Abstract
The interactions between cAMP-response element-binding protein (CREB)-binding protein (CBP) and gene-specific transcription factors play an important role in activation of transcription from numerous genes. Cooperative interactions between CBP and multiple transcriptional activators may provide a mechanism for synergistic increases in transcriptional activation. Here we report the characterization of ternary complexes formed by the KIX domain of CBP and the transactivation domain of the trithorax group protein mixed lineage leukemia protein (MLL), together with either the phosphorylated kinase-inducible domain (pKID) of CREB or the activation domain from c-Myb. Isothermal titration calorimetry experiments show that KIX in complex with the MLL activation domain binds the c-Myb activation domain and pKID domain with 2-fold higher affinity than does the KIX domain alone. Thus, the activation domains of Myb and MLL or of pKID and MLL bind cooperatively to KIX. The thermodynamics of these interactions imply different mechanisms of binding cooperativity for the two ternary complexes; the KIX.MLL.pKID complex is stabilized by entropy increases, whereas the enhancement of Myb binding in the presence of the MLL activation domain is due to more favorable enthalpy. NMR experiments show that the MLL-binding site on KIX is distinct from the surface that binds the pKID and c-Myb activation domains. Our data indicate that KIX can directly mediate cooperative interactions between pairs of transcriptional regulatory proteins. In the case of MLL and c-Myb, both proteins are involved in proliferation of hematopoietic cells and leukemogenesis, and synergistic interactions mediated by CBP may play a functional role.
Highlights
The interactions between cAMP-response elementbinding protein (CREB)-binding protein (CBP) and gene-specific transcription factors play an important role in activation of transcription from numerous genes
Binding of mixed lineage leukemia protein (MLL) to KIX Measured by isothermal titration calorimetry (ITC)—To determine the thermodynamic parameters of MLL binding to the KIX domain of CBP, a 19-mer peptide that encompasses residues 2840 – 2858 (MLL19) was synthesized from the MLL activation domain
ITC experiments revealed that a phosphorylated peptide encompassing CREB residues 116 –149 binds to KIX with high affinity in the presence of nearly saturating amounts of MLL19
Summary
Sample Preparation—All peptides under 40 residues were prepared by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid phase peptide synthesis using a Pioneer Peptide Synthesis System (Perspective Biosystems). pKID34 (CREB residues 116 –149) is phosphorylated at Ser-133. Titration of pKID34, Myb, or MLL19 (ϳ0.6 mM) into KIX (50 M) was performed at °C using an MCS titration calorimeter from MicroCal Inc. Two injections of 5 l were followed by injections of 10 l until a molar ratio of ϳ2.5 peptide/protein was obtained. Ternary complex formation was monitored with a larger than 2-fold molar excess of MLL19 to KIX, ensuring that Ͼ90% of KIX was bound to MLL19 before titration of pKID34 or Myb. Complete backbone 15N, 1HN, 13C␣, and 13C chemical shift assignments (excluding the amide resonance from the N-terminal Gly-586) were obtained for each complex by NMRview analysis [37] of HNCACB and CBCA(CO)NH spectra [38] and by comparison with chemical shift assignments from free [33] and Myb25-bound [6] KIX. Complete backbone 15N, 1HN, 13C␣, and 13C chemical shift assignments (excluding the amide resonance from the N-terminal Gly-586) were obtained for each complex by NMRview analysis [37] of HNCACB and CBCA(CO)NH spectra [38] and by comparison with chemical shift assignments from free [33] and Myb25-bound [6] KIX. 1H, 13C, and 15N chemical shifts were referenced using a 2,2-dimethyl-2-silapentane-5sulfonate standard, and C␣ secondary shifts from peptide-derived random coil values [39] were calculated using NMRview
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