Enhanced Kinase-Activity in Sv40-Transformed Cells May Be Compensated by Enhanced Phosphatase-Activity in Revertants as Reflected by Phosphorylation of P53

Scheidtmann, Kh; Landsberg, G; Graessmann, A.

doi:10.3892/ijo.5.6.1353

Cited by 4 publications

(7 citation statements)

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“…The effect of okadaic acid was dose-dependent and affected mainly those phosphorylation sites which exhibit a high turnover in vivo (Scheidtmann et al, 1994) and which are preferentially dephosphorylated by phosphatase 2A in vitro (Scheidtmann et al, 1991). Since okadaic acid inhibits phosphatases 1 and 2A to a similar extent, in vivo, we cannot deduce with certainty which enzyme is responsible for the high phosphatase activity in insect cells.…”

Section: Discussionmentioning

confidence: 88%

“…This was particularly striking since in rat cells peptide 1 is efficiently phosphorylated only in the presence of a functional LT (Scheidtmann and Haber, 1990). It should be pointed out that the relative decrease of peptides 2 and 3 upon okadaic acid treatment is not due to dephosphorylation of these sites, as they exhibit a low turnover (Scheidtmann et al, 1994). Rather, phosphorylation of the other sites becomes so strong that the respective peptide maps had to be exposed for much shorter times.…”

Section: Resultsmentioning

confidence: 99%

“…However, our studies on phosphorylation of p53 showed that the C-terminal casein kinase I1 site is efficiently phosphorylated without okadaic acid (Scheidtmann and Haber, 1990; this study). Moreover, this site exhibits a low turnover in vivo (Scheidtmann et al, 1994) and is relatively resistant to phosphatase treatment in vitro (Scheidtmann et al, 1991). Thus, it appears unlikely that (a) okadaic acid treatment substantially enhanced phosphorylation of this site and (b) that this was responsible for enhanced DNA binding.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System. Manipulation of the Phosphorylation State with Okadaic Acid and Influence on DNA Binding

1995

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To elucidate the role of phosphorylation of p53 we used the baculovims expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two-dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although some sites were underrepresented. Coexpression of simian virus 40 (SV40) large T antigen or cyclin-dependent kinases, cdc2 or cdk2, had only marginal effects on the phosphorylation state of p53. However, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose-dependent manner and resembled that of p53 from SV40-transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid treatment, except for a mutant at the cdk site which failed to do so. These data show that: (a) insect cells contain all the protein kinases necessary for phosphorylation of a mammalian protein, p53 ; (b) in insect cells the ratio of kinase/phosphatase activities differs from that in mammalian cells so that underphosphorylation of recombinant proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate ; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA-binding activity of p53.

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Section: Discussionmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System. Manipulation of the Phosphorylation State with Okadaic Acid and Influence on DNA Binding

1995

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“…Since CK1δ phosphorylates p53 in vivo and co-immunoprecipitates with T-Ag/p53 complexes, it is most likely that CK1δ also targets T-Ag within the T-Ag/p53 complex [8], [49]–[56]. Indeed, in vitro kinase assays revealed that CK1δ is able to phosphorylate baculovirus-expressed T-Ag (figure 1A), suggesting that the altered phosphorylation of T-Ag and p53 [57] in Rev2 cells might result from an altered CK1δ activity. We therefore analyzed the activity of CK1δ in fractionated extracts of SV40 transformed SV-52 (maximal transformed) and Rev2 cells (minimal transformed).…”

Section: Resultsmentioning

confidence: 99%

Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

et al. 2012

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Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.

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“…T antigen binds to the growth suppressor proteins p53 and Rb and this binding seems to contribute considerably to the transforming potential of T antigen (Figge et al, 1988;DeCaprio et al, 1988;. Other protein/protein interactions, such as with the replication protein A (RPA) or with DNA polymerase primase complex, seem to be essential for virus replication (Fanning, 1994;Schneider et al, 1994;Fanning, 1992). The association of large T antigen with a phosphorylating enzyme has been known for years.…”

Section: Discussionmentioning

confidence: 99%

A Casein‐Kinase‐2‐Related Protein Kinase is Tightly Associated with the Large T Antigen of Simian Virus 40

Götz

Koenig

Issinger

et al. 1995

European Journal of Biochemistry

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The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we show that immunopurified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation of T antigen by the associated kinase is reduced whereas a p34d"-kinase-specific peptide has no influence. In addition, the Tantigen-associated protein kinase can use GTP and ATP as phosphate donors. These properties together with the observation that immunopurified T antigen can be phosphorylated by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a proteinkinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitm binding studies. Experiments with temperature-sensitive SV40-transformed cells provide evidence for a close correlation between cell transformation and phosphorylation of T antigen by the associated protein kinase.

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Enhanced Kinase-Activity in Sv40-Transformed Cells May Be Compensated by Enhanced Phosphatase-Activity in Revertants as Reflected by Phosphorylation of P53

Cited by 4 publications

References 0 publications

Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System. Manipulation of the Phosphorylation State with Okadaic Acid and Influence on DNA Binding

Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System. Manipulation of the Phosphorylation State with Okadaic Acid and Influence on DNA Binding

Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

A Casein‐Kinase‐2‐Related Protein Kinase is Tightly Associated with the Large T Antigen of Simian Virus 40

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