¹Department of Biophysics, Boston University School of Medicine, Boston MA, USA;
²Oregon Health Sciences University, Portland,OR, USA;
³Protein Structure Facility, Shriners Hospital for Children, Portland, OR, USA;
⁴Department of Biochemistry and Molecular Biology, Rockefeller University, New York NY, USA
 

Abstract
      CREB-binding protein (CBP) and its homologues p300 are common or global transcription coactivators with histone/protein-acetyltransferase activity (HAT). They are tumor suppressors playing regulatory roles in cell growth, differentiation, development, and apoptosis.
      Phosphorylation is a major posttranslational modification that leads regulation of protein function. CBP/p300 is phosphorylated dynamically during cell cycle. It is essential to know the nature of the phosphorylation in order to understand the regulation of CBP/p300 function.
      Here presents the first identification of a major in vivo phosphorylation site in p300. In this work we performed a systemic study, combining traditional methods (2-D peptide mapping and point mutation analysis) with a novel approach (automatic sequencing of phosphorylated peptide followed by manual sequencing to identify phospho-residue) plus a new technique, using LC/ESI/MS/MS analysis, to determine the phospho-residue within the endogenous p300 protein directly.

¹Istituto Biologia Cellulare and ²Istituto Tecnologie Biomediche, CNR, Rome, Italy;
³Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, USA;
⁴Istituto Regina Elena, Rome, Italy
 

Abstract
      We have demonstrated that the muscle-specific transcription factor MyoD induces the retinoblastoma gene (Rb1) promoter.  In the present work, we demonstrate that the MyoD-mediated induction of the Rb1 promoter does not require new protein synthesis.  Moreover, we identified the minimal Rb1 promoter region targeted by MyoD as a 50-bp sequence containing the binding sites of three families of transcription factors: E2F, ATF/CREB and E4TF1.  Site-directed mutagenesis of these sites indicated that: 1. the E4TF1 binding site is required for the promoter basal activity; 2. the ATF/CREB site is important for both the basal and MyoD-induced promoter activity; 3. the E2F site modulates the basal promoter activity
      By means of band shift experiments, we determined that the transcription factor CREB binds the ATF/CREB site present in the Rb1 promoter and that both the CBP and p300 transcriptional coactivators participate to the DNA-bound complex.  These results togather with our previous finding that MyoD and p300 can physically and functionally interact, suggest that MyoD might induce the activity of CREB through binding to the p300 transcriptional coactivator.