Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Apr;19(4):2455-64.
doi: 10.1128/MCB.19.4.2455.

The J domain of papovaviral large tumor antigen is required for synergistic interaction with the POU-domain protein Tst-1/Oct6/SCIP

E Sock  1 , J Enderich, M Wegner
Affiliations

The J domain of papovaviral large tumor antigen is required for synergistic interaction with the POU-domain protein Tst-1/Oct6/SCIP

E Sock et al. Mol Cell Biol. 1999 Apr.

Abstract

Large T antigens from polyomaviruses are multifunctional proteins with roles in transcriptional regulation, viral DNA replication, and cellular transformation. They have been shown to enhance the activity of various cellular transcription factors. In the case of the POU protein Tst-1/Oct6/SCIP, this enhancement involves a direct physical interaction between the POU domain of the transcription factor and the amino-terminal region of large T antigen. Here we have analyzed the structural requirements for synergistic interaction between the two proteins in greater detail. Tst-1/Oct6/SCIP and the related POU protein Brn-1 were both capable of direct physical interaction with large T antigen. Nevertheless, only Tst-1/Oct6/SCIP functioned synergistically with large T antigen. This differential behavior was due to differences in the amino-terminal regions of the proteins, as evident from chimeras between Tst-1/Oct6/SCIP and Brn-1. Synergy was specifically observed for constructs containing the amino-terminal region of Tst-1/Oct6/SCIP. Large T antigen, on the other hand, functioned synergistically with Tst-1/Oct6/SCIP only when the integrity of its J-domain-containing amino terminus was maintained. Mutations that disrupted the J domain concomitantly abolished the ability to enhance the function of Tst-1/Oct6/SCIP. The J domain of T antigen was also responsible for the physical interaction with Tst-1/Oct6/SCIP and could be replaced in this property by other J domains. Intriguingly, a heterologous J domain from a human DnaJ protein partially substituted for the amino terminus of T antigen even with regard to the synergistic enhancement of Tst-1/Oct6/SCIP function. Given the general role of J domains, we propose chaperone activity as the underlying mechanism for synergy between Tst-1/Oct6/SCIP and large T antigens.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Physical interaction of JC viral large T antigen with POU domains. (A) GST or GST fusions were immobilized on glutathione-agarose resins, purified, and analyzed on a Coomassie blue-stained SDS-polyacrylamide gel. GST fusions were prepared from the following proteins: wild-type Tst-1/Oct6/SCIP (Tst-1), the isolated POU domain of Tst-1/Oct6/SCIP (Tst-1 Pou), a mutant Tst-1/Oct6/SCIP with tryptophane-to-cysteine and phenylalanine-to-serine mutations in helix 3 of the POU homeodomain (Tst-1 Pou mt), and the isolated POU domain of Brn-1 (Brn-1 POU). (B) Purified large T antigen (T ag) was incubated with glutathione-agarose resins carrying GST or GST fusions. Resin-bound T antigen was detected after SDS-polyacrylamide gel electrophoresis by Western blotting with an anti-JC viral T-antigen antiserum. One-fifth of the amount of T antigen incubated with the resins is shown for comparison (input). The sizes of molecular mass markers are indicated in kilodaltons.
FIG. 2
FIG. 2
Functional comparison between Tst-1/Oct6/SCIP and Brn-1. Luciferase reporter plasmids pJCearly-luc (A) and siteA-luc (B) were cotransfected with pCMV/Tst-1, pCMV/Brn-1, pRSV/JCT, or combinations thereof in U138 cells as indicated. Luciferase activities were determined in three independent experiments, each performed in duplicate. Values from transfections with luciferase reporter and empty expression plasmids were arbitrarily set to 1. Data from all other transfections are presented as fold induction above this level. Error bars indicate standard deviations.
FIG. 3
FIG. 3
Requirement for the amino-terminal domain of Tst-1/Oct6/SCIP. (A and D) Schematic representation of the TNBP and BNTP chimeras and the Tst-1 mutants Δ13-137 and Δ144-197 in relation to Tst-1/Oct6/SCIP and Brn-1. (B and E) Expression of TNBP and BNTP chimeric proteins and the Tst-1 mutants Δ13-137 and Δ144-197 in transfected cells, controlled by Western blot analyses of CV-1 whole-cell extracts with polyclonal antisera against Tst-1/Oct6/SCIP (α Tst-1) and Brn-1 (α Brn-1). Numbers on the left indicate sizes of molecular mass markers in kilodaltons. (C and F) Synergy of TNBP and BNTP chimeras as well as the Tst-1 mutants Δ13-137 and Δ144-197 with JC viral T antigen. The luciferase reporter plasmid siteA-luc was cotransfected with pCMV/Tst-1, pCMV/Brn-1, pCMV/TNBP, pCMV/BNTP, pCMV/Tst-1Δ13-137, pCMV/Tst-1Δ144-197, pRSV/JCT, or combinations thereof in U138 cells as indicated. Luciferase activities were determined in three independent experiments, each performed in duplicate. Values from transfections with luciferase reporter and empty expression plasmids were arbitrarily set to 1. Data from all other transfections are presented as fold induction above this level. Error bars indicate standard deviations.
