Characterization of a CREB gain-of-function mutant with constitutive transcriptional activity in vivo

The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene express ion, following its phosphorylation at Ser133, via recruitment of the coacti vator paralogs CREB-binding protein (CBP) and p300, CBP and p300, in turn, appear to mediate target gene induction via their association with RNA poly merase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide varie ty of stimuli, including hypoxia, UV irradiation, and growth factor additio n, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of p romoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-C BP complex formation or in the subsequent recruitment of the transcriptiona l apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wil d-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene e xpression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the K-m for PKA phosp horylation and thereby induces high levels of constitutive Ser133 phosphory lation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB s trongly promoted differentiation of PC12 cells in concert with suboptimal d oses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter.