BONE-SPECIFIC EXPRESSION OF THE ALPHA-CHAIN OF THE NASCENT POLYPEPTIDE-ASSOCIATED COMPLEX, A COACTIVATOR POTENTIATING C-JUN-MEDIATED TRANSCRIPTION

The alpha chain of the nascent polypeptide-associated complex (alpha-N AC) coactivator was shown to potentiate the activity of the homodimeri c c-Jun activator, while transcription mediated by the c-Fos/c-Jun het erodimer was unaffected. The use of deletion mutants in pull down assa ys revealed that alpha-NAC interacted with amino acids 1 to 89 of the c-Jun protein and that the coactivator could interact with both the un phosphorylated and the serine 73-phosphorylated form of c-Jun. N-termi nal-deleted c-Jun protein failed to interact with alpha-NAC in mammali an two-hybrid assays, while mutant c-Jun proteins lacking the leucine zipper or the basic domain retained interaction with alpha-NAC in vivo . Kinetics studies with purified c-Jun homodimer and recombinant alpha -NAC proteins allowed determination of the mechanism of coactivation b y alpha-NAC: the coactivator stabilized the AP-1 complex formed by the c-Jun homodimer on its DNA recognition sequence through an eightfold reduction in the dissociation constant (k(d)) of the complex. This eff ect of alpha-NAC was specific, because alpha-NAC could not stabilize t he interactions of JunB or Sp1 with their cognate binding sites, Inter estingly, the expression of alpha-NAC was first detected at 14.5 to 15 days postconception, concomitantly with the onset of ossification dur ing embryogenesis. The alpha-NAC protein was specifically expressed in differentiated osteoblasts at the centers of ossification, Thus, the alpha-NAC gene product exhibits the properties of a developmentally re gulated, bone-specific transcriptional coactivator.