FIG. 4
FIG. 4
Requirement for J-domain function of large T antigen. (A) Schematic representation of SV40 dl1135, 5110, K67P, and 3213 large-T-antigen mutants. DNA-bdg, DNA-binding domain. (B) The luciferase reporter plasmid siteA-luc was cotransfected in U138 cells with pCMV/Tst-1, pRSV/JCT, or pRSV-B-neo (SV40T) and its mutants (dl1135, 5110, K67P, and 3213) in the indicated combinations. Luciferase activities were determined in three independent experiments, each performed in duplicate. Values from transfections with luciferase reporter and empty expression plasmids were arbitrarily set to 1. Data from all other transfections are presented as fold induction above this level. Error bars indicate standard deviations.
FIG. 5
FIG. 5
Requirement for physical interactions between the J domain of large T antigen and Tst-1/Oct6/SCIP. (A) CV1 nuclear extract containing Tst-1/Oct6/SCIP was incubated with glutathione-agarose resins carrying GST or GST fusions with SV40 T antigen (GST-SV40 T). The GST fusions contained the following T-antigen sequences: amino acids 1 to 82, 1 to 65, and 66 to 82 of wild-type T antigen (1-82, 1-65, and 66-82, respectively) and amino acids 1 to 82 of the dl1135 mutant (1-82 dl1135), of the 5110 mutant (1-82 5110), and of the K67P mutant (1-82 K67P). Resin-bound Tst-1/Oct6/SCIP was detected after SDS-polyacrylamide gel electrophoresis by Western blotting with an anti-Tst-1 antiserum. One-fifth of the amount of Tst-1/Oct6/SCIP incubated with the resins is shown for comparison (input). (B) Coomassie blue-stained SDS-polyacrylamide gel of GST proteins. The sizes of molecular mass markers are indicated in kilodaltons.
FIG. 6
FIG. 6
Effects of DnaJ on synergy between Tst-1/Oct6/SCIP and large T antigen. (A) Purified DnaJ was incubated with glutathione-agarose resins carrying GST or GST/Tst-1. Resin-bound DnaJ was detected after SDS-polyacrylamide gel electrophoresis by Western blotting with a polyclonal antiserum against DnaJ. (B) Nuclear (NE) and cytosolic (Cyt) extracts from CV-1 cells ectopically expressing DnaJ were analyzed by Western blotting with the DnaJ antiserum. Numbers on the right in panel A and on the left in panel B indicate sizes of molecular mass markers in kilodaltons. (C) The luciferase reporter plasmid siteA-luc was cotransfected with pCMV/Tst-1, pCMV/DnaJ, pRSV/JCT, or combinations thereof in U138 cells as indicated. (D) The estrogen-responsive luciferase reporter 2xERE-luc was cotransfected with pRSV/hER, pCMV/DnaJ, or both in estrogen (E2)-stimulated U138 cells as indicated. Luciferase activities for panels C and D were determined in three independent experiments, each performed in duplicate. Values from transfections with luciferase reporter and empty expression plasmids were arbitrarily set to 1. Data from all other transfections are presented as fold induction above this level. Error bars indicate standard deviations.
FIG. 7
FIG. 7
Effects of a heterologous J domain on synergy between Tst-1/Oct6/SCIP and large T antigen. The luciferase reporter plasmid siteA-luc was cotransfected in U138 cells with pCMV/Tst-1, pSG5-HSJ1-T, or pSG5-HSJ1-HQ in the indicated combinations. Luciferase activities were determined in three independent experiments, each performed in duplicate. Values from transfections with luciferase reporter and empty expression plasmids were arbitrarily set to 1. Data from all other transfections are presented as fold induction above this level. Error bars indicate standard deviations.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alvarez B G, Rosenfeld M G, Swanson L W. Model of forebrain regionalization based on spatiotemporal patterns of POU-III homeobox gene expression, birthdates, and morphological features. J Comp Neurol. 1995;355:237–295. - PubMed
    1. Berger L, Smith D B, Davidson I, Hwang J-J, Fanning E, Wildeman A G. Interaction between T antigen and TEA domain of the factor TEF-1 derepresses simian virus 40 late promoter in vitro: identification of T-antigen domains important for transcriptional control. J Virol. 1996;70:1203–1212. - PMC - PubMed
    1. Bermingham J R, Scherer S S, O’Connell S, Arroyo E, Kalla K A, Powell F L, Rosenfeld M G. Tst-1/Oct6/SCIP regulates a unique step in peripheral myelination and is required for normal respiration. Genes Dev. 1996;10:1751–1762. - PubMed
    1. Campbell K S, Mullane K P, Aksoy I A, Stubdal H, Zalvide J, Pipas J M, Silver P A, Roberts T M, Schaffhausen B S, DeCaprio J A. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient DNA replication. Genes Dev. 1997;11:1098–1110. - PubMed
    1. Casaz P, Sundseth R, Hansen U. trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1. J Virol. 1991;65:6535–6543. - PMC - PubMed

Publication types

LinkOut - more resources