Abstract
Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) is mediated by the viral oncoprotein Tax, which utilizes cellular transcriptional machinery to perform this function. The viral promoter carries three cyclic AMP-response elements (CREs), which are recognized by the cellular transcription factor cAMP-response element-binding protein (CREB). Tax binds to GC-rich sequences that immediately flank the CREs. The coactivator CREB-binding protein (CBP)/p300 binds to this promoter-bound ternary complex, which promotes the initiation of HTLV-1 transcription. Protein kinase A phosphorylation of CREB at serine 133 facilitates transcription from cellular CREs by recruiting CBP/p300 via its KIX domain. However, it remains controversial whether CREB phosphorylation plays a role in Tax transactivation. In this study, we biochemically characterized the quaternary complex formed by Tax, CREB, KIX, and the viral CRE by examining the individual molecular interactions that contribute to Tax stabilization in the complex. Our data show KIX, Ser(133)-phosphorylated CREB, and vCRE DNA are all required for stable Tax incorporation into the complex in vitro. Consonant with a fundamental role for CREB phosphorylation in Tax recruitment to the complex, we found that CREB is highly phosphorylated in a panel of HTLV-1-infected human T-cell lines. Significantly, we show that Tax is directly responsible for promoting elevated levels of CREB phosphorylation. Together, these data support a model in which Tax promotes CREB phosphorylation in vivo to ensure availability for Tax transactivation. Because pCREB has been implicated in leukemogenesis, enhancement of CREB phosphorylation by the virus may play a role in the etiology of adult T-cell leukemia.
Highlights
The Viral cyclic AMP-response elements (CREs) and phosphorylated CREB (pCREB) Enhance Tax Binding to the KIX Domain of CREB-binding protein (CBP)—Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) requires, in part, the formation of a viral CRE-bound complex composed of Tax, cAMP-response element-binding protein (CREB), and CBP/p300
To carry out this study, we first examined whether Tax binding to the KIX domain of CBP is affected by viral cyclic AMP response elements (vCREs) DNA and/or pCREB
We found that (i) the viral CRE DNA and strongly enhanced Tax binding to KIX, (ii) pCREB and strongly enhanced Tax binding to KIX, (iii) KIX and full-length p300 enhanced Tax binding to the pCREB1⁄7vCRE1⁄7DNA complex, and (iv) Ser133phosphorylated CREB is required for efficient Tax recruitment into the quaternary complex
Summary
Expression and Purification of Recombinant Proteins—Bacterially expressed CREB A [17], CREB S133A, Tax-His6 [18], and GST-KIX [8] proteins were purified to Ͼ95% homogeneity as previously described [8]. Bound proteins were resolved by electrophoresis on 10 or 12% SDS-polyacrylamide gels and transferred to nitrocellulose for subsequent Western blot analysis. Biotinylated double-stranded oligonucleotides containing a single CRE element were bound to streptavidin-agarose beads by incubating 90 min at 25 °C according to the manufacturer’s directions. DNA-bound proteins were separated by electrophoresis on a 10 or a 10 –20% gradient SDS gel and transferred to nitrocellulose for detection by Western blot analysis. Electrophoretic Mobility Shift Assays (EMSA)—EMSAs were performed by incubation of the indicated amount of purified CREB, Tax, or GST-KIX-(588 – 683) in 12.5 mM HEPES, pH 7.9, 75 mM KCl, 6.25 mM MgCl2, 10% (v/v) glycerol, 5 M ZnSO4, 0.05% (v/v) Nonidet P-40, and 0.5 mM EDTA containing 32P-end-labeled viral CRE probe and 250 ng/ml poly(dA)1⁄7poly(dT) in a 20-l reaction volume. All experiments presented in this report were shown to be reproducible in at least three independent trials
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