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.

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 Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

1986 ◽  
Vol 6 (6) ◽  
pp. 2033-2040 ◽  
Author(s):  
H Piwnica-Worms ◽  
D R Kaplan ◽  
M Whitman ◽  
T M Roberts
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Shuttle Vector ◽  
T Antigen ◽  
Protein Kinase Activity ◽  
Polyomavirus Middle T Antigen ◽  
Nih 3T3 ◽  
Middle T Antigen

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.

scholarly journals Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase

2013 ◽  
Vol 33 (6) ◽  
Author(s):  
James M. J. Dickson ◽  
Woo-Jeong Lee ◽  
Peter R. Shepherd ◽  
Christina M. Buchanan
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Cell Signalling ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
His Tag ◽  
The Impact

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

scholarly journals Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase

2002 ◽  
Vol 368 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Pauline DOUGLAS ◽  
Gopal P. SAPKOTA ◽  
Nick MORRICE ◽  
Yaping YU ◽  
Aaron A. GOODARZI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Sequencing ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

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.

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo

1986 ◽  
Vol 6 (6) ◽  
pp. 2033-2040
Author(s):  
H Piwnica-Worms ◽  
D R Kaplan ◽  
M Whitman ◽  
T M Roberts
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Shuttle Vector ◽  
T Antigen ◽  
Protein Kinase Activity ◽  
Polyomavirus Middle T Antigen ◽  
Nih 3T3 ◽  
Middle T Antigen

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.

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