scholarly journals Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.

1990 ◽  
Vol 10 (12) ◽  
pp. 6472-6481 ◽  
Author(s):  
S P Lees-Miller ◽  
Y R Chen ◽  
C W Anderson
Keyword(s):  
Protein Kinase ◽  
Gel Filtration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mouse Tumor ◽  
Dependent Manner ◽  
Large Tumor ◽  
Ku Autoantigen

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen

1990 ◽  
Vol 10 (12) ◽  
pp. 6472-6481
Author(s):  
S P Lees-Miller ◽  
Y R Chen ◽  
C W Anderson
Keyword(s):  
Protein Kinase ◽  
Gel Filtration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mouse Tumor ◽  
Dependent Manner ◽  
Large Tumor ◽  
Ku Autoantigen

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

scholarly journals A DNA-activated protein kinase from HeLa cell nuclei.

1990 ◽  
Vol 10 (12) ◽  
pp. 6460-6471 ◽  
Author(s):  
T Carter ◽  
I Vancurová ◽  
I Sun ◽  
W Lou ◽  
S DeLeon
Keyword(s):  
Protein Kinase ◽  
Thermal Inactivation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Nuclei ◽  
Exogenous Substrates

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.

scholarly journals Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3)

2010 ◽  
Vol 431 (2) ◽  
pp. 267-275 ◽  
Author(s):  
Véronique Mouton ◽  
Louise Toussaint ◽  
Didier Vertommen ◽  
Marie-Agnès Gueuning ◽  
Liliane Maisin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dominant Negative ◽  
Rat Cardiomyocytes ◽  
Kinase Activation ◽  
Dependent Protein

On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBβ-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBα/β siRNA or PKBα siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBα.

scholarly journals Intestinal Hyperplasia Induced by Simian Virus 40 Large Tumor Antigen Requires E2F2

2007 ◽  
Vol 81 (23) ◽  
pp. 13191-13199 ◽  
Author(s):  
M. Teresa Sáenz-Robles ◽  
Jennifer A. Markovics ◽  
Jean-Leon Chong ◽  
Rene Opavsky ◽  
Robert H. Whitehead ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
Large Tumor ◽  
Intestinal Hyperplasia

ABSTRACT The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.

A DNA-activated protein kinase from HeLa cell nuclei

1990 ◽  
Vol 10 (12) ◽  
pp. 6460-6471
Author(s):  
T Carter ◽  
I Vancurová ◽  
I Sun ◽  
W Lou ◽  
S DeLeon
Keyword(s):  
Protein Kinase ◽  
Thermal Inactivation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Nuclei ◽  
Exogenous Substrates

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.

scholarly journals Stimulation of the Human Heat Shock Protein 70 Promoter in Vitro by Simian Virus 40 Large T Antigen

1989 ◽  
Vol 264 (27) ◽  
pp. 16160-16164
Author(s):  
I C Taylor ◽  
W Solomon ◽  
B M Weiner ◽  
E Paucha ◽  
M Bradley ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Human Heat Shock Protein ◽  
Stimulation Of

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

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