Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300. with Tax for KIX binding, suggesting a novel mechanism of Tax oncogenesis in which normal MLL function is disrupted by Tax. found that MLL binds a distinctly separate surface of KIX than the c-Myb/pKID binding surface.17 These observations suggest the pCREB/Tax/KIX ternary complex may be analogous to the c-Myb/MLL/KIX ternary complex. Another transcription factor, c-Jun, has been shown to occupy the MLL binding site on KIX. 19 Tax was found to compete with c-Jun for KIX binding, 18 providing further support for a two-site binding model in which Tax and pCREB simultaneously bind separate surfaces of KIX. c-Myb and MLL have also been shown to assemble cooperatively with KIX,20 while similarly, KIX binds Tax most effectively when it is assembled into a complex with full-length pCREB and viral CRE DNA.21 While both the pKID domain of CREB and a small N-terminal Tax peptide22 have been shown to interact with separate surfaces on KIX, the full-length proteins have not previously been used to characterize these binding sites, nor have they been examined together. In this report we sought to better characterize the interaction of Tax and KIX in the context of the physiologically relevant quaternary complex that also contains pCREB and vCRE DNA. We show that full-length pCREB and Tax bind KIX at two separate sites. MLL competition with Tax for KIX binding demonstrates Tax and MLL share the same interaction surface of KIX. In addition, KIX constructs carrying mutations in one of the two transcription factor binding sites have enabled us to distinguish independent Tax and pCREB binding to KIX. These findings support a model in which KIX binds the ternary complex composed of full-length Tax, pCREB, and vCRE DNA through simultaneous interaction with both Tax and pCREB on separate interfaces. Our observations also suggest a novel mechanism for Tax oncogenesis through disruption of MLL function. Results Tax and pCREB simultaneously bind the KIX domain of CBP Our first objective was to determine whether full-length pCREB and Tax bind KIX both simultaneously and independently. We reasoned that if Tax and pCREB simultaneously bind to two sites on KIX, saturated Dimethyl phthalate binding of one protein to KIX would have no effect on the binding of the second protein. Purified GST-KIX (CBP aa 588C683) was bound to glutathione-agarose beads and used in a GST pull-down assay. Increasing amounts of purified Tax were added to binding reactions until saturation of GST-KIX was achieved (Figure 1A). We performed several experiments to establish that this concentration of Tax saturated GST-KIX (data not shown). pCREB was then titrated into reactions containing the highest concentration of Tax (Figure 1A). pCREB bound to GST-KIX without displacing Tax. The converse experiment was then performed Dimethyl phthalate in which immobilized GST-KIX was incubated with increasing amounts of pCREB until saturation was achieved DNAJC15 (Figure 1B). As before, several experiments were performed to determine the concentration of Dimethyl phthalate pCREB that saturated GST-KIX (data not shown). vCRE DNA was also included in these binding reactions as we were interested in examining the binding of Tax to GST-KIX in the context of the more physiologically-relevant ternary complex (pCREB/Tax/vCRE DNA) (Figure 1C). For simplicity, figure 1C depicts one KIX molecule binding to the ternary complex though it is unknown whether one or two KIX molecules bind the complex. Tax was then titrated into reactions containing the highest concentration of pCREB (Figure 1B). Tax bound to GST-KIX with no.
Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300
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