CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter

Abstract

The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.

FIG. 1

Specific association of Tax with CREB-2. (A) Analysis of the interaction between Tax and CREB-2 by β-galactosidase assay. The HF7c yeast cells were transformed with an expression vector containing the CREB-2 cDNA clone fused to the GAL4 activation domain (pGAD-CREB-2) together with a plasmid expressing either the GAL4 DNA binding domain alone (pGBT9) or with plasmid pGBT9 fused to Tax (pGBT-Tax) or fused to Lamin (pGBT-Lamin). pGBT-Tax was also cotransformed with a plasmid expressing the GAL4 activation domain alone (pGAD10). The β-galactosidase assay with O-nitrophenyl-β-d-galactoside (ONPG) as the substrate was performed on three independent colonies per transformation as described in the Clontech protocol. The mean values expressed in Miller units are shown. (B) In vitro binding assays of wild-type Tax, Tax-M22, and Tax-M47 to CREB-2. The GST-CREB-2 and GST-AS fixed to glutathione Sepharose beads were mixed with either in vitro-translated wild-type ³⁵S-Tax, ³⁵S-Tax-M22, or ³⁵S-Tax-M47 (lanes Tax, Tax-M22, and Tax-M47). Bound Tax was analyzed by SDS-PAGE (see the lanes corresponding to Tax + GST-CREB-2, Tax + GST-AS, Tax-M22 + GST-CREB-2, Tax-M22 + GST-AS, Tax-M47 + GST-CREB-2, and Tax-M47 + GST-AS).

FIG. 1

Specific association of Tax with CREB-2. (A) Analysis of the interaction between Tax and CREB-2 by β-galactosidase assay. The HF7c yeast cells were transformed with an expression vector containing the CREB-2 cDNA clone fused to the GAL4 activation domain (pGAD-CREB-2) together with a plasmid expressing either the GAL4 DNA binding domain alone (pGBT9) or with plasmid pGBT9 fused to Tax (pGBT-Tax) or fused to Lamin (pGBT-Lamin). pGBT-Tax was also cotransformed with a plasmid expressing the GAL4 activation domain alone (pGAD10). The β-galactosidase assay with O-nitrophenyl-β-d-galactoside (ONPG) as the substrate was performed on three independent colonies per transformation as described in the Clontech protocol. The mean values expressed in Miller units are shown. (B) In vitro binding assays of wild-type Tax, Tax-M22, and Tax-M47 to CREB-2. The GST-CREB-2 and GST-AS fixed to glutathione Sepharose beads were mixed with either in vitro-translated wild-type ³⁵S-Tax, ³⁵S-Tax-M22, or ³⁵S-Tax-M47 (lanes Tax, Tax-M22, and Tax-M47). Bound Tax was analyzed by SDS-PAGE (see the lanes corresponding to Tax + GST-CREB-2, Tax + GST-AS, Tax-M22 + GST-CREB-2, Tax-M22 + GST-AS, Tax-M47 + GST-CREB-2, and Tax-M47 + GST-AS).

FIG. 2

Activation of the HTLV-1 promoter by CREB-2. CEM cells were cotransfected with 2 μg of HTLV-1 LTR-CAT, 5 μg of pACβ1 (β-galactosidase-containing reference plasmid), 1 μg of pSG-Tax (+) or empty pSG-5 vector (−), and 0 to 10 μg of pCI-CREB-2 or empty pCI-neo. CAT values were normalized for β-galactosidase activity and are expressed as fold increase relative to that of cells cotransfected with pSG-5, pCI-neo, and HTLV-1 LTR CAT. CAT assays were conducted by using a CAT antigen capture assay kit (Boehringer Mannheim). Values are the means ± standard deviations (n = 4).

FIG. 3

Tax interacts with the bZIP domain of CREB-2. (A) In vitro binding assays of Tax to CREB-2, CREB-2_263–351, and CREB-2_1–262. GST-CREB-2, GST-CREB-2_263–351 (lane GST-bZIP), and GST-CREB-2_1–262 (lane GST-ΔbZIP) fixed to glutathione Sepharose beads were mixed with ³⁵S-Tax, and bound proteins were analyzed by SDS-PAGE. The first lane corresponds to in vitro-translated ³⁵S-Tax. (B) Activation of the HTLV-1 promoter by the bZIP domain of CREB-2. CEM cells were cotransfected as described in the legend to Fig. 2 with pSG-Tax and 0.1 to 1 μg of pCDM7-CREB-2_262–351, a CREB-2 bZIP domain expression vector. Values are the means ± standard deviations (n = 4).

FIG. 3

Tax interacts with the bZIP domain of CREB-2. (A) In vitro binding assays of Tax to CREB-2, CREB-2_263–351, and CREB-2_1–262. GST-CREB-2, GST-CREB-2_263–351 (lane GST-bZIP), and GST-CREB-2_1–262 (lane GST-ΔbZIP) fixed to glutathione Sepharose beads were mixed with ³⁵S-Tax, and bound proteins were analyzed by SDS-PAGE. The first lane corresponds to in vitro-translated ³⁵S-Tax. (B) Activation of the HTLV-1 promoter by the bZIP domain of CREB-2. CEM cells were cotransfected as described in the legend to Fig. 2 with pSG-Tax and 0.1 to 1 μg of pCDM7-CREB-2_262–351, a CREB-2 bZIP domain expression vector. Values are the means ± standard deviations (n = 4).

FIG. 4

Confocal microscopy analysis of the colocalization of Tax and CREB-2 in vivo. Cos7 cells were cotransfected with pSG-Tax and pEGFP-CREB-2_263–351. Analysis of the red (A), green (B), and merged (C) fluorescence was performed with a Bio-Rad MRC 1024 confocal microscope. The Tax protein was detected by using culture supernatant of the anti-Tax 168A51-42 hybridoma and goat anti-mouse immunoglobulin G antibody coupled to rhodamine (Pierce).