Characterization of HTLV-1 Tax molecular interaction with the KIX domain of CBP

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of a rare, aggressive form of cancer called adult T-cell leukemia (ATL). The HTLV-1 oncoprotein Tax and the cellular transcription factor CREB bind to viral cyclic AMP response elements (vCREs) located in the viral promoter. 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. PKA-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. We biochemically characterized the quaternary complex formed by Tax, CREB, the KIX domain of CBP, and the viral CRE by examining the individual molecular interactions that contribute to Tax stabilization in the complex. Our data show Ser133-phosphorylated CREB, KIX, and vCRE DNA are all required for stable Tax incorporation into the complex. While the interaction between the phosphorylated kinase inducible domain (pKID) of pCREB and the KIX domain of CBP/p300 has been well-characterized, the molecular interactions between KIX, full-length Tax, and pCREB have not been examined. We further characterized the quaternary complex by examining the interaction between Tax and KIX in a physiologically relevant complex containing pCREB and vCRE DNA. Our data show that Tax and pCREB simultaneously and independently bind two distinct surfaces on the KIX domain: Tax binds KIX at the previously-characterized mixed-lineage leukemia (MLL) protein interaction surface while pCREB binds KIX at the well-known pKID-KIX interface. These results provide evidence for a model in which Tax and pCREB bind distinct surfaces of KIX for effective CBP/p300 recruitment to the HTLV-1 promoter. During investigation of the dual binding site model, we found that a small peptide representing the minimal MLL activation domain competed with Tax for KIX binding. Tax may therefore compete with MLL for CBP/p300 binding, affecting transcription of MLL-responsive promoters in HTLV-1 infected cells expressing Tax. Chromosomal rearrangements causing MLL dysfunction characterize certain types of leukemia, suggesting a novel mechanism of Tax leukemogenesis in which normal MLL function is disrupted by Tax.