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.

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.

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.

Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

1976 ◽  
Vol 35 (03) ◽  
pp. 635-642 ◽  
Author(s):  
M Steiner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Qualitative Change ◽  
Protein Kinase Activity ◽  
Protein Substrates ◽  
Phosphoprotein Phosphatase ◽  
Progressive Loss ◽  
Platelet Membranes ◽  
Endogenous Protein ◽  
Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

scholarly journals Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases

2000 ◽  
Vol 350 (2) ◽  
pp. 353-359 ◽  
Author(s):  
Carolyn A. BEETON ◽  
Edwin M. CHANCE ◽  
Lazaros C. FOUKAS ◽  
Peter R. SHEPHERD
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Membrane Lipid ◽  
Kinetic Properties ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
Functional Roles ◽  
Wide Range ◽  
High Concentrations

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.

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.

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