◽  
Disease Process ◽  
Kinase C ◽  
Parasitic Burden ◽  
Infected Cells

ABSTRACT The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 h. In this study, we investigated the role of MIP-1α and MCP-1 in the regulation of impaired PKC activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.

scholarly journals Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

2000 ◽  
Vol 20 (16) ◽  
pp. 5858-5864 ◽  
Author(s):  
Gregory J. Reynard ◽  
William Reynolds ◽  
Rati Verma ◽  
Raymond J. Deshaies
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Multiple Substrates ◽  
Cell Extracts

ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

scholarly journals Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src.

1986 ◽  
Vol 6 (3) ◽  
pp. 751-757 ◽  
Author(s):  
M P Kamps ◽  
B M Sefton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Atp Binding Site ◽  
Rous Sarcoma ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
Dependent Protein ◽  
Infected Cells

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

scholarly journals Testis-specific mak protein kinase is expressed specifically in the meiotic phase in spermatogenesis and is associated with a 210-kilodalton cellular phosphoprotein.

1993 ◽  
Vol 13 (7) ◽  
pp. 4146-4156 ◽  
Author(s):  
A Jinno ◽  
K Tanaka ◽  
H Matsushime ◽  
T Haneji ◽  
M Shibuya
Keyword(s):  
Protein Kinase ◽  
Cellular Protein ◽  
Kinase Assay ◽  
Soluble Form ◽  
Protein Kinase Activity ◽  
Cell Fractionation ◽  
Testicular Germ Cell ◽  
Fusion Products

The mak gene encodes a new protein kinase distantly related to cdc2 kinase, and its transcripts are expressed exclusively in testicular germ cells at and after meiosis (H. Matsushime, A. Jinno, N. Takagi, and M. Shibuya, Mol. Cell. Biol. 10:2261-2268, 1990). In this study, we prepared a series of antibodies against synthetic peptides and fusion products of the mak gene and characterized the subcellular localization, protein kinase activity, and association with other cellular proteins of Mak. Mak products were identified as 66- and 60-kDa proteins that specifically appeared in rat testes after puberty. Testicular germ cell fractionation revealed that Mak products were most abundant in the fraction of the late pachytene stage and that their levels were dramatically decreased in postmeiotic haploid cells. Mak products were localized mostly in the cytoplasm as a soluble form. [35S]methionine labelling demonstrated that Mak products were associated with a 210-kDa cellular protein; in an in vitro kinase assay with immunoprecipitates of Mak products, the 210-kDa cellular protein was efficiently phosphorylated on serine and threonine residues. Furthermore, in a testicular cell culture system with 32Pi, the 210-kDa molecule associated with Mak was phosphorylated in vivo on serine and threonine residues. These results strongly suggest that the Mak complex may play a role in meiosis during spermatogenesis and that a phosphorylated 210-kDa protein is one of the physiological substrates for this protein kinase.

